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Sample records for active efflux transport

  1. Neuroinflammation activates efflux transport by NFκB

    PubMed Central

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

    2009-01-01

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

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

    PubMed

    Shennan, D B; McNeillie, S A

    1995-04-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

    Sajja, Ravi K; Cucullo, Luca

    2015-06-26

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

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

    PubMed

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

    2017-01-03

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

  6. Effluxing ABC Transporters in Human Corneal Epithelium

    PubMed Central

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

    2010-01-01

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

  7. 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.

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    2007-03-01

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

  10. 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

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

    PubMed

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

    2015-08-13

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

  12. 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

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

    PubMed Central

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

    2015-01-01

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

  14. 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

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

    PubMed Central

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

    2013-01-01

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

  16. 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

  17. 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.

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

    PubMed

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

    2002-01-15

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

  19. Getting pumped: membrane efflux transporters for enhanced biomolecule production.

    PubMed

    Boyarskiy, Sergey; Tullman-Ercek, Danielle

    2015-10-01

    Small molecule production in microbial hosts is limited by the accumulation of the product inside the cell. Efflux transporters show promise as a solution to removal of the often-toxic products. Recent advances in transporter identification through expression profiling, heterologous expression, and knockout studies have identified transporters capable of secreting compounds of biotechnological interest. In addition, engineering of well-studied transporters has shown that substrate specificity in these transporters is malleable. Future work in identification, engineering, and expression of small molecule exporters can be instrumental in expanding the biocatalysis portfolio.

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

    PubMed

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

    2008-06-15

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

  1. Heavy metal transport by the CusCFBA efflux system

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-03-10

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins.

  3. Pregnancy Represses Induction of Efflux Transporters in Livers of Type I Diabetic Mice

    PubMed Central

    Aleksunes, Lauren M.; Xu, Jialin; Lin, Eugenia; Wen, Xia; Goedken, Michael J.; Slitt, Angela L.

    2013-01-01

    Purpose The purpose of this study was to determine whether down-regulation of transcription factor signaling during pregnancy disrupts the induction of efflux transporters in type I diabetic mice. Methods Type I diabetes was induced in female C57BL/6 mice with multiple low dose intraperitoneal injections of streptozotocin (STZ) at least 2 weeks prior to mating with normoglycemic male mice. On gestation day 14, livers were collected from vehicle- and STZ-treated non-pregnant and pregnant mice for quantification of efflux transporter and transcription factor signaling. Results STZ treatment up-regulated expression of Mrp1–5, Mdr1, Abcg5, Abcg8, Bcrp, and Bsep mRNA and/or protein in the livers of non-pregnant mice. Interestingly, little to no change in transporter expression was observed in STZ-treated mice that became pregnant. Enhanced PPARγ, Nrf2, and FXR transcription factor signaling, as quantified by nuclear binding and target gene regulation, was also observed in non-pregnant mice treated with STZ. Similar to efflux transporter expression, activation of these transcriptional pathways was diminished by pregnancy in STZ-treated mice. Conclusions This study demonstrates the opposing regulation of hepatobiliary efflux transporters in response to diabetes and pregnancy and points to PPARγ, Nrf2, and FXR as candidate pathways underlying the differential expression of transporters. PMID:23319174

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

    PubMed Central

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

    2015-01-01

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

  5. Intestinal permeability and P-glycoprotein-mediated efflux transport of ticagrelor in Caco-2 monolayer cells.

    PubMed

    Marsousi, Niloufar; Doffey-Lazeyras, Fabienne; Rudaz, Serge; Desmeules, Jules A; Daali, Youssef

    2016-12-01

    Ticagrelor is the unique reversible oral antiplatelet drug commercialized today. During this study, the intestinal permeability of ticagrelor and its potential P-glycoprotein (P-gp)-mediated active transport were assessed. To this end, bidirectional transport of ticagrelor was performed across Caco-2 (human epithelial colorectal adenocarcinoma) monolayer model in the presence and absence of potent P-gp inhibitor valspodar. Ticagrelor presented an apical-basolateral apparent permeability coefficient (Papp ) of 6.0 × 10(-6) cm/s. On the other hand, mean efflux ratio (ER) of 2.71 was observed for ticagrelor describing a higher efflux permeability compared to the influx component. Valspodar showed a significant inhibitory effect on the efflux of ticagrelor suggesting involvement of P-gp in its oral disposition. Co-incubation of the P-gp inhibitor decreased the efflux Papp of ticagrelor from 1.60 × 10(-5) to 1.13 × 10(-5) cm/s and decreased its ER by 70%. Results suggest a modest active transport of ticagrelor by P-gp across the Caco-2 cell monolayer. The co-administration of ticagrelor with a P-gp inhibitor seems altogether unlikely to have an extended impact on pharmacokinetics of ticagrelor and cause bleeding events in patients.

  6. 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.

  7. 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

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

    PubMed

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

    2013-02-01

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

  9. 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.

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

    PubMed

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

    2015-02-01

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

  11. Human paraoxonase 1 overexpression in mice stimulates HDL cholesterol efflux and reverse cholesterol transport

    PubMed Central

    Ikhlef, Souade; Berrougui, Hicham; Kamtchueng Simo, Olivier; Zerif, Echarki

    2017-01-01

    This study was aimed to investigate the effect of human PON1 overexpression in mice on cholesterol efflux and reverse cholesterol transport. PON1 overexpression in PON1-Tg mice induced a significant 3-fold (p<0.0001) increase in plasma paraoxonase activity and a significant ~30% (p<0.0001) increase in the capacity of HDL to mediate cholesterol efflux from J774 macrophages compared to wild-type mice. It also caused a significant 4-fold increase (p<0.0001) in the capacity of macrophages to transfer cholesterol to apoA-1, a significant 2-fold (p<0.0003) increase in ABCA1 mRNA and protein expression, and a significant increase in the expression of PPARγ (p<0.0003 and p<0.04, respectively) and LXRα (p<0.0001 and p<0.01, respectively) mRNA and protein compared to macrophages from wild-type mice. Moreover, transfection of J774 macrophages with human PON1 also increased ABCA1, PPARγ and LXRα protein expression and stimulates macrophages cholesterol efflux to apo A1. In vivo measurements showed that the overexpression of PON1 significantly increases the fecal elimination of macrophage-derived cholesterol in PON1-Tg mice. Overall, our results suggested that the overexpression of PON1 in mice may contribute to the regulation of the cholesterol homeostasis by improving the capacity of HDL to mediate cholesterol efflux and by stimulating reverse cholesterol transport. PMID:28278274

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

    PubMed Central

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

    2010-01-01

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

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

    SciTech Connect

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

    2010-09-23

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

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

    PubMed

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

    2016-01-01

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

  15. 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

  16. 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

  17. ATP-Binding Cassette Efflux Transporters in Human Placenta

    PubMed Central

    Ni, Zhanglin; Mao, Qingcheng

    2010-01-01

    Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

  18. Amphipathic polyproline peptides stimulate cholesterol efflux by the ABCA1 transporter.

    PubMed

    Sviridov, D O; Drake, S K; Freeman, L A; Remaley, A T

    2016-03-18

    ApoA-I mimetics are short synthetic peptides that contain an amphipathic α-helix and stimulate cholesterol efflux by the ABCA1 transporter in a detergent-like extraction mechanism. We investigated the use of amphipathic peptides with a polypro helix for stimulating cholesterol efflux by ABCA1. Polypro peptides were synthesized with modified prolines, containing either a hydrophobic phenyl group (Prop) or a polar N-acetylgalactosamine (Prog) attached to the pyrrolidine ring and were designated as either PP-2, 3, 4, or 5, depending on the number of 3 amino acid repeat units (Prop-Prog-Prop). Based on molecular modeling, these peptides were predicted to be relatively rigid and to bind to a phospholipid bilayer. By CD spectroscopy, PP peptides formed a Type-II polypro helix in an aqueous solution. PP-2 was inactive in promoting cholesterol efflux, but peptides with more than 2 repeat units were active. PP-4 showed a similar Vmax as a much longer amphipathic α-helical peptide, containing 37 amino acids, but had a Km that was approximately 20-fold lower. PP peptides were specific in that they did not stimulate cholesterol efflux from cells not expressing ABCA1 and were also non-cytotoxic. Addition of PP-3, 4 and 5 to serum promoted the formation of smaller size HDL species (7 nM) and increased its capacity for ABCA1-dependent cholesterol efflux by approximately 20-35% (p < 0.05). Because of their relatively small size and increased potency, amphipathic peptides with a polypro helix may represent an alternative structural motif for the development of apoA-I mimetic peptides.

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

    PubMed Central

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

    2007-01-01

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

  20. Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport

    PubMed Central

    Kuteykin-Teplyakov, Konstantin; Luna-Tortós, Carlos; Ambroziak, Kamila; Löscher, Wolfgang

    2010-01-01

    Background and purpose: P-glycoprotein (Pgp) efflux assays are widely used to identify Pgp substrates. The kidney cell lines Madin-Darby canine kidney (MDCK)-II and LLC-PK1, transfected with human MDR1 (ABCB1) are used to provide recombinant models of drug transport. Endogenous transporters in these cells may contribute to the activities of recombinant transporters, so that drug transport in MDR1-transfected cells is often corrected for the transport obtained in parental (wildtype) cells. However, expression of endogenous transporters may vary between transfected and wildtype cells, so that this correction may cause erroneous data. Here, we have measured the expression of endogenous efflux transporters in transfected and wildtype MDCK-II or LLC cells and the consequences for Pgp-mediated drug transport. Experimental approach: Using quantitative real-time RT-PCR, we determined the expression of endogenous Mdr1 mRNA and other efflux transporters in wildtype and MDR1-transfected MDCK-II and LLC cells. Transcellular transport was measured with the test substrate vinblastine. Key results: In MDR1-transfected MDCK cells, expression of endogenous (canine) Mdr1 and Mrp2 (Abcc2) mRNA was markedly lower than in wildtype cells, whereas MDR1-transfected LLC cells exhibited comparable Mdr1 but strikingly higher Mrp2 mRNA levels than wildtype cells. As a consequence, transport of vinblastine by human Pgp in efflux experiments was markedly underestimated when transport in MDR1-transfected MDCK cells was corrected for transport obtained in wildtype cells. This problem did not occur in LLC cells. Conclusions and implications: Differences in the expression of endogenous efflux transporters between transfected and wildtype MDCK cells provide a potential bias for in vitro studies on Pgp-mediated drug transport. PMID:20590635

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

    PubMed

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

    2010-03-01

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

  2. In Vivo Exposure of Kaempferol Is Driven by Phase II Metabolic Enzymes and Efflux Transporters.

    PubMed

    Zheng, Liang; Zhu, Lijun; Zhao, Min; Shi, Jian; Li, Yuhuan; Yu, Jia; Jiang, Huangyu; Wu, Jinjun; Tong, Yunli; Liu, Yuting; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2016-09-01

    Kaempferol is a well-known flavonoid; however, it lacks extensive pharmacokinetic studies. Phase II metabolic enzymes and efflux transporters play an important role in the disposition of flavonoids. This study aimed to investigate the mechanism by which phase II metabolic enzymes and efflux transporters determine the in vivo exposure of kaempferol. Pharmacokinetic analysis in Sprague-Dawley rats revealed that kaempferol was mostly biotransformed to conjugates, namely, kaempferol-3-glucuronide (K-3-G), kaempferol-7-glucuronide (K-7-G), and kaempferol-7-sulfate, in plasma. K-3-G represented the major metabolite. Compared with that in wild-type mice, pharmacokinetics in knockout FVB mice demonstrated that the absence of multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP) significantly increased the area under the curve (AUC) of the conjugates. The lack of MRP1 resulted in a much lower AUC of the conjugates. Intestinal perfusion in rats revealed that the glucuronide conjugates were mainly excreted in the small intestine, but 7-sulfate was mainly excreted in the colon. In Caco-2 monolayers, K-7-G efflux toward the apical (AP) side was significantly higher than K-3-G efflux. In contrast, K-3-G efflux toward the basolateral (BL) side was significantly higher than K-7-G efflux. The BL-to-AP efflux was significantly reduced in the presence of the MRP2 inhibitor LTC4. The AP-to-BL efflux was significantly decreased in the presence of the BL-side MRPs inhibitor MK571. The BCRP inhibitor Ko143 decreased the glucuronide conjugate efflux. Therefore, kaempferol is mainly exposed as K-3-G in vivo, which is driven by phase II metabolic enzymes and efflux transporters (i.e., BCRP and MRPs).

  3. Identification of Transport Proteins Involved in Free Fatty Acid Efflux in Escherichia coli

    PubMed Central

    Lennen, Rebecca M.; Politz, Mark G.; Kruziki, Max A.

    2013-01-01

    Escherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB+ strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts. PMID:23104810

  4. 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

  5. Effect of drug efflux transporters on placental transport of antiretroviral agent abacavir.

    PubMed

    Neumanova, Zuzana; Cerveny, Lukas; Greenwood, Susan L; Ceckova, Martina; Staud, Frantisek

    2015-11-01

    Abacavir is as a frequent part of combination antiretroviral therapy used in pregnant women. The aim of this study was to investigate, using in vitro, in situ and ex vivo experimental approaches, whether the transplacental pharmacokinetics of abacavir is affected by ATP-binding cassette (ABC) efflux transporters functionally expressed in the placenta: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), multidrug resistance-associated protein 2 (ABCC2) and multidrug resistance-associated protein 5 (ABCC5). In vitro transport assays revealed that abacavir is a substrate of human ABCB1 and ABCG2 transporters but not of ABCC2 or ABCC5. In addition, in situ experiments using dually perfused rat term placenta confirmed interactions of abacavir with placental Abcb1/Abcg2. In contrast, uptake studies in human placental villous fragments did not reveal any interaction of abacavir with efflux transporters suggesting a large contribution of passive diffusion and/or influx mechanisms to net transplacental abacavir transfer.

  6. 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

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

    PubMed

    Hasdemir, Ufuk

    2007-04-01

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

  8. Role of human placental apical membrane transporters in the efflux of glyburide, rosiglitazone, and metformin

    PubMed Central

    HEMAUER, Sarah J.; PATRIKEEVA, Svetlana L.; NANOVSKAYA, Tatiana N.; HANKINS, Gary D.V.; AHMED, Mahmoud S.

    2010-01-01

    Objective Substrates of placental efflux transporters could compete for a single transporter, which could result in an increase in the transfer of each substrate to the fetal circulation. Our aim was to determine the role of placental transporters in the biodisposition of oral hypoglycemic drugs that could be used as monotherapy or in combination therapy for gestational diabetes. Study design Inside-out brush border membrane vesicles from term placentas were used to determine the efflux of glyburide, rosiglitazone, and metformin by P-gp, Breast Cancer Resistance Protein (BCRP), and Multidrug Resistance Protein (MRP1). Results Glyburide was transported by MRP1 (43 ± 4%); BCRP (25 ± 5%); and P-gp (9 ± 5%). Rosiglitazone was transported predominantly by P-gp (71 ± 26%). Metformin was transported by P-gp (58 ± 20%) and BCRP (25 ± 14%). Conclusion Multiple placental transporters contribute to efflux of glyburide, rosiglitazone, and metformin. Administration of drug combinations could lead to their competition for efflux transporters. PMID:20350646

  9. Establishment of optimized MDCK cell lines for reliable efflux transport studies.

    PubMed

    Gartzke, Dominik; Fricker, Gert

    2014-04-01

    Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results.

  10. The Phytosiderophore Efflux Transporter TOM2 Is Involved in Metal Transport in Rice*

    PubMed Central

    Nozoye, Tomoko; Nagasaka, Seiji; Kobayashi, Takanori; Sato, Yuki; Uozumi, Nobuyuki; Nakanishi, Hiromi; Nishizawa, Naoko K.

    2015-01-01

    Iron is an essential metal element for all living organisms. Graminaceous plants produce and secrete mugineic acid family phytosiderophores from their roots to acquire iron in the soil. Phytosiderophores chelate and solubilize insoluble iron hydroxide in the soil. Subsequently, plants take up iron-phytosiderophore complexes through specific transporters on the root cell membrane. Phytosiderophores are also thought to be important for the internal transport of various transition metals, including iron. In this study, we analyzed TOM2 and TOM3, rice homologs of transporter of mugineic acid family phytosiderophores 1 (TOM1), a crucial efflux transporter directly involved in phytosiderophore secretion into the soil. Transgenic rice analysis using promoter-β-glucuronidase revealed that TOM2 was expressed in tissues involved in metal translocation, whereas TOM3 was expressed only in restricted parts of the plant. Strong TOM2 expression was observed in developing tissues during seed maturation and germination, whereas TOM3 expression was weak during seed maturation. Transgenic rice in which TOM2 expression was repressed by RNA interference showed growth defects compared with non-transformants and TOM3-repressed rice. Xenopus laevis oocytes expressing TOM2 released 14C-labeled deoxymugineic acid, the initial phytosiderophore compound in the biosynthetic pathway in rice. In onion epidermal and rice root cells, the TOM2-GFP fusion protein localized to the cell membrane, indicating that the TOM2 protein is a transporter for phytosiderophore efflux to the cell exterior. Our results indicate that TOM2 is involved in the internal transport of deoxymugineic acid, which is required for normal plant growth. PMID:26432636

  11. Suppression of asymmetric acid efflux and gravitropism in maize roots treated with auxin transport inhibitors of sodium orthovanadate

    NASA Technical Reports Server (NTRS)

    Mulkey, T. J.; Evans, M. L.

    1982-01-01

    In gravitropically stimulated roots of maize (Zea mays L., hybrid WF9 x 38MS), there is more acid efflux on the rapidly growing upper side than on the slowly growing lower side. In light of the Cholodny/Went hypothesis of gravitropism which states that gravitropic curvature results from lateral redistribution of auxin, the effects of auxin transport inhibitors on the development of acid efflux asymmetry and curvature in gravistimulated roots were examined. All the transport inhibitors tested prevented both gravitropism and the development of asymmetric acid efflux in gravistimulated roots. The results indicate that auxin redistribution may cause the asymmetry of acid efflux, a finding consistent with the Cholodny/Went hypothesis of gravitropism. As further evidence that auxin-induced acid efflux asymmetry may mediate gravitropic curvature, sodium orthovanadate, an inhibitor of auxin-induced H+ efflux was found to prevent both gravitropism and the development of asymmetric acid efflux in gravistimulated roots.

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

    PubMed

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

    2012-05-01

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

  13. Bardoxolone methyl modulates efflux transporter and detoxifying enzyme expression in cisplatin-induced kidney cell injury.

    PubMed

    Atilano-Roque, Amandla; Aleksunes, Lauren M; Joy, Melanie S

    2016-09-30

    Cisplatin is prescribed for the treatment of solid tumors and elicits toxicity to kidney tubules, which limits its clinical use. Nuclear factor erythroid 2-related factor 2 (Nrf2, NFE2L2) is a critical transcription factor that has been shown to protect against kidney injury through activation of antioxidant mechanisms. We aimed to evaluate the ability of short-term treatment with the Nrf2 activator bardoxolone methyl (CDDO-Me) to protect against cisplatin-induced kidney cell toxicity. Cell viability was assessed in human kidney proximal tubule epithelial cells (hPTCs) exposed to low, intermediate, and high cisplatin concentrations in the presence and absence of CDDO-Me, administered either prior to or after cisplatin. Treatment with cisplatin alone resulted in reductions in hPTC viability, while CDDO-Me administered prior to or after cisplatin exposure yielded significantly higher cell viability (17%-71%). Gene regulation (mRNA expression) studies revealed the ability of CDDO-Me to modify protective pathways including Nrf2 induced detoxifying genes [GCLC (increased 1.9-fold), NQO1 (increased 9.3-fold)], and an efflux transporter [SLC47A1 (increased 4.5-fold)] at 12h. Protein assessments were in agreement with gene expression. Immunofluorescence revealed localization of GCLC and NQO1 to the nucleus and cytosol, respectively, with CDDO-Me administered prior to or after cisplatin exposure. The findings of enhanced cell viability and increased expression of detoxifying enzymes (GCLC and NQO1) and the multidrug and toxin extrusion protein 1 (MATE1) efflux transporter (SLC47A1) in hPTCs exposed to CDDO-Me, suggest that intermittent treatment with CDDO-Me prior to or after cisplatin exposure may be a promising approach to mitigate acute kidney injury.

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

    PubMed Central

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

    2015-01-01

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

  15. Transporter-mediated Efflux Influences CNS Side Effects: ABCB1, from Antitarget to Target

    PubMed Central

    Broccatelli, Fabio; Carosati, Emanuele; Cruciani, Gabriele; Oprea, Tudor I.

    2012-01-01

    We examined the relationship between sedation and orthostatic hypotension, two central side effects and ABCB1 transporter-mediated efflux for a set of 64 launched drugs that are documented as histamine H1 receptor antagonists. This relationship was placed in the context of passive diffusion (estimated using LogP, the octanol/water partition coefficient), receptor affinity, and the adjusted therapeutic daily dose, in order to account for side effect variability. Within this set, CNS permeability was not dependent on passive diffusion, as no significant differences were found for LogP and its pH-corrected equivalent, LogD74. Sedation and orthostatic hypotension can be explained within the framework of ABCB1-mediated efflux and adjusted dose, while target potency has less influence. ABCB1, an antitarget for anti-cancer agents, acts in fact as a drug target for non-sedating antihistamines. An empirical set of rules, based on the incidence of these two side-effects, target affinity and dose was used to predict efflux effects for a number of drugs. Among them, azelastine and mizolastine are predicted to be effluxed via ABCB1-mediated transport, whereas aripiprazole, clozapine, cyproheptadine, iloperidone, olanzapine, and ziprasidone are likely to be non-effluxed. PMID:22347894

  16. Bacterial glyphosate resistance conferred by overexpression of an E. coli membrane efflux transporter.

    PubMed

    Staub, Jeffrey M; Brand, Leslie; Tran, Minhtien; Kong, Yifei; Rogers, Stephen G

    2012-04-01

    Glyphosate herbicide-resistant crop plants, introduced commercially in 1994, now represent approximately 85% of the land area devoted to transgenic crops. Herbicide resistance in commercial glyphosate-resistant crops is due to expression of a variant form of a bacterial 5-enolpyruvylshikimate-3-phosphate synthase with a significantly decreased binding affinity for glyphosate at the target site of the enzyme. As a result of widespread and recurrent glyphosate use, often as the only herbicide used for weed management, increasing numbers of weedy species have evolved resistance to glyphosate. Weed resistance is most often due to changes in herbicide translocation patterns, presumed to be through the activity of an as yet unidentified membrane transporter in plants. To provide insight into glyphosate resistance mechanisms and identify a potential glyphosate transporter, we screened Escherichia coli genomic DNA for alternate sources of glyphosate resistance genes. Our search identified a single non-target gene that, when overexpressed in E. coli and Pseudomonas, confers high-level glyphosate resistance. The gene, yhhS, encodes a predicted membrane transporter of the major facilitator superfamily involved in drug efflux. We report here that an alternative mode of glyphosate resistance in E. coli is due to reduced accumulation of glyphosate in cells that overexpress this membrane transporter and discuss the implications for potential alternative resistance mechanisms in other organisms such as plants.

  17. Transplacental pharmacokinetics of glyburide, rhodamine 123, and BODIPY FL prazosin: effect of drug efflux transporters and lipid solubility.

    PubMed

    Cygalova, Lenka Hahnova; Hofman, Jakub; Ceckova, Martina; Staud, Frantisek

    2009-12-01

    Breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp) are the most abundantly expressed ATP-binding cassette (ABC) drug transporters in the placenta. They recognize a large, partly overlapping spectrum of chemically unrelated compounds and affect their transplacental passage. In this study we investigate the effect of Bcrp and P-gp on the transplacental pharmacokinetics of their specific and common substrates employing the technique of dually perfused rat placenta. We show that the clearance of rhodamine 123 (P-gp substrate), glyburide (BCRP substrate) and BODIPY FL prazosin (P-gp and BCRP substrate) in fetal-to-maternal direction is 11, 11.2 and 4 times higher, respectively, than that in the maternal-to-fetal direction. In addition, all of these substances were found to be transported from the fetal compartment even against concentration gradient. We thus demonstrate the ability of placental ABC transporters to hinder maternal-to-fetal and accelerate fetal-to-maternal transport in a concentration-dependent manner. However, by means of pharmacokinetic modeling we describe the inverse correlation between lipid solubility of a molecule and its active transport by placental ABC efflux transporters. Therefore, in the case of highly lipophilic substrates, such as BODIPY FL prazosin in this study, the efficacy of efflux transporters to pump the molecule back to the maternal circulation is markedly limited.

  18. Influence of Efflux Transporters on the Accumulation and Efflux of Four Quinolones (Ciprofloxacin, Levofloxacin, Garenoxacin, and Moxifloxacin) in J774 Macrophages

    PubMed Central

    Michot, Jean-Michel; Seral, Cristina; Van Bambeke, Françoise; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M.

    2005-01-01

    Ciprofloxacin is subject to efflux from J774 macrophages through a multidrug resistance-related protein-like transporter (J. M. Michot, F. Van Bambeke, M. P. Mingeot-Leclercq, and P. M. Tulkens, Antimicrob. Agents Chemother. 48:2673-2682, 2004). Here, we compare ciprofloxacin to levofloxacin, garenoxacin, and moxifloxacin for transport. At 4 mg/liter, an apparent steady state in accumulation was reached after 30 to 60 min for all quinolones but to quite different levels (approximately 3, 5, 10, and 16 fold). Accumulation of ciprofloxacin was increased (to about 16 to 20 fold) by ATP depletion, increase in extracellular concentration, and the addition of probenecid, gemfibrozil, or MK571 (but not verapamil or GF120918). These treatments did not affect the accumulation of moxifloxacin. Levofloxacin and garenoxacin showed an intermediate behavior. Efflux of ciprofloxacin was slowed down by probenecid (half-life, 7.2 versus 1.6 min). Moxifloxacin efflux was faster and unaffected by probenecid (half-lifes, 0.27 versus 0.33 min). Efflux of levofloxacin and garenoxacin was modestly decreased by probenecid (1.5 and 2.1 fold). Accumulation of 14C-labeled ciprofloxacin was increased by unlabeled ciprofloxacin and moxifloxacin, but moxifloxacin was two times less potent. Accumulation of moxifloxacin at 4°C was almost identical to that at 37°C, whereas that of ciprofloxacin was minimal (levofloxacin and garenoxacin showed intermediate behaviors). Cells subjected to thermal shock (56°C; 10 min) accumulated all quinolones at a similar level (16 to 23 fold). We conclude that moxifloxacin is apparently not subject to efflux from J774 macrophages, even though it can interact with the ciprofloxacin transporter. Levofloxacin and garenoxacin are partially effluxed. Data suggest that efflux plays an important role in the differential accumulation of quinolones by J774 macrophages. PMID:15917543

  19. Evidence for regulation of polar auxin transport at the efflux carrier in maize coleoptile sections

    SciTech Connect

    Vesper, M.J. )

    1989-04-01

    Previously we have shown that conditions which result in an increased auxin-induced growth response in maize (Zea mays L.) coleoptile sections also result in a decrease in the velocity of polar auxin transport. Coleoptile sections given conditions which result in slower transport of IAA have different kinetics for net IAA accumulation compared to sections given conditions which result in faster transport. In further experiments, sections were loaded with 30 nM ({sup 3}H)IAA in the presence of increasing unlabeled IAA at low pH. Efflux of ({sup 3}H)IAA was then followed as a function of unlabeled IAA. Saturation of efflux appears to occur at a lower conc. of IAA in sections showing slower transport.

  20. Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter.

    PubMed

    Sviridov, D O; Ikpot, I Z; Stonik, J; Drake, S K; Amar, M; Osei-Hwedieh, D O; Piszczek, G; Turner, S; Remaley, A T

    2011-07-08

    Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.

  1. 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

  2. Switch-Loop Flexibility Affects Transport of Large Drugs by the Promiscuous AcrB Multidrug Efflux Transporter

    PubMed Central

    Cha, Hi-jea; Müller, Reinke T.

    2014-01-01

    Multidrug efflux transporters recognize a variety of structurally unrelated compounds for which the molecular basis is poorly understood. For the resistance nodulation and cell division (RND) inner membrane component AcrB of the AcrAB-TolC multidrug efflux system from Escherichia coli, drug binding occurs at the access and deep binding pockets. These two binding areas are separated by an 11-amino-acid-residue-containing switch loop whose conformational flexibility is speculated to be essential for drug binding and transport. A G616N substitution in the switch loop has a distinct and local effect on the orientation of the loop and on the ability to transport larger drugs. Here, we report a distinct phenotypical pattern of drug recognition and transport for the G616N variant, indicating that drug substrates with minimal projection areas of >70 Å2 are less well transported than other substrates. PMID:24914123

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

    PubMed

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

    2014-06-19

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

  4. The boron efflux transporter ROTTEN EAR is required for maize inflorescence development and fertility.

    PubMed

    Chatterjee, Mithu; Tabi, Zara; Galli, Mary; Malcomber, Simon; Buck, Amy; Muszynski, Michael; Gallavotti, Andrea

    2014-07-01

    Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs.

  5. Quantitative evaluation of the combination between cytotoxic drug and efflux transporter inhibitors based on a tumour growth inhibition model.

    PubMed

    Sostelly, Alexandre; Payen, Léa; Guitton, Jérôme; Di Pietro, Attilio; Falson, Pierre; Honorat, Mylène; Boumendjel, Ahcène; Gèze, Annabelle; Freyer, Gilles; Tod, Michel

    2014-04-01

    ATP-Binding Cassette transporters such as ABCG2 confer resistance to various anticancer drugs including irinotecan and its active metabolite, SN38. Early quantitative evaluation of efflux transporter inhibitors-cytotoxic combination requires quantitative drug-disease models. A proof-of-concept study has been carried out for studying the effect of a new ABCG2 transporter inhibitor, MBLI87 combined to irinotecan in mice xenografted with cells overexpressing ABCG2. Mice were treated with irinotecan alone or combined to MBLI87, and tumour size was periodically measured. To model those data, a tumour growth inhibition model was developed. Unperturbed tumour growth was modelled using Simeoni's model. Drug effect kinetics was accounted for by a Kinetic-Pharmacodynamic approach. Effect of inhibitor was described with a pharmacodynamic interaction model where inhibitor enhances activity of cytotoxic. This model correctly predicted tumour growth dynamics from our study. MBLI87 increased irinotecan potency by 20% per μmol of MBLI87. This model retains enough complexity to simultaneously describe tumour growth and effect of this type of drug combination. It can thus be used as a template to early evaluate efflux transporter inhibitors in-vivo.

  6. PPAR-α, a lipid-sensing transcription factor, regulates blood-brain barrier efflux transporter expression.

    PubMed

    More, Vijay R; Campos, Christopher R; Evans, Rebecca A; Oliver, Keith D; Chan, Gary Ny; Miller, David S; Cannon, Ronald E

    2017-04-01

    Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR- α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood-brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR- α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR- α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood-brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain.

  7. Efflux transporters- and cytochrome P-450-mediated interactions between drugs of abuse and antiretrovirals☆

    PubMed Central

    Pal, Dhananjay; Kwatra, Deep; Minocha, Mukul; Paturi, Durga K.; Budda, Balasubrahmanyam; Mitra, Ashim K.

    2010-01-01

    Multidrug regimens and corresponding drug interactions cause many adverse reactions and treatment failures. Drug efflux transporters: P-gp, MRP, BCRP in conjunction with metabolizing enzymes (CYPs) are major factors in such interactions. Most effective combination antiretrovirals (ARV) therapy includes a PI or a NNRTI or two NRTI. Coadministration of such ARV may induce efflux transporters and/or CYP3A4 resulting in sub-therapeutic blood levels and therapeutic failure due to reduced absorption and/or increased metabolism. A similar prognosis is true for ARV-compounds and drugs of abuse combinations. Morphine and nicotine enhance CYP3A4 and MDR1 expression in vitro. A 2.5 fold rise of cortisol metabolite was evident in smokers relative to nonsmokers. Altered functions of efflux transporters and CYPs in response to ARV and drugs of abuse may result in altered drug absorption and metabolism. Appropriate in vitro models can be employed to predict such interactions. Influence of genetic polymorphism, SNP and inter-individual variation in drug response has been discussed. Complexity underlying the relationship between efflux transporters and CYP makes it difficult to predict the outcome of HAART as such, particularly when HIV patients taking drugs of abuse do not adhere to HAART regimens. HIV+ pregnant women on HAART medications, indulging in drugs of abuse, may develop higher viral load due to such interactions and lead to increase in mother to child transmission of HIV. A multidisciplinary approach with clear understanding of mechanism of interactions may allow proper selection of regimens so that desired therapeutic outcome of HAART can be reached without any side effects. PMID:20932495

  8. Importance of nondiffusive transport for soil CO2 efflux in a temperate mountain grassland.

    PubMed

    Roland, Marilyn; Vicca, Sara; Bahn, Michael; Ladreiter-Knauss, Thomas; Schmitt, Michael; Janssens, Ivan A

    2015-03-01

    Soil respiration and its biotic and abiotic drivers have been an important research topic in recent years. While the bulk of these efforts has focused on the emission of CO2 from soils, the production and subsequent transport of CO2 from soil to atmosphere received far less attention. However, to understand processes underlying emissions of CO2 from terrestrial ecosystems, both processes need to be fully evaluated. In this study, we tested to what extent the transport of CO2 in a grassland site in the Austrian Alps could be modeled based on the common assumption that diffusion is the main transport mechanism for trace gases in soils. Therefore, we compared the CO2 efflux calculated from the soil CO2 concentration gradient with the CO2 efflux from chamber measurements. We used four commonly used diffusion-driven models for the flux-gradient approach. Models generally underestimated the soil chamber effluxes and their amplitudes, indicating that processes other than diffusion were responsible for the transport of CO2. We further observed that transport rates correlated well with irradiation and, below a soil moisture content of 33%, with wind speed. This suggests that mechanisms such as bulk soil air transport, due to pressure pumping or thermal expansion of soil air due to local surface heating, considerably influence soil CO2 transport at this site. Our results suggest that nondiffusive transport may be an important mechanism influencing diel and day-to-day dynamics of soil CO2 emissions, leading to a significant mismatch (10-87% depending on the model used) between the two approaches at short time scales.

  9. Influence of the gastrointestinal microflora and efflux transporters on the absorption of scutellarin and scutellarein.

    PubMed

    You, Hai-Sheng; Xing, Jian-Feng; Lu, Jun; Dong, Wei-Hua; Liu, Jun-Tian; Dong, Ya-Lin

    2014-09-01

    Scutellarin (SG) and its aglycone, Scutellarein (S), are flavonoids of therapeutic cardiocerebrovascular disease. SG was hydrolyzed by bacterial enzyme into S which was absorbed in the intestine. The aim of this study was to determine the effects of the microflora in the intestinal lumen and the efflux transporter of intestinal epithelial cells on the absorption process of SG and S. After oral administration of antibiotics in Sprague-Dawley rats, the reduced bacterial enzyme formation significantly hinders the absorption of SG, whereas scarcely that of S. The absorption study in situ single-pass intestinal perfusion revealed that S could be absorbed throughout the intestine of rats. The effective intestinal permeability of S in the jejunum was much lower than in the other sections of the GI tract. The efflux transporter promoted SG secretion into lumen from enterocytes, which hindered the absorption of both SG and S into the bloodstream. The efflux transporter protein inhibitor (verapamil, probenecid and reserpine) remarkably enhanced the absorption of S and the bioconversion of S into SG in both the rat intestine and Caco-2-monolayer models.

  10. The H2 receptor antagonist nizatidine is a P-glycoprotein substrate: characterization of its intestinal epithelial cell efflux transport.

    PubMed

    Dahan, Arik; Sabit, Hairat; Amidon, Gordon L

    2009-06-01

    The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H(2) receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05-10 mM in both apical-basolateral (AP-BL) and BL-AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC(50) of verapamil on nizatidine P-gp secretion was 1.2 x 10(-2) mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J(max) = 5.7 x 10(-3) nmol cm(-2) s(-1) and K(m) = 2.2 mM) and one nonsaturable component (K(d) = 7 x 10(-4) microL cm(-2) s(-1)). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V(max) and K(m) estimated for nizatidine P-gp-mediated secretion were 4 x 10(-3) nmol cm(-2) s(-1) and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug-drug interactions.

  11. Roles of pollen-specific boron efflux transporter, OsBOR4, in the rice fertilization process.

    PubMed

    Tanaka, Nobuhiro; Uraguchi, Shimpei; Saito, Akihiro; Kajikawa, Masataka; Kasai, Koji; Sato, Yutaka; Nagamura, Yoshiaki; Fujiwara, Toru

    2013-12-01

    Arabidopsis thaliana BOR1 was the first boron (B) transporter identified in living systems. There are four AtBOR1-like genes, OsBOR1, 2, 3 and 4, present in the rice genome. We characterized the activity, expression and physiological function of OsBOR4. OsBOR4 is an active efflux transporter of B. Quantitative PCR analysis and OsBOR4 promoter-green fluorescent protein (GFP) fusion revealed that OsBOR4 was both highly and specifically expressed in pollen. We obtained five Tos17 insertion mutants of osbor4. The pollen grains were viable and development of floral organs was normal in the homozygous osbor4 mutants. We observed that in all Tos17 insertion lines tested, the frequency of osbor4 homozygous plants was lower than expected in the progeny of self-fertilized heterozygous plants. These results establish that OsBOR4 is essential for normal reproductive processes. Pollen from osbor4 homozygous plants elongated fewer tubes on wild-type stigmas, and tube elongation of mutant pollen was less efficient compared with the wild-type pollen, suggesting reduced competence of osbor4 mutant pollen. The reduced competence of mutant pollen was further supported by the crosses of independent Tos17-inserted alleles of OsBOR4. Our results suggest that OsBOR4, a boron efflux transporter, is required for normal pollen germination and/or tube elongation.

  12. Effects of natural nuclear factor-kappa B inhibitors on anticancer drug efflux transporter human P-glycoprotein.

    PubMed

    Nabekura, Tomohiro; Hiroi, Takashi; Kawasaki, Tatsuya; Uwai, Yuichi

    2015-03-01

    Drug efflux transporter P-glycoprotein plays an important role in cancer chemotherapy. The nuclear factor-κB (NF-κB) transcription factors play critical roles in development and progression of cancer. In this study, the effects of natural compounds that can inhibit NF-κB activation on the function of P-glycoprotein were investigated using human MDR1 gene-transfected KB/MDR1 cells. The accumulation of daunorubicin or rhodamine 123, fluorescent substrates of P-glycoprotein, in KB/MDR1 cells increased in the presence of caffeic acid phenetyl ester (CAPE), licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol in a concentration-dependent manner. In contrast, lupeol, zerumbone, thymoquinone, emodin, and anethol had no effects. The ATPase activities of P-glycoprotein were stimulated by CAPE, licochalcone A, anacardic acid, celastrol, xanthohumol, magnolol, and honokiol. Tumor necrosis factor (TNF)-α stimulated NF-κB activation was inhibited by CAPE, licochalcone A, anacardic acid, and xanthohumol. KB/MDR1 cells were sensitized to vinblastine cytotoxicity by CAPE, licochalcone A, anacardic acid, xanthohumol, magnolol, and honokiol, showing that these natural NF-κB inhibitors reverse multidrug resistance. These results suggest that natural compounds, such as CAPE, licochalcone A, and anacardic acid, have dual inhibitory effects on the anticancer drug efflux transporter P-glycoprotein and NF-κB activation, and may become useful to enhance the efficacy of cancer chemotherapy.

  13. Caveolin-1 and ATP binding cassette transporter A1 and G1-mediated cholesterol efflux.

    PubMed

    Wang, Faqi; Gu, Hong-mei; Zhang, Da-wei

    2014-01-01

    Atherosclerosis is one major cause of cardiovascular diseases, the leading cause of death in industrialized countries. Reverse cholesterol transport (RCT) is thought to be one primary pathway to protect against atherosclerosis. The first and rate-limiting step of RCT is ATP-binding cassette transport A1 (ABCA1) and ABCG1-mediated cholesterol efflux from the cells. Recently, caveolin-1 (CAV1), a scaffolding protein that organizes and concentrates certain caveolin-interacting signaling molecules and receptors within caveolae membranes, has been shown to regulate ABCA1 and ABCG1-mediated cholesterol efflux probably via interacting with them. In the present review, we summarize the current knowledge and views on the regulatory role of CAV1 on the cholesterol homeostasis with emphasis on the association of CAV1 with ABCA1 and ABCG1. We conclude that the dominance of the positive regulation by CAV1 on the ABCA1 and ABCG1-mediated cholesterol efflux is depending on the species, cell types, as well as the levels of CAV1 expression.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2012-06-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2010-04-22

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

  19. Bafetinib (INNO-406) reverses multidrug resistance by inhibiting the efflux function of ABCB1 and ABCG2 transporters

    NASA Astrophysics Data System (ADS)

    Zhang, Yun-Kai; Zhang, Guan-Nan; Wang, Yi-Jun; Patel, Bhargav A.; Talele, Tanaji T.; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-05-01

    ATP-Binding Cassette transporters are involved in the efflux of xenobiotic compounds and are responsible for decreasing drug accumulation in multidrug resistant (MDR) cells. Discovered by structure-based virtual screening algorithms, bafetinib, a Bcr-Abl/Lyn tyrosine kinase inhibitor, was found to have inhibitory effects on both ABCB1- and ABCG2-mediated MDR in this in-vitro investigation. Bafetinib significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates and increased the intracellular accumulation of anticancer drugs, particularly doxorubicin and [3H]-paclitaxel in ABCB1 overexpressing cells; mitoxantrone and [3H]-mitoxantrone in ABCG2 overexpressing cells, respectively. Bafetinib stimulated ABCB1 ATPase activities while inhibited ABCG2 ATPase activities. There were no significant changes in the expression level or the subcellular distribution of ABCB1 and ABCG2 in the cells exposed to 3 μM of bafetinib. Overall, our study indicated that bafetinib reversed ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings might be useful in developing combination therapy for MDR cancer treatment.

  20. A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

    PubMed Central

    Kim, Do-Geun; Bynoe, Margaret S.

    2016-01-01

    The blood-brain barrier (BBB) protects the brain from toxic substances within the peripheral circulation. It maintains brain homeostasis and is a hurdle for drug delivery to the CNS to treat neurodegenerative diseases, including Alzheimer’s disease and brain tumors. The drug efflux transporter P-glycoprotein (P-gp) is highly expressed on brain endothelial cells and blocks the entry of most drugs delivered to the brain. Here, we show that activation of the A2A adenosine receptor (AR) with an FDA-approved A2A AR agonist (Lexiscan) rapidly and potently decreased P-gp expression and function in a time-dependent and reversible manner. We demonstrate that downmodulation of P-gp expression and function coincided with chemotherapeutic drug accumulation in brains of WT mice and in primary mouse and human brain endothelial cells, which serve as in vitro BBB models. Lexiscan also potently downregulated the expression of BCRP1, an efflux transporter that is highly expressed in the CNS vasculature and other tissues. Finally, we determined that multiple pathways, including MMP9 cleavage and ubiquitinylation, mediated P-gp downmodulation. Based on these data, we propose that A2A AR activation on BBB endothelial cells offers a therapeutic window that can be fine-tuned for drug delivery to the brain and has potential as a CNS drug-delivery technology. PMID:27043281

  1. Rethinking Drug Treatment Approaches in ALS by Targeting ABC Efflux Transporters

    DTIC Science & Technology

    2012-10-01

    disease  progression,  and  found  that   chronic  treatment  with  either   the...down   disease  progression  as  reported.     In  addition  to  evaluating  the  toxicity  of   chronic  Elacridar...far underestimated issue of disease -driven pharmacoresistance mediated by the multi-drug resistance (mdr) efflux transporter, P-glycoprotein

  2. Substrate-dependent dynamics of the multidrug efflux transporter AcrB of Escherichia coli.

    PubMed

    Yamamoto, Kentaro; Tamai, Rei; Yamazaki, Megumi; Inaba, Takehiko; Sowa, Yoshiyuki; Kawagishi, Ikuro

    2016-02-26

    The resistance-nodulation-cell division (RND)-type xenobiotic efflux system plays a major role in the multidrug resistance of gram-negative bacteria. The only constitutively expressed RND system of Escherichia coli consists of the inner membrane transporter AcrB, the membrane fusion protein AcrA, and the outer membrane channel TolC. The latter two components are shared with another RND-type transporter AcrD, whose expression is induced by environmental stimuli. Here, we demonstrate how RND-type ternary complexes, which span two membranes and the cell wall, form in vivo. Total internal reflection fluorescence (TIRF) microscopy revealed that most fluorescent foci formed by AcrB fused to green fluorescent protein (GFP) were stationary in the presence of TolC but showed lateral displacements when tolC was deleted. The fraction of stationary AcrB-GFP foci decreased with increasing levels of AcrD. We propose that the AcrB-containing complex becomes unstable upon the induction of AcrD, which presumably replaces AcrB, a process we call "transporter exchange." This instability is suppressed by AcrB-specific substrates, suggesting that the ternary complex is stabilised when it is in action. These results suggest that the assembly of the RND-type efflux system is dynamically regulated in response to external stimuli, shedding new light on the adaptive antibiotic resistance of bacteria.

  3. 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.

  4. Interaction Studies of Resolvin E1 Analog (RX-10045) with Efflux Transporters

    PubMed Central

    Cholkar, Kishore; Trinh, Hoang M.; Vadlapudi, Aswani Dutt; Wang, Zhiying; Pal, Dhananjay

    2015-01-01

    Abstract Purpose: Screening interactions of a resolvin E1 analog (RX-10045) with efflux transporters (P-glycoprotein [P-gp], multidrug resistance-associated protein [MRP2], and breast cancer-resistant protein [BCRP]). Methods: Madin-Darby canine kidney cells transfected with P-gp, MRP2, and BCRP genes were selected for this study. [3H]-Digoxin, [3H]-vinblastine and [3H]-abacavir were selected as model substrates for P-gp, MRP2, and BCRP. Uptake and permeability studies across cell monolayer in both apical to basal (AP–BL) and BL–AP of these substrates were conducted in the presence of specific efflux pump inhibitors and RX-10045. Cell viability studies were conducted with increasing concentrations of RX-10045. Results: Uptake studies showed a higher accumulation in the presence of inhibitors (GF120918 and ketoconazole for P-gp; MK571 for MRP2; and β-estradiol for BCRP) as well as RX-10045. Similarly, dose-dependent inhibition studies demonstrated higher accumulation of various substrates ([3H]-digoxin, [3H]-vinblastine, and [3H]-abacavir) in the presence of RX-10045. IC50 values of dose-dependent inhibition of RX-10045 for P-gp, MRP2, and BCRP were 239±11.2, 291±79.2, and 300±42 μM, respectively. Cell viability assay indicated no apparent toxicity up to 350 μM concentration. Enhanced permeability for model substrates was observed in the presence of RX-10045. Uptake studies in human corneal epithelial cells suggest that RX-10045 is a strong inhibitor of organic cation transporter-1 (OCT-1). Conclusions: In summary, the resolvin analog (RX-10045) was identified as a substrate/inhibitor for efflux transporters (MRP2 and BCRP). Also, RX-10045 appears to be a strong inhibitor/substrate of OCT-1. Novel formulation strategies such as nanoparticles, nanomicelles, and liposomes for circumventing efflux barriers and delivering higher drug concentrations leading to a higher therapeutic efficacy may be employed. PMID:25844889

  5. Mitochondrial stress-induced dopamine efflux and neuronal damage by malonate involves the dopamine transporter.

    PubMed

    Moy, Lily Y; Wang, Sheng-Ping; Sonsalla, Patricia K

    2007-02-01

    Endogenous striatal dopamine (DA) overflow has been associated with neuropathological conditions resulting from ischemia, psychostimulants, and metabolic inhibition. Malonate, a reversible inhibitor of succinate dehydrogenase, models the effects of energy impairment in neurodegenerative disorders. We have previously reported that the striatal DA efflux and damage to DA nerve terminals resulting from intrastriatal malonate infusions is prevented by prior DA depletion, suggesting that DA plays a role in the neuronal damage. We presently report that the malonate-induced DA efflux is partially mediated by reverse transport of DA from the cytosol to the extracellular space via the DA transporter (DAT). Pharmacological blockade of the DAT with a series of structurally different inhibitors [cocaine, mazindol, 1-(2-(bis(4-fluophenyl methoxy) ethyl)-4-(3-(4-fluorophenyl)-propyl)piperazine) dimethane sulfonate (GBR 13098) and methyl(-)-3beta-(p-fluorophenyl)-1alphaH,5alphaH-tropane-2beta-carboxylate1,5-naphthalene (Win 35,428)] attenuated malonate-induced DA overflow in vivo and protected mice against subsequent damage to DA nerve terminals. Consistent with these findings, the DAT inhibitors prevented malonate-induced damage to DA neurons in mesencephalic cultures and also protected against the loss of GABA neurons in this system. The DAT inhibitors did not modify malonate-induced formation of reactive oxygen species or lactate production, indicating that the DAT inhibitors neither exert antioxidant effects nor interfere with the actions of malonate. Taken together, these findings provide direct evidence that mitochondrial impairment and metabolic stress cause striatal DA efflux via the DAT and suggest that disruptions in DA homeostasis resulting from energy impairment may contribute to the pathogenesis of neurodegenerative diseases.

  6. Application of microdialysis to evaluate the efflux transport of estradiol 17-beta glucuronide across the rat blood-retinal barrier.

    PubMed

    Katayama, Kazunori; Ohshima, Yuki; Tomi, Masatoshi; Hosoya, Ken-ichi

    2006-09-30

    The purpose of the present study was to evaluate vitreous humor/retina-to-blood efflux transport in rats and determine the efflux transport of estradiol 17-beta glucuronide (E17betaG) across the blood-retinal barrier (BRB) by the use of microdialysis. [(3)H]E17betaG and [(14)C]D-mannitol, which were used as a model compound for amphipathic organic anions and a bulk flow marker, respectively, were injected into the vitreous humor of rat eye, and a microdialysis probe was placed in the vitreous humor. [(3)H]E17betaG and [(14)C]D-mannitol were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]E17betaG during the terminal phase was 1.9-fold greater than that of [(14)C]D-mannitol and reduced the level of [(14)C]D-mannitol in the retinal presence of 0.3 mM E17betaG, suggesting that [(3)H]E17betaG is transported via a carrier-mediated efflux transport process across the BRB. The efflux transport of [(3)H]E17betaG was significantly inhibited by organic anions, such as probenecid, sulfobromophthalein, digoxin, and dehydroepiandrosterone sulfate, whereas it was not inhibited by p-aminohippuric acid. In conclusion, the efflux transport of [(3)H]E17betaG across the rat BRB was evaluated by microdialysis and its inhibition by organic anions suggests organic anion transporting polypeptide 1a4-mediated E17betaG efflux transport at the BRB.

  7. 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

  8. A Barley Efflux Transporter Operates in a Na+-Dependent Manner, as Revealed by a Multidisciplinary Platform[OPEN

    PubMed Central

    Nagarajan, Yagnesh; Rongala, Jay; Luang, Sukanya; Shadiac, Nadim; Sutton, Tim; Tyerman, Stephen D.; McPhee, Gordon; Voelcker, Nicolas H.; Lee, Jung-Goo

    2016-01-01

    Plant growth and survival depend upon the activity of membrane transporters that control the movement and distribution of solutes into, around, and out of plants. Although many plant transporters are known, their intrinsic properties make them difficult to study. In barley (Hordeum vulgare), the root anion-permeable transporter Bot1 plays a key role in tolerance to high soil boron, facilitating the efflux of borate from cells. However, its three-dimensional structure is unavailable and the molecular basis of its permeation function is unknown. Using an integrative platform of computational, biophysical, and biochemical tools as well as molecular biology, electrophysiology, and bioinformatics, we provide insight into the origin of transport function of Bot1. An atomistic model, supported by atomic force microscopy measurements, reveals that the protein folds into 13 transmembrane-spanning and five cytoplasmic α-helices. We predict a trimeric assembly of Bot1 and the presence of a Na+ ion binding site, located in the proximity of a pore that conducts anions. Patch-clamp electrophysiology of Bot1 detects Na+-dependent polyvalent anion transport in a Nernstian manner with channel-like characteristics. Using alanine scanning, molecular dynamics simulations, and transport measurements, we show that conductance by Bot1 is abolished by removal of the Na+ ion binding site. Our data enhance the understanding of the permeation functions of Bot1. PMID:26672067

  9. From evolution to revolution: miRNAs as pharmacological targets for modulating cholesterol efflux and reverse cholesterol transport.

    PubMed

    Dávalos, Alberto; Fernández-Hernando, Carlos

    2013-09-01

    There has been strong evolutionary pressure to ensure that an animal cell maintains levels of cholesterol within tight limits for normal function. Imbalances in cellular cholesterol levels are a major player in the development of different pathologies associated to dietary excess. Although epidemiological studies indicate that elevated levels of high-density lipoprotein (HDL)-cholesterol reduce the risk of cardiovascular disease, recent genetic evidence and pharmacological therapies to raise HDL levels do not support their beneficial effects. Cholesterol efflux as the first and probably the most important step in reverse cholesterol transport is an important biological process relevant to HDL function. Small non-coding RNAs (microRNAs), post-transcriptional control different aspects of cellular cholesterol homeostasis including cholesterol efflux. miRNA families miR-33, miR-758, miR-10b, miR-26 and miR-106b directly modulates cholesterol efflux by targeting the ATP-binding cassette transporter A1 (ABCA1). Pre-clinical studies with anti-miR therapies to inhibit some of these miRNAs have increased cellular cholesterol efflux, reverse cholesterol transport and reduce pathologies associated to dyslipidemia. Although miRNAs as therapy have benefits from existing antisense technology, different obstacles need to be solved before we incorporate such research into clinical care. Here we focus on the clinical potential of miRNAs as therapeutic target to increase cholesterol efflux and reverse cholesterol transport as a new alternative to ameliorate cholesterol-related pathologies.

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

    PubMed

    Vehniäinen, Eeva-Riikka; Kukkonen, Jussi V K

    2015-04-01

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

  11. Aryl hydrocarbon receptor-mediated up-regulation of ATP-driven xenobiotic efflux transporters at the blood-brain barrier.

    PubMed

    Wang, Xueqian; Hawkins, Brian T; Miller, David S

    2011-02-01

    Many widespread and persistent organic pollutants, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), activate the aryl hydrocarbon receptor (AhR), causing it to translocate to the cell nucleus, where it transactivates target genes. AhR's ability to target the blood-brain barrier is essentially unexplored. We show here that exposing isolated rat brain capillaries to 0.05-0.5 nM TCDD roughly doubled transport activity and protein expression of P-glycoprotein, an ATP-driven drug efflux pump and a critical determinant of drug entry into the CNS. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists resveratrol and α-naphthoflavone. Brain capillaries from TCDD-dosed rats (1-5 μg/kg, i.p.) exhibited increased transport activity and protein expression of 3 xenobiotic efflux pumps, P-glycoprotein, multidrug resistance-associated protein 2, and breast cancer resistance polypeptide, as well as expression of Cyp1a1 and Cyp1b1, both AhR target genes. Consistent with increased P-glycoprotein expression in capillaries from TCDD-dosed rats, in situ brain perfusion indicated significantly reduced brain accumulation of verapamil, a P-glycoprotein substrate. These findings suggest a new paradigm for the field of environmental toxicology: toxicants acting through AhR to target xenobiotic efflux transporters at the blood-brain barrier and thus reduce brain accumulation of CNS-acting therapeutic drugs.

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

    PubMed Central

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

    2014-01-01

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

  13. In vitro characterization of axitinib interactions with human efflux and hepatic uptake transporters: implications for disposition and drug interactions.

    PubMed

    Reyner, Eric L; Sevidal, Samantha; West, Mark A; Clouser-Roche, Andrea; Freiwald, Sascha; Fenner, Katherine; Ullah, Mohammed; Lee, Caroline A; Smith, Bill J

    2013-08-01

    Axitinib is an inhibitor of tyrosine kinase vascular endothelin growth factor receptors 1, 2, and 3. The ATP-binding cassette (ABC) and solute carrier (SLC) transport properties of axitinib were determined in selected cellular systems. Axitinib exhibited high passive permeability in all cell lines evaluated (Papp ≥ 6 × 10(-6) cm/s). Active efflux was observed in Caco-2 cells, and further evaluation in multidrug resistance gene 1 (MDR1) or breast cancer resistance protein (BCRP) transfected Madin-Darby canine kidney cells type 2 (MDCK) cells indicated that axitinib is at most only a weak substrate for P-glycoprotein (P-gp) but not BCRP. Axitinib showed incomplete inhibition of P-gp-mediated transport of digoxin in Caco-2 cells and BCRP transport of topotecan in BCRP-transfected MDCK cells with IC50 values of 3 μM and 4.4 μM, respectively. Axitinib (10 mg) did not pose a risk for systemic drug interactions with P-gp or BCRP per regulatory guidance. A potential risk for drug interactions through inhibition of P-gp and BCRP in the gastrointestinal tract was identified because an axitinib dose of 10 mg divided by 250 mL was greater than 10-fold the IC50 for each transporter. However, a GastroPlus simulation that considered the low solubility of axitinib resulted in lower intestinal concentrations and suggested a low potential for gastrointestinal interactions with P-gp and BCRP substrates. Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 transfected human embryonic kidney 293 (HEK293) cells transported axitinib to a minor extent but uptake into suspended hepatocytes was not inhibited by rifamycin SV suggesting that high passive permeability predominates. Mouse whole-body autoradiography revealed that [(14)C]axitinib-equivalents showed rapid absorption and distribution to all tissues except the brain. This suggests that efflux transport of axitinib may occur at the mouse blood-brain barrier.

  14. Characterization of multidrug transporter-mediated efflux of avermectins in human and mouse neuroblastoma cell lines.

    PubMed

    Dalzell, Abigail M; Mistry, Pratibha; Wright, Jayne; Williams, Faith M; Brown, Colin D A

    2015-06-15

    ABC transporters play an important role in the disposition of avermectins in several animal species. In this study the interactions of three key avermectins, abamectin, emamectin and ivermectin, with human and mouse homologues of MDR1 (ABCB1/Abcb1a) and MRP (ABCC/Abcc), transporters endogenously expressed by human SH-SY5Y and mouse N2a neuroblastoma cells were investigated. In both cell lines, retention of the fluorescent dye H33342 was found to be significantly increased in the presence of avermectins and cyclosporin A. These effects were shown to be unresponsive to the BCRP inhibitor Ko-143 and therefore MDR1/Mdr1-dependent. Avermectins inhibited MDR1/Mdr1a-mediated H33342 dye efflux, with apparent Ki values of 0.24±0.08 and 0.18±0.02μM (ivermectin); 0.60±0.07 and 0.56±0.02μM (emamectin) and 0.95±0.08 and 0.77±0.25μM (abamectin) in SH-SY5Y and N2a cells, respectively. There were some apparent affinity differences for MDR1 and Mdr1a within each cell line (affinity for ivermectin>emamectin≥abamectin, P<0.05 by One-Way ANOVA), but importantly, the Ki values for individual avermectins for human MDR1 or mouse Mdr1a were not significantly different. MK571-sensitive retention of GSMF confirmed the expression of MRP/Mrp efflux transporters in both cell lines. Avermectins inhibited MRP/Mrp-mediated dye efflux with IC50 values of 1.58±0.51 and 1.94±0.72μM (ivermectin); 1.87±0.57 and 2.74±1.01μM (emamectin) and 2.25±0.01 and 1.68±0.63μM (abamectin) in SH-SY5Y and N2a cells, respectively. There were no significant differences in IC50 values between individual avermectins or between human MRP and mouse Mrp. Kinetic data for endogenous human MDR1/MRP isoforms in SH-SY5Y cells and mouse Mdr1a/b/Mrp isoforms in N2a cells are comparable for the selected avermectins. All are effluxed at concentrations well above 0.05-0.1μM ivermectin detected in plasma (Ottesen and Campbell, 1994; Ottesen and Campbell, 1994) This is an important finding in the light of

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

    PubMed

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

    2013-02-01

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

  16. 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.

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

    PubMed

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

    2011-10-01

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

  18. Prominent expression of xenobiotic efflux transporters in mouse extraembryonic fetal membranes compared with placenta.

    PubMed

    Aleksunes, Lauren M; Cui, Yue; Klaassen, Curtis D

    2008-09-01

    Fetal exposure to xenobiotics can be restricted by transporters at the interface between maternal and fetal circulation. Previous work identified transporters in the placenta; however, less is known about the presence of these transporters in the fetal membranes (i.e., yolk sac and amniotic membranes). The purpose of this study was to quantify mRNA and protein expression of xenobiotic transporters in mouse placenta and fetal membranes during mid to late gestation. Concepti (placenta and fetal membranes, gestation day 11) or placenta and fetal membranes (gestation days 14 and 17) were collected from pregnant mice and analyzed for expression of multidrug resistance-associated proteins (Mrps), multidrug resistance proteins (Mdrs), multidrug and toxin extrusion proteins (Mates), breast cancer resistance protein (Bcrp), and organic anion-transporting polypeptides (Oatps). Maternal liver and kidneys were also collected at day 14 for mRNA and immunohistochemical analysis. mRNA expression of Mrp, Mdr, Bcrp, Mate-1, and Oatp isoforms was detected at day 11. The uptake carriers Oatp2a1, 3a1, 4a1, and 5a1 showed placenta-predominant expression. At days 14 and 17, fetal membranes expressed higher mRNA levels of the efflux transporters Mrp2 (7-fold), Mrp4 (5-fold), Mrp5 (3-fold), Mrp6 (12-fold), Bcrp (2-fold), and Mate-1 (7-fold) than placenta. Western blot analysis of Mrp2, Mrp4, Mrp6, and Bcrp confirmed higher expression in fetal membranes. Immunostaining revealed apical (Mrp2 and Bcrp) and basolateral (Mrp4, 5, and 6) cellular localization in epithelial cells of the yolk sac. In conclusion, xenobiotic transporters in the fetal membranes may provide an additional route to protect the fetus against endogenous chemicals and xenobiotics.

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

    PubMed Central

    Wang, Tianli; Agarwal, Sagar

    2012-01-01

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

  20. From Evolution to Revolution: miRNAs as Pharmacological Targets for Modulating Cholesterol Efflux and Reverse Cholesterol Transport

    PubMed Central

    Dávalos, Alberto; Fernández-Hernando, Carlos

    2013-01-01

    There has been strong evolutionary pressure to ensure that an animal cell maintain levels of cholesterol within tight limits for normal function. Imbalances in cellular cholesterol levels are a major player in the development of different pathologies associated to dietary excess. Although epidemiological studies indicate that elevated levels of high-density lipoprotein (HDL)-cholesterol reduce the risk of cardiovascular disease, recent genetic evidence and pharmacological therapies to raise HDL levels do not support their beneficial effects. Cholesterol efflux as the first and probably the most important step in reverse cholesterol transport is an important biological process relevant to HDL function. Small non-coding RNAs (microRNAs), post-transcriptional control different aspects of cellular cholesterol homeostasis including cholesterol efflux. miRNA families miR-33, miR-758, miR-10b, miR-26 and miR-106b directly modulates cholesterol efflux by targeting the ATP-binding cassette transporter A1 (ABCA1). Pre-clinical studies with anti-miR therapies to inhibit some of these miRNAs have increased cellular cholesterol efflux, reverse cholesterol transport and reduce pathologies associated to dyslipidemia. Although miRNAs as therapy have benefits from existing antisense technology, different obstacles need to be solved before we incorporate such research into clinical care. Here we focus on the clinical potential of miRNAs as therapeutic target to increase cholesterol efflux and reverse cholesterol transport as a new alternative to ameliorate cholesterol-related pathologies. PMID:23435093

  1. Multidrug efflux transporters limit accumulation of inorganic, but not organic, mercury in sea urchin embryos.

    PubMed

    Bosnjak, Ivana; Uhlinger, Kevin R; Heim, Wesley; Smital, Tvrtko; Franekić-Colić, Jasna; Coale, Kenneth; Epel, David; Hamdoun, Amro

    2009-11-01

    Mercuric compounds are persistent global pollutants that accumulate in marine organisms and in humans who consume them. While the chemical cycles and speciation of mercury in the oceans are relatively well described, the cellular mechanisms that govern which forms of mercury accumulate in cells and why they persist are less understood. In this study we examined the role of multidrug efflux transport in the differential accumulation of inorganic (HgCl(2)) and organic (CH(3)HgCl) mercury in sea urchin (Strongylocentrotus purpuratus) embryos. We found that inhibition of MRP/ABCC-type transporters increases intracellular accumulation of inorganic mercury but had no effect on accumulation of organic mercury. Similarly, pharmacological inhibition of metal conjugating enzymes by ligands GST/GSH significantly increases this antimitotic potency of inorganic mercury, but had no effect on the potency of organic mercury. Our results point to MRP-mediated elimination of inorganic mercury conjugates as a cellular basis for differences in the accumulation and potency of the two major forms of mercury found in marine environments.

  2. The Boron Efflux Transporter ROTTEN EAR Is Required for Maize Inflorescence Development and Fertility[C][W][OPEN

    PubMed Central

    Chatterjee, Mithu; Tabi, Zara; Galli, Mary; Malcomber, Simon; Buck, Amy; Muszynski, Michael; Gallavotti, Andrea

    2014-01-01

    Although boron has a relatively low natural abundance, it is an essential plant micronutrient. Boron deficiencies cause major crop losses in several areas of the world, affecting reproduction and yield in diverse plant species. Despite the importance of boron in crop productivity, surprisingly little is known about its effects on developing reproductive organs. We isolated a maize (Zea mays) mutant, called rotten ear (rte), that shows distinct defects in vegetative and reproductive development, eventually causing widespread sterility in its inflorescences, the tassel and the ear. Positional cloning revealed that rte encodes a membrane-localized boron efflux transporter, co-orthologous to the Arabidopsis thaliana BOR1 protein. Depending on the availability of boron in the soil, rte plants show a wide range of phenotypic defects that can be fully rescued by supplementing the soil with exogenous boric acid, indicating that rte is crucial for boron transport into aerial tissues. rte is expressed in cells surrounding the xylem in both vegetative and reproductive tissues and is required for meristem activity and organ development. We show that low boron supply to the inflorescences results in widespread defects in cell and cell wall integrity, highlighting the structural importance of boron in the formation of fully fertile reproductive organs. PMID:25035400

  3. The arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier

    NASA Technical Reports Server (NTRS)

    Chen, R.; Hilson, P.; Sedbrook, J.; Rosen, E.; Caspar, T.; Masson, P. H.

    1998-01-01

    Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996) Current Biol. 6, 1589-1591]. Here we show that mutations in the Arabidopsis thaliana AGRAVITROPIC 1 (AGR1) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that AGR1 encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in Saccharomyces cerevisiae, AGR1 promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound. AGR1 transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several AGR1-related genes in Arabidopsis, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.

  4. Drug Efflux Transporters Are Overexpressed in Short-Term Tamoxifen-Induced MCF7 Breast Cancer Cells

    PubMed Central

    Krisnamurti, Desak Gede Budi; Louisa, Melva; Anggraeni, Erlia; Wanandi, Septelia Inawati

    2016-01-01

    Tamoxifen is the first line drug used in the treatment of estrogen receptor-positive (ER+) breast cancer. The development of multidrug resistance (MDR) to tamoxifen remains a major challenge in the treatment of cancer. One of the mechanisms related to MDR is decrease of drug influx via overexpression of drug efflux transporters such as P-glycoprotein (P-gp/MDR1), multidrug resistance associated protein (MRP), or BCRP (breast cancer resistance protein). We aimed to investigate whether the sensitivity of tamoxifen to the cells is maintained through the short period and whether the expressions of several drug efflux transporters have been upregulated. We exposed MCF7 breast cancer cells with tamoxifen 1 μM for 10 passages (MCF7 (T)). The result showed that MCF7 began to lose their sensitivity to tamoxifen from the second passage. MCF7 (T) also showed a significant increase in all transporters examined compared with MCF7 parent cells. The result also showed a significant increase of CC50 in MCF7 (T) compared to that in MCF7 (97.54 μM and 3.04 μM, resp.). In conclusion, we suggest that the expression of several drug efflux transporters such as P-glycoprotein, MRP2, and BCRP might be used and further studied as a marker in the development of tamoxifen resistance. PMID:26981116

  5. The Role of Monocarboxylate Transporters and Their Chaperone CD147 in Lactate Efflux Inhibition and the Anticancer Effects of Terminalia chebula in Neuroblastoma Cell Line N2-A

    PubMed Central

    Messeha, S. S.; Zarmouh, N. O.; Taka, E.; Gendy, S. G.; Shokry, G. R.; Kolta, M. G.; Soliman, K. F. A.

    2016-01-01

    Aims In the presence of oxygen, most of the synthesized pyruvate during glycolysis in the cancer cell of solid tumors is released away from the mitochondria to form lactate (Warburg Effect). To maintain cell homeostasis, lactate is transported across the cell membrane by monocarboxylate transporters (MCTs). The major aim of the current investigation is to identify novel compounds that inhibit lactate efflux that may lead to identifying effective targets for cancer treatment. Study Design In this study, 900 ethanol plant extracts were screened for their lactate efflux inhibition using neuroblastoma (N2-A) cell line. Additionally, we investigated the mechanism of inhibition for the most potent plant extract regarding monocarboxylate transporters expression, and consequences effects on viability, growth, and apoptosis. Methodology The potency of lactate efflux inhibition of ethanol plant extracts was evaluated in N2-A cells by measuring extracellular lactate levels. Caspase 3- activity and acridine orange/ethidium bromide staining were performed to assess the apoptotic effect. The antiproliferative effect was measured using WST assay. Western blotting was performed to quantify protein expression of MCTs and their chaperone CD147 in treated cells lysates. Results Terminalia chebula plant extract was the most potent lactate efflux inhibitor in N2-A cells among the 900 - tested plant extracts. The results obtained show that extract of Terminalia chebula fruits (TCE) significantly (P = 0.05) reduced the expression of the MCT1, MCT3, MCT4 and the chaperone CD147. The plant extract was more potent (IC50 of 3.59 ± 0.26 μg/ml) than the MCT standard inhibitor phloretin (IC50 76.54 ± 3.19 μg/ml). The extract also showed more potency and selective cytotoxicity in cancer cells than DI-TNC1 primary cell line (IC50 7.37 ± 0.28 vs. 17.35 ± 0.19 μg/ml). Moreover, TCE Inhibited N2-A cell growth (IG50 = 5.20 ± 0.30 μg/ml) and induced apoptosis at the 7.5 μg/ml concentration

  6. Down-regulation of a single auxin efflux transport protein in tomato induces precocious fruit development

    PubMed Central

    Mounet, Fabien; Kowalczyk, Mariusz; Aoki, Koh; Lemaire-Chamley, Martine

    2012-01-01

    The PIN-FORMED (PIN) auxin efflux transport protein family has been well characterized in the model plant Arabidopsis thaliana, where these proteins are crucial for auxin regulation of various aspects of plant development. Recent evidence indicates that PIN proteins may play a role in fruit set and early fruit development in tomato (Solanum lycopersicum), but functional analyses of PIN-silenced plants failed to corroborate this hypothesis. Here it is demonstrated that silencing specifically the tomato SlPIN4 gene, which is predominantly expressed in tomato flower bud and young developing fruit, leads to parthenocarpic fruits due to precocious fruit development before fertilization. This phenotype was associated with only slight modifications of auxin homeostasis at early stages of flower bud development and with minor alterations of ARF and Aux/IAA gene expression. However, microarray transcriptome analysis and large-scale quantitative RT-PCR profiling of transcription factors in developing flower bud and fruit highlighted differentially expressed regulatory genes, which are potential targets for auxin control of fruit set and development in tomato. In conclusion, this work provides clear evidence that the tomato PIN protein SlPIN4 plays a major role in auxin regulation of tomato fruit set, possibly by preventing precocious fruit development in the absence of pollination, and further gives new insights into the target genes involved in fruit set. PMID:22844095

  7. Statistical thermodynamics for functionally rotating mechanism of the multidrug efflux transporter AcrB.

    PubMed

    Mishima, Hirokazu; Oshima, Hiraku; Yasuda, Satoshi; Kinoshita, Masahiro

    2015-02-26

    AcrB, a homotrimer, is the pivotal part of a multidrug efflux pump. A "functionally rotating" picture has been proposed for the drug transport by AcrB, but its mechanism remains unresolved. Here, we investigate the energetics of the whole functional rotation cycle using our theoretical methods. We find that the packing efficiency of AcrB is ununiform, and this ununiformity plays imperative roles primarily through the solvent-entropy effect. When a proton binds to or dissociates from a protomer, the packing properties of this protomer and its two interfaces are perturbed overall in the direction that the solvent translational entropy is lowered. The packing properties of the other two protomers are then reorganized with the recovery or maintenance of closely packed interfaces, so that the solvent-entropy loss can be compensated. The functional structural change by an isolated protomer would cause a seriously large free-energy increase. By forming a trimer, any free-energy increase caused by a protomer is always canceled out by the free-energy decrease brought by the other two protomers via the mechanism mentioned above. The functional structural rotation is thus accomplished using the free-energy decrease arising from the transfer of only a single proton per cycle. The similarities to F1-ATPase are also discussed.

  8. Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

    PubMed Central

    2009-01-01

    Background Burkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear. Results To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed. Conclusion Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium. PMID:19761586

  9. 3D structure of AcrB: the archetypal multidrug efflux transporter of Escherichia coli likely captures substrates from periplasm.

    PubMed

    Elkins, Christopher A; Nikaido, Hiroshi

    2003-02-01

    Recent advances in structural biology have extended our understanding of the multiple drug efflux complex, AcrAB-TolC, of Escherichia coli. This tripartite complex and its homologs are the major mechanisms that give most Gram-negative bacteria their characteristic intrinsic resistance to a variety of lipophilic drugs, dyes, and detergents. Most recently, the structure of the transporter AcrB was elucidated at high resolution [Nature 419(2002)587]. It is a particularly significant achievement since integral membrane proteins are notoriously elusive structures for crystallography. The striking features of this trimeric pump, such as the presence of potential substrate-binding sites in the periplasmic domain and the possibility of direct interaction with the end of TolC tunnel, refine our understanding of the mode of action of this tripartite efflux transport complex.

  10. Mycobacterium tuberculosis efpA encodes an efflux protein of the QacA transporter family.

    PubMed Central

    Doran, J L; Pang, Y; Mdluli, K E; Moran, A J; Victor, T C; Stokes, R W; Mahenthiralingam, E; Kreiswirth, B N; Butt, J L; Baron, G S; Treit, J D; Kerr, V J; Van Helden, P D; Roberts, M C; Nano, F E

    1997-01-01

    The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species. PMID:9008277

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

    PubMed

    Mercer, Susan L; Coop, Andrew

    2011-01-01

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

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

    PubMed Central

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  14. 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

  15. 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.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  18. Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

    PubMed

    Marchitti, Satori A; Mazur, Christopher S; Dillingham, Caleb M; Rawat, Swati; Sharma, Anshika; Zastre, Jason; Kenneke, John F

    2017-01-01

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [((3)H)-paclitaxel and ((3)H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

  19. A DTX/MATE-type transporter facilitates abscisic acid efflux and modulates ABA sensitivity and drought tolerance in Arabidopsis.

    PubMed

    Zhang, Haiwen; Zhu, Huifen; Pan, Yajun; Yu, Yuexuan; Luan, Sheng; Li, Legong

    2014-10-01

    Abscisic acid (ABA) regulates numerous physiological and developmental processes in plants. Recent studies identify intracellular ABA receptors, implicating the transport of ABA across cell membranes as crucial for ABA sensing and response. Here, we report that a DTX/Multidrug and Toxic Compound Extrusion (MATE) family member in Arabidopsis thaliana, AtDTX50, functions as an ABA efflux transporter. When expressed heterologously in both an Escherichia coli strain and Xenopus oocyte cells, AtDTX50 was found to facilitate ABA efflux. Furthermore, dtx50 mutant mesophyll cells preloaded with ABA released less ABA compared with the wild-type (WT). The AtDTX50 gene was expressed mainly in the vascular tissues and guard cells and its expression was strongly up-regulated by exogenous ABA. The AtDTX50::GFP fusion protein was localized predominantly to the plasma membrane. The dtx50 mutant plants were observed to be more sensitive to ABA in growth inhibition. In addition, compared with the WT, dtx50 mutant plants were more tolerant to drought with lower stomatal conductance, consistent with its function as an ABA efflux carrier in guard cells.

  20. Effect of glucose transport inhibitors on vincristine efflux in multidrug-resistant murine erythroleukaemia cells overexpressing the multidrug resistance-associated protein (MRP) and two glucose transport proteins, GLUT1 and GLUT3.

    PubMed Central

    Martell, R. L.; Slapak, C. A.; Levy, S. B.

    1997-01-01

    The relationship between mammalian facilitative glucose transport proteins (GLUT) and multidrug resistance was examined in two vincristine (VCR)-selected murine erythroleukaemia (MEL) PC4 cell lines. GLUT proteins, GLUT1 and GLUT3, were constitutively coexpressed in the parental cell line and also in the VCR-selected cell lines. Increased expression of the GLUT1 isoform was noted both in the PC-V40 (a non-P-glycoprotein, mrp-overexpressing subline) and in the more resistant PC-V160 (overexpressing mrp and mdr3) cell lines. Overexpression of GLUT3 was detected only in the PC-V160 subline. An increased rate of facilitative glucose transport (Vmax) and level of plasma membrane GLUT protein expression paralleled increased VCR resistance, active VCR efflux and decreased VCR steady-state accumulation in these cell lines. Glucose transport inhibitors (GTIs), cytochalasin B (CB) and phloretin blocked the active efflux and decreased steady-state accumulation of VCR in the PC-V40 subline. GTIs did not significantly affect VCR accumulation in the parental or PC-V160 cells. A comparison of protein sequences among GLUT1, GLUT3 and MRP revealed a putative cytochalasin B binding site in MRP, which displayed 44% sequence similarity/12% identity with that previously identified in GLUT1 and GLUT3; these regions also exhibited a similar hydropathy plot pattern. The findings suggested that CB bound to MRP and directly or indirectly lowered VCR efflux and/or CB bound to one or both GLUT proteins, which acted to lower the VCR efflux mediated by MRP. This is the first report of a non-neuronal murine cell line that expressed GLUT3. Images Figure 3 PMID:9010020

  1. Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

    PubMed Central

    Bakhsheshian, Joshua; Wei, Bih-Rong; Chang, Ki-Eun; Shukla, Suneet; Ambudkar, Suresh V.; Simpson, R. Mark; Gottesman, Michael M.; Hall, Matthew D.

    2013-01-01

    ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood–brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that d-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter. PMID:24297888

  2. 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.

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

    PubMed

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

    2015-05-01

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

  4. Importance of Peptide Transporter 2 on the Cerebrospinal Fluid Efflux Kinetics of Glycylsarcosine Characterized by Nonlinear Mixed Effects Modeling

    PubMed Central

    Huh, Yeamin; Hynes, Scott M.; Smith, David E.

    2013-01-01

    Purpose To develop a population pharmacokinetic model to quantitate the distribution kinetics of glycylsarcosine (GlySar), a substrate of peptide transporter 2 (PEPT2), in blood, CSF and kidney in wild-type and PEPT2 knockout mice. Methods A stepwise compartment modeling approach was performed to describe the concentration profiles of GlySar in blood, CSF, and kidney simultaneously using nonlinear mixed effects modeling (NONMEM). The final model was selected based on the likelihood ratio test and graphical goodness-of-fit. Results The profiles of GlySar in blood, CSF, and kidney were best described by a four-compartment model. The estimated systemic elimination clearance, volume of distribution in the central and peripheral compartments were 0.236 vs 0.449 ml/min, 3.79 vs 4.75 ml, and 5.75 vs 9.18 ml for wild-type versus knockout mice. Total CSF efflux clearance was 4.3 fold higher for wild-type compared to knockout mice. NONMEM parameter estimates indicated that 77% of CSF efflux clearance was mediated by PEPT2 and the remaining 23% was mediated by the diffusional and bulk clearances. Conclusions Due to the availability of PEPT2 knockout mice, we were able to quantitatively determine the significance of PEPT2 in the efflux kinetics of GlySar at the blood-cerebrospinal fluid barrier. PMID:23371515

  5. Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminum (Al) toxicity is a primary limitation to crop productivity on acid soils throughout the plant. Root efflux of organic acid anions constitutes a mechanism by which plants cope with toxic aluminum (Al) ions on acid soils. In this study, we have characterized ZmALMT2 (a member of aluminum-acti...

  6. PatA and PatB form a functional heterodimeric ABC multidrug efflux transporter responsible for the resistance of Streptococcus pneumoniae to fluoroquinolones.

    PubMed

    Boncoeur, Emilie; Durmort, Claire; Bernay, Benoît; Ebel, Christine; Di Guilmi, Anne Marie; Croizé, Jacques; Vernet, Thierry; Jault, Jean-Michel

    2012-10-02

    All bacterial multidrug ABC transporters have been shown to work as either homodimers or heterodimers. Two possibly linked genes, patA and patB from Streptococcus pneumococcus, that encode half-ABC transporters have been shown previously to be involved in fluoroquinolone resistance. We showed that the ΔpatA, ΔpatB, or ΔpatA/ΔpatB mutant strains were more sensitive to unstructurally related compounds, i.e., ethidium bromide or fluoroquinolones, than the wild-type R6 strain. Inside-out vesicles prepared from Escherichia coli expressing PatA and/or PatB transported Hoechst 33342, a classical substrate of multidrug transporters, only when both PatA and PatB were coexpressed. This transport was inhibited either by orthovanadate or by reserpine, and mutation of the conserved Walker A lysine residue of either PatA or PatB fully abrogated Hoechst 33342 transport. PatA, PatB, and the PatA/PatB heterodimer were purified from detergent-solubilized E. coli membrane preparations. Protein dimers were identified in all cases, albeit in different proportions. In contrast to the PatA/PatB heterodimers, homodimers of PatA or PatB failed to show a vanadate-sensitive ATPase activity. Thus, PatA and PatB need to interact together to make a functional drug efflux transporter, and they work only as heterodimers.

  7. 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

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

    PubMed Central

    Bhattacharya, Somanon; Sobel, Jack D.

    2016-01-01

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

  9. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, Chih-Chia; Long, Feng; McDermott, Gerry; Shafer, William M.; Yu, Edward W.

    2008-04-01

    The multidrug efflux transporter NorM from N. gonorrhoeae has been crystallized and X-ray diffraction data have been collected to a resolution of 6.5 Å. The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 Å and diffraction data were complete to 6.5 Å resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 Å.

  10. A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: Evidence for passive and facilitated transport

    PubMed Central

    Stott, Lucy C.; Schnell, Sabine; Hogstrand, Christer; Owen, Stewart F.; Bury, Nic R.

    2015-01-01

    The gill is the principle site of xenobiotic transfer to and from the aqueous environment. To replace, refine or reduce (3Rs) the large numbers of fish used in in vivo uptake studies an effective in vitro screen is required that mimics the function of the teleost gill. This study uses a rainbow trout (Oncorhynchus mykiss) primary gill cell culture system grown on permeable inserts, which tolerates apical freshwater thus mimicking the intact organ, to assess the uptake and efflux of pharmaceuticals across the gill. Bidirectional transport studies in media of seven pharmaceuticals (propranolol, metoprolol, atenolol, formoterol, terbutaline, ranitidine and imipramine) showed they were transported transcellularly across the epithelium. However, studies conducted in water showed enhanced uptake of propranolol, ranitidine and imipramine. Concentration-equilibrated conditions without a concentration gradient suggested that a proportion of the uptake of propranolol and imipramine is via a carrier-mediated process. Further study using propranolol showed that its transport is pH-dependent and at very low environmentally relevant concentrations (ng L−1), transport deviated from linearity. At higher concentrations, passive uptake dominated. Known inhibitors of drug transport proteins; cimetidine, MK571, cyclosporine A and quinidine inhibited propranolol uptake, whilst amantadine and verapamil were without effect. Together this suggests the involvement of specific members of SLC and ABC drug transporter families in pharmaceutical transport. PMID:25544062

  11. Type I secretion and multidrug efflux: transport through the TolC channel-tunnel.

    PubMed

    Buchanan, S K

    2001-01-01

    The crystal structure of TolC from Escherichia coli was recently determined to 2.1-A resolution and shows a unique type of channel architecture: a 12-stranded beta-barrel spans the outer membrane and is attached to a long alpha-helical channel that penetrates far into the periplasm. The structure suggests a mechanism for its role in secretion of proteins and in efflux of toxic small molecules. The TolC export pathway is compared with several import pathways of gram-negative bacteria where the outer membrane protein structures are also known.

  12. 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

  13. Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux.

    PubMed

    Buracco, Simona; Peracino, Barbara; Cinquetti, Raffaella; Signoretto, Elena; Vollero, Alessandra; Imperiali, Francesca; Castagna, Michela; Bossi, Elena; Bozzaro, Salvatore

    2015-09-01

    The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and the transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, and electrophysiological and radiochemical assays, we show that Nramp1, similar to rat DMT1 (also known as SLC11A2), transports Fe(2+) and manganese, not Fe(3+) or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe(2+) in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.

  14. Simultaneous Semimechanistic Population Analyses of Levofloxacin in Plasma, Lung, and Prostate To Describe the Influence of Efflux Transporters on Drug Distribution following Intravenous and Intratracheal Administration.

    PubMed

    Zimmermann, Estevan Sonego; Laureano, João Victor; Dos Santos, Camila Neris; Schmidt, Stephan; Lagishetty, Chakradhar V; de Castro, Whocely Victor; Dalla Costa, Teresa

    2016-02-01

    Levofloxacin (LEV) is a broad-spectrum fluoroquinolone used to treat pneumonia, urinary tract infections, chronic bacterial bronchitis, and prostatitis. Efflux transporters, primarily P-glycoprotein (P-gp), are involved in LEV's tissue penetration. In the present work, LEV free lung and prostate interstitial space fluid (ISF) concentrations were evaluated by microdialysis in Wistar rats after intravenous (i.v.) and intratracheal (i.t.) administration (7 mg/kg of body weight) with and without coadministration of the P-gp inhibitor tariquidar (TAR; 15 mg/kg administered i.v.). Plasma and tissue concentration/time profiles were evaluated by noncompartmental analysis (NCA) and population pharmacokinetics (popPK) analysis. The NCA showed significant differences in bioavailability (F) for the control group (0.4) and the TAR group (0.86) after i.t. administration. A four-compartment model simultaneously characterized total plasma and free lung (compartment 2) and prostate (compartment 3) ISF concentrations. Statistically significant differences in lung and prostate average ISF concentrations and levels of kidney active secretion in the TAR group from those measured for the control group (LEV alone) were observed. The estimated population means were as follows: volume of the central compartment (V1), 0.321 liters; total plasma clearance (CL), 0.220 liters/h; TAR plasma clearance (CLTAR), 0.180 liters/h. The intercompartmental distribution rate constants (K values) were as follows: K12, 8.826 h(-1); K21, 7.271 h(-1); K13, 0.047 h(-1); K31, 7.738 h(-1); K14, 0.908 h(-1); K41, 0.409 h(-1); K21 lung TAR (K21LTAR), 8.883 h(-1); K31 prostate TAR (K31PTAR), 4.377 h(-1). The presence of P-gp considerably impacted the active renal secretion of LEV but had only a minor impact on the efflux from the lung following intratracheal dosing. Our results strongly support the idea of a role of efflux transporters other than P-gp contributing to LEV's tissue penetration into the prostrate.

  15. Characterization of gastrointestinal absorption of digoxin involving influx and efflux transporter in rats: application of mdr1a knockout (-/-) rats into absorption study of multiple transporter substrate.

    PubMed

    Suzuki, Motoya; Komura, Hiroshi; Yoshikawa, Tomonori; Enya, Seiji; Nagao, Akemi; Takubo, Hiroaki; Kogayu, Motohiro

    2014-11-01

    1.  This study was aimed to characterize gastrointestinal absorption of digoxin using wild-type (WT) and multidrug resistance protein 1a [mdr1a; P-glycoprotein (P-gp)] knockout (-/-) rats. 2.  In WT rats, the area under the plasma concentration-time curve (AUC) of oral digoxin increased after oral pretreatment with quinidine at 30 mg/kg compared with non-treatment, but the increasing ratio tended to decrease at a high dose of 100 mg/kg. In mdr1a (-/-) rats, however, quinidine pretreatment caused a dose-dependent decrease in the AUC. 3.  Quinidine pretreatment did not alter the hepatic availability of digoxin, indicating that the changes in the digoxin AUC were attributable to inhibition of the absorption process by quinidine; i.e. inhibition of influx by quinidine in mdr1a (-/-) rats and inhibition of efflux and influx by quinidine in WT rats. 4.  An in situ rat intestinal closed loop study using naringin implied that organic anion transporting peptide (Oatp) 1a5 may be a responsible transporter in the absorption of digoxin. 5.  These findings imply that the rat absorption behavior of digoxin is possibly governed by Oatp1a5-mediated influx and P-gp-mediated efflux. The mdr1a (-/-) rat is therefore a useful in vivo tool to investigate drug absorption associated with multiple transporters including P-gp.

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

    PubMed

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

    2004-08-01

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

  17. SWEET sugar transporters for phloem transport and pathogen nutrition.

    PubMed

    Chen, Li-Qing

    2014-03-01

    Many intercellular solute transport processes require an apoplasmic step, that is, efflux from one cell and subsequent uptake by an adjacent cell. Cellular uptake transporters have been identified for many solutes, including sucrose; however, efflux transporters have remained elusive for a long time. Cellular efflux of sugars plays essential roles in many processes, such as sugar efflux as the first step in phloem loading, sugar efflux for nectar secretion, and sugar efflux for supplying symbionts such as mycorrhiza, and maternal efflux for filial tissue development. Furthermore, sugar efflux systems can be hijacked by pathogens for access to nutrition from hosts. Mutations that block recruitment of the efflux mechanism by the pathogen thus cause pathogen resistance. Until recently, little was known regarding the underlying mechanism of sugar efflux. The identification of sugar efflux carriers, SWEETs (Sugars Will Eventually be Exported Transporters), has shed light on cellular sugar efflux. SWEETs appear to function as uniporters, facilitating diffusion of sugars across cell membranes. Indeed, SWEETs probably mediate sucrose efflux from putative phloem parenchyma into the phloem apoplasm, a key step proceeding phloem loading. Engineering of SWEET mutants using transcriptional activator-like effector nuclease (TALEN)-based genomic editing allowed the engineering of pathogen resistance. The widespread expression of the SWEET family promises to provide insights into many other cellular efflux mechanisms.

  18. Regulation of ATP-binding cassette transporters and cholesterol efflux by glucose in primary human monocytes and murine bone marrow-derived macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. Two models were used...

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

    PubMed Central

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

    2013-01-01

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

  20. Cellular and regional specific changes in multidrug efflux transporter expression during recovery of vasogenic edema in the rat hippocampus and piriform cortex.

    PubMed

    Kim, Yeon-Joo; Kim, Ji-Eun; Choi, Hui-Chul; Song, Hong-Ki; Kang, Tae-Cheon

    2015-06-01

    In the present study, we investigated the characteristics of drug efflux transporter expressions following status epilepticus (SE). In the hippocampus and piriform cortex (PC), vasogenic edema peaked 3-4 days after SE. The expression of breast cancer resistance protein (BCRP), multidrug resistance protein-4 (MRP4), and p-glycoprotein (p-GP) were decreased 4 days after SE when vasogenic edema was peaked, but subsequently increased 4 weeks after SE. Multidrug resistance protein-1 (MRP1) expression gradually decreased in endothelial cells until 4 weeks after SE. These findings indicate that SE-induced vasogenic edema formation transiently reduced drug efflux pump expressions in endothelial cells. Subsequently, during recovery of vasogenic edema drug efflux pump expressions were differentially upregulated in astrocytes, neuropils, and endothelial cells. Therefore, we suggest that vasogenic edema formation may be a risk factor in pharmacoresistent epilepsy.

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

    PubMed

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

    2014-05-01

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

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

    PubMed

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

    2011-09-01

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

  4. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    PubMed

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  5. Moving out: from sterol transport to drug resistance - the ABCG subfamily of efflux pumps.

    PubMed

    Moitra, Karobi; Silverton, Latoya; Limpert, Katy; Im, Kate; Dean, Michael

    2011-01-01

    The ATP binding cassette (ABC) proteins are typically ATP-driven transmembrane pumps that have been evolutionarily conserved from bacteria to humans. In humans these transporters are subdivided into seven subfamilies, ranging from A to G. The ABCG subfamily of transporters is the primary focus of this review. This subfamily of proteins has been conserved throughout evolution and plays a central role in several cellular processes, such as sterol homeostasis and multidrug resistance. Functional polymorphisms/mutations in some of these G-subfamily transporters have clinical consequences in humans.

  6. 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

  7. The gut microbiota ellagic acid-derived metabolite urolithin A and its sulfate conjugate are substrates for the drug efflux transporter breast cancer resistance protein (ABCG2/BCRP).

    PubMed

    González-Sarrías, Antonio; Miguel, Verónica; Merino, Gracia; Lucas, Ricardo; Morales, Juan C; Tomás-Barberán, Francisco; Alvarez, Ana I; Espín, Juan C

    2013-05-08

    The breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter that can affect the pharmacological and toxicological properties of many molecules. Urolithins, metabolites produced by the gut microbiota from ellagic acid (EA) and ellagitannins, have been acknowledged with in vivo anti-inflammatory and cancer chemopreventive properties. This study evaluated whether urolithins (Uro-A, -B, -C, and -D) and their main phase II metabolites Uro-A sulfate, Uro-A glucuronide, and Uro-B glucuronide as well as their precursor EA were substrates for ABCG2/BCRP. Parental and Bcrp1-transduced MDCKII cells were used for active transport assays. Uro-A and, to a lesser extent, Uro-A sulfate showed a significant increase in apically directed translocation in Bcrp1-transduced cells. Bcrp1 did not show affinity for the rest of the tested compounds. Data were confirmed for murine, human, bovine, and ovine BCRP-transduced subclones as well as with the use of the selective BCRP inhibitor Ko143. The transport inhibition by Uro-A was analyzed by flow cytometry compared to Ko143 using the antineoplastic agent mitoxantrone as a model substrate. Results showed that Uro-A was able to inhibit mitoxantrone transport in a dose-dependent manner. This study reports for the first time that Uro-A and its sulfate conjugate are ABCG2/BCRP substrates. The results suggest that physiologically relevant concentrations of these gut microbiota-derived metabolites could modulate ABCG2/BCRP-mediated transport processes and mechanisms of cancer drug resistance. Further in vivo investigations are warranted.

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

    PubMed

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

    2017-03-01

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

  9. Transport of gemifloxacin, a 4th generation quinolone antibiotic, in the Caco-2 and engineered MDCKII cells, and potential involvement of efflux transporters in the intestinal absorption of the drug.

    PubMed

    Jin, Hyo-Eon; Song, Boran; Kim, Sang-Bum; Shim, Won-Sik; Kim, Dae-Duk; Chong, Saeho; Chung, Suk-Jae; Shim, Chang-Koo

    2013-04-01

    The oral (po) bioavailability of gemifloxacin mesylate in rats and its possible association with efflux transporters was investigated. The apparent permeabilities (Papp) of gemifloxacin across the Caco-2 cell monolayer were 1.20 ± 0.09 × 10(-5) cm/s for apical to basal (absorptive) transport, and 2.13 ± 0.6 × 10(-5) cm/s for basal to apical (secretory) transport for a 5-500 μM concentration range, suggesting the involvement of a carrier-mediated efflux in the secretory transport. The secretory transport in Caco-2 cells was significantly decreased by MRP2 (MK571) and BCRP (Ko143) inhibitors. The secretory transport was distinct in MDCKII/P-gp, MDCKII/MRP2 and MDCKII/BCRP cells, and the affinity was highest for MRP2, followed by BCRP and P-gp. The efflux was significantly decreased by verapamil and Ko143, but not significantly by MK571. The comparative po bioavailability in rats was increased by the preadministration of Ko143 (four-fold), MK571 (two-fold) and verapamil (two-fold). Efflux transporters appeared to significantly limit the bioavailability of gemifloxacin in rats, suggesting their possible contribution to the low bioavailability of the drug in the human (70%).

  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. Efficiency of transcellular transport and efflux of flavonoids with different glycosidic units from flavonoids of Litsea coreana L. in a MDCK epithelial cell monolayer model.

    PubMed

    Chen, Zhaolin; Ma, Taotao; Huang, Cheng; Zhang, Lei; Zhong, Jian; Han, Jingwen; Hu, Tingting; Li, Jun

    2014-03-12

    Although there is strong evidence to suggest that beneficial effects of the flavonoids in human health, the extent to which flavonoids are absorbed and the mechanisms involved are controversial. The objective of this study was to determine the bi-directional permeability and efflux characters of the four main flavonoids with different glycosidic units isolated from flavonoids of Litsea coreana L. and to discuss the transport mechanisms using the epithelial cell model MDCK. The transport of the four main flavonoid glycosides at concentration of 40, 80, 160 μM was concentration-dependent in both apical to basolateral and the reverse direction. Contemporary, the influx and efflux of the flavonoid glycosides were temperature-dependent and pH-dependent at concentration of 80 μM, and transport of flavonoid glycosides was obviously decreased when experiments performed in the presence of 1mM sodium azide (an ATP inhibitor). Uptake of quercetin-3-O-β-D-glucoside or kaempferol-3-O-β-D-glucoside was inhibited by 50 μM phloridzin, a specific and competitive inhibitor of SGLT. Moreover, the flavonoids exhibited significantly larger basolateral to apical Papp than that of the reverse direction, suggesting the existence of efflux mechanisms. The 50 μM verapamil, a chemical inhibitor of P-glycoprotein (P-gp), had no effect on the transport of four flavonoid glycosides. However, 50 μM MK-571 or 1 mM probenecid, MRP2 inhibitors, led to an apparently decrease in the efflux of flavonoid glycosides. Therefore, MRP2 but P-gp may be involved in the transport of the four flavonoid glycosides. Taken together, the experimental observations in our study provide useful information for pharmacological applications of flavonoids with different glycosidic units from flavonoids of L. coreana L.

  12. Crystallization and preliminary X-ray diffraction analysis of the multidrug efflux transporter NorM from Neisseria gonorrhoeae

    SciTech Connect

    Su, C.C.; Long, F.; McDermott, G.; Shafer, W.M.; Yu, E.W.

    2008-06-03

    The crystallization and preliminary X-ray data analysis of the NorM multidrug efflux pump produced by Neisseria gonorrhoeae are reported. NorM is a cytoplasmic membrane protein that consists of 459 amino-acid residues. It is a member of the recently classified multidrug and toxic compound extrusion (MATE) family of transporters and recognizes a number of cationic toxic compounds such as ethidium bromide, acriflavin, 2-N-methylellipticinium and ciprofloxacin. Recombinant NorM protein was expressed in Escherichia coli and purified by metal-affinity and gel-filtration chromatography. The protein was crystallized using hanging-drop vapor diffusion. X-ray diffraction data were collected from cryocooled crystals at a synchrotron light source. The best crystal diffracted anisotropically to 3.8 {angstrom} and diffraction data were complete to 6.5 {angstrom} resolution. The space group was determined to be C2, with unit-cell parameters a = 81.5, b = 164.4, c = 111.5 {angstrom}.

  13. Effect of Efflux Transporter Inhibition on the Distribution of Fluconazole in the Rat Brain.

    PubMed

    Wang, Wei; Zheng, Na; Zhang, Jiatang; Huang, Xusheng; Yu, Shengyuan

    2017-03-24

    Multidrug resistance-associated proteins (MRPs) and organic anion transporters (OATs) are expressed on the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB), preventing the entry of or the pumping out of numerous molecules. Fluconazole is widely used to treat fungal meningoencephalitis. The effect of these transporters on the distribution of fluconazole in the brain is unclear. We used microdialysis to compare the distribution of fluconazole in the rat brain with and without co-administration of probenecid, a MRP and OAT inhibitor. Additionally, we also observed the difference in fluconazole distribution between the two barriers. The results showed that probenecid increased the penetration of fluconazole into the BBB but did not alter the penetration of fluconazole into the BCSFB of rats. The penetration of the BBB and BCSFB by fluconazole did not statistically differ according to physiological condition. These results demonstrate that transporters that can be inhibited by probenecid may be involved in fluconazole resistance at the BBB and provide a laboratory basis for predicting brain extracellular fluid (ECF) concentration using the cerebrospinal fluid (CSF) concentration of fluconazole.

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

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    1997-01-01

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

  16. 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

  17. Interactions of attention-deficit/hyperactivity disorder therapeutic agents with the efflux transporter P-glycoprotein

    PubMed Central

    Zhu, Hao-Jie; Wang, Jun-Sheng; Donovan, Jennifer L.; Jiang, Yan; Gibson, Bryan B.; DeVane, C. Lindsay; Markowitz, John S.

    2009-01-01

    The objective of this study was to assess the potential interactions of the drug transporter P-glycoprotein with attention-deficit/hyperactivity disorder (ADHD) therapeutic agents atomoxetine — and the individual isomers of methylphenidate, amphetamine, and modafinil utilizing established in vitro assay. An initial ATPase assay indicated that both d- and l-methylphenidate have weak affinity for P-glycoprotein. The intracellular accumulation of P-glycoprotein substrates doxorubicin and rhodamine123 in the P-glycoprotein overexpressing cell line LLC-PK1/MDR1 was determined to evaluate potential inhibitory effects on P-glycoprotein. The results demonstrated that all compounds, except both modafinil isomers, significantly increased doxorubicin and rhodamine123 accumulation in LLC-PK1/MDR1 cells at higher concentrations. To investigate the P-glycoprotein substrate properties, the intracellular concentrations of the tested compounds in LLC-PK1/MDR1 and P-glycoprotein negative LLC-PK1 cells were measured in the presence and absence of the P-glycoprotein inhibitor PSC833. The results indicate that the accumulation of d-methylphenidate in LLC-PK1 cells was 32.0% higher than in LLC-PK1/MDR1 cells. Additionally, coadministration of PSC833 leads to 52.9% and 45.6% increases in d-modafinil and l-modafinil accumulation, respectively, in LLC-PK1/MDR1 cells. Further studies demonstrated that l-modafinil transport across LLC-PK1/MDR1 cell monolayers in the basolateral-to-apical (B–A) direction was significantly higher than in the apical-to-basolateral (A–B) direction. PSC833 treatment significantly decreased the transport of l-modafinil in B–A direction. In conclusion, our results suggest that all tested agents with the exception of modafinil isomers are relatively weak P-glycoprotein inhibitors. Furthermore, P-glycoprotein may play a minor role in the transport of d-methylphenidate, d-modafinil, and l-modafinil. PMID:17963743

  18. Improving soil CO2 efflux estimates from in-situ soil CO2 sensors with gas transport measurements

    NASA Astrophysics Data System (ADS)

    Sanchez-Canete, E. P.; Barron-Gafford, G.; Van Haren, J. L. M.; Scott, R. L.

    2015-12-01

    Correctly estimating soil carbon dioxide (CO2) fluxes emitted to the atmosphere is essential because they are a large component of the ecosystem carbon balance. Continuous estimates of soil CO2 flux, especially when paired with eddy covariance measurements of whole-ecosystem CO­2 exchange, help to disaggregate net ecosystem CO2 exchange. Most researchers estimate soil CO2 fluxes by applying the gradient method; however, this is only appropriate in the absence of advective or convective processes. Given the rarity of such static states, we must move toward measurement techniques that will allow us to quantify the dynamic soil efflux system with gas transport by convective, advective and molecular diffusion processes. Convective processes are mainly relevant in caves, where values of relative humidity, temperature and CO2 molar fraction determine the buoyancy of the external-internal air masses. These convective processes also are important in large fractures when temperature differences between surface and depth can generate convection, transporting CO2 from deep layers to the atmosphere. Advective processes occur both in caves and in soils, and the CO2 exchanges are mainly due to three factors: wind, changes in atmospheric pressure, and changes in the water table. Molecular diffusion processes are being widely applied in the determination of soil-atmosphere gas exchanges by applying the gradient method. However, the use of the gradient method can yield inappropriate flux estimates due to the uncertainties mainly associated with the inappropriate determination of the soil diffusion coefficient. Therefore, in-situ methods to determine diffusion coefficient are necessary to obtain accurate CO2 fluxes. If this is resolved, the gradient method has great potential to become the most used technique to monitor atmosphere-soil CO2 exchanges within the next few years. Here we review the state of the science and describe a series of field measurements for significantly

  19. 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

  20. Efflux Transport Characterization of Resveratrol Glucuronides in UDP-Glucuronosyltransferase 1A1 Transfected HeLa Cells: Application of a Cellular Pharmacokinetic Model to Decipher the Contribution of Multidrug Resistance-Associated Protein 4.

    PubMed

    Wang, Shuai; Li, Feng; Quan, Enxi; Dong, Dong; Wu, Baojian

    2016-04-01

    Resveratrol undergoes extensive metabolism to form biologically active glucuronides in humans. However, the transport mechanisms for resveratrol glucuronides are not fully established. Here, we aimed to characterize the efflux transport of resveratrol glucuronides using UGT1A1-overexpressing HeLa cells (HeLa1A1 cells), and to determine the contribution of multidrug resistance-associated protein (MRP) 4 to cellular excretion of the glucuronides. Two glucuronide isomers [i.e., resveratrol 3-O-glucuronide (R3G) and resveratrol 4'-O-glucuronide (R4'G)] were excreted into the extracellular compartment after incubation of resveratrol (1-100 μM) with HeLa1A1 cells. The excretion rate was linearly related to the level of intracellular glucuronide, indicating that glucuronide efflux was a nonsaturable process. MK-571 (a dual inhibitor of UGT1A1 and MRPs) significantly decreased the excretion rates of R3G and R4'G while increasing their intracellular levels. Likewise, short-hairpin RNA (shRNA)-mediated silencing of MRP4 caused a significant reduction in glucuronide excretion but an elevation in glucuronide accumulation. Furthermore, β-glucuronidase expressed in the cells catalyzed the hydrolysis of the glucuronides back to the parent compound. A cellular pharmacokinetic model integrating resveratrol transport/metabolism with glucuronide hydrolysis/excretion was well fitted to the experimental data, allowing derivation of the efflux rate constant values in the absence or presence of shRNA targeting MRP4. It was found that a large percentage of glucuronide excretion (43%-46%) was attributed to MRP4. In conclusion, MRP4 participated in cellular excretion of R3G and R4'G. Integration of mechanistic pharmacokinetic modeling with transporter knockdown was a useful method to derive the contribution percentage of an exporter to overall glucuronide excretion.

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

    PubMed

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

    2015-01-01

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

  2. 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

  3. Multidrug Resistance-Associated Protein 4 (MRP4/ABCC4) Controls Efflux Transport of Hesperetin Sulfates in Sulfotransferase 1A3-Overexpressing Human Embryonic Kidney 293 Cells.

    PubMed

    Sun, Hua; Wang, Xiao; Zhou, Xiaotong; Lu, Danyi; Ma, Zhiguo; Wu, Baojian

    2015-10-01

    Sulfonation is an important metabolic pathway for hesperetin. However, the mechanisms for the cellular disposition of hesperetin and its sulfate metabolites are not fully established. In this study, disposition of hesperetin via the sulfonation pathway was investigated using human embryonic kidney (HEK) 293 cells overexpressing sulfotransferase 1A3. Two monosulfates, hesperetin-3'-O-sulfate (H-3'-S) and hesperetin-7-O-sulfate (H-7-S), were rapidly generated and excreted into the extracellular compartment upon incubation of the cells with hesperetin. Regiospecific sulfonation of hesperetin by the cell lysate followed the substrate inhibition kinetics (Vmax = 0.66 nmol/min per mg, Km = 12.9 μM, and Ksi= 58.1 μM for H-3'-S; Vmax = 0.29 nmol/min per mg, Km = 14.8 μM, and Ksi= 49.1 μM for H-7-S). The pan-multidrug resistance-associated protein (MRP) inhibitor MK-571 at 20 μM essentially abolished cellular excretion of both H-3'-S and H-7-S (the excretion activities were only 6% of the control), whereas the breast cancer resistance protein-selective inhibitor Ko143 had no effects on sulfate excretion. In addition, knockdown of MRP4 led to a substantial reduction (>47.1%; P < 0.01) in sulfate excretion. Further, H-3'-S and H-7-S were good substrates for transport by MRP4 according to the vesicular transport assay. Moreover, sulfonation of hesperetin and excretion of its metabolites were well characterized by a two-compartment pharmacokinetic model that integrated drug uptake and sulfonation with MRP4-mediated sulfate excretion. In conclusion, the exporter MRP4 controlled efflux transport of hesperetin sulfates in HEK293 cells. Due to significant expression in various organs/tissues (including the liver and kidney), MRP4 should be a determining factor for the elimination and body distribution of hesperetin sulfates.

  4. Effects of stachyose on absorption and transportation of tea catechins in mice: possible role of Phase II metabolic enzymes and efflux transporters inhibition by stachyose

    PubMed Central

    Li, Wenfeng; Lu, Yalong; Huang, Di; Han, Xiao; Yang, Xingbin

    2016-01-01

    Background Nutritional and absorption-promoting properties of stachyose combined with tea catechins (TC) have been revealed. However, the mechanism involved in non-digestible oligosaccharides-mediated enhancement of flavonoid absorption has largely remained elusive. Methods This study was designed to investigate the molecular mechanism of stachyose in enhancing absorption and transportation of TC in mice. Mice were orally pre-treated with stachyose (50, 250, and 500 mg/kg·bw) for 0–8 weeks, and 1 h before sacrifice, mice were treated with TC (250 mg/kg·bw). Results Gas chromatography-mass spectrometry analysis showed that serum concentrations of epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate were dose- and time-dependently elevated with stachyose pre-treatment in mice. Furthermore, pre-treatment with stachyose in mice reduced intestinal sulfotransferase and uridine diphosphate-glucuronosyltransferase levels by 3.3–43.2% and 23.9–30.4%, relative to control mice, respectively. Moreover, intestinal P-glycoprotein and multidrug resistance-associated protein-1 contents were decreased in mice by pre-administration of stachyose in dose- and time-dependent manner. Conclusions This is the first time to demonstrate that suppression of Phase II metabolic enzymes and efflux transporters of TC in the intestine can play a major role in increasing absorption of TC by stachyose feeding. PMID:27782875

  5. 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...

  6. Unraveling carbohydrate transport mechanisms in young beech trees (Fagus sylvatica f. purpurea) by 13CO2 efflux measurements from stem and soil

    NASA Astrophysics Data System (ADS)

    Thoms, Ronny; Muhr, Jan; Keitel, Claudia; Kayler, Zachary; Gavrichkova, Olga; Köhler, Michael; Gessler, Arthur; Gleixner, Gerd

    2016-04-01

    Transport mechanisms of soluble carbohydrates and diurnal CO2 efflux from tree stems and surrounding soil are well studied. However, the effect of transport carbohydrates on respiration and their interaction with storage processes is largely unknown. Therefore, we performed a set of 13CO2 pulse labeling experiments on young trees of European beech (Fagus sylvatica f. purpurea). We labeled the whole tree crowns in a closed transparent plastic chamber with 99% 13CO2 for 30 min. In one experiment, only a single branch was labeled and removed 36 hours after labeling. In all experiments, we continuously measured the 13CO2 efflux from stem, branch and soil and sampled leaf and stem material every 3 h for 2 days, followed by a daily sampling of leaves in the successive 5 days. The compound specific δ 13C value of extracted soluble carbohydrates from leaf and stem material was measured by high-performance liquid chromatography linked with an isotope ratio mass spectrometer (HPLC-IRMS). The 13CO2 signal from soil respiration occurred only few hours after labeling indicating a very high transport rate of carbohydrates from leaf to roots and to the rhizosphere. The label was continuously depleted within the next 5 days. In contrast, we observed a remarkable oscillating pattern of 13CO2 efflux from the stem with maximum 13CO2 enrichment at noon and minima at night time. This oscillation suggests that enriched carbohydrates are respired during the day, whereas in the night the enriched sugars are not respired. The observed oscillation in stem 13CO2 enrichment remained unchanged even when only single branches were labelled and cut right afterwards. Thus, storage and conversion of carbohydrates only occurred within the stem. The δ13C patterns of extracted soluble carbohydrates showed, that a transformation of transitory starch to carbohydrates and vice versa was no driver of the oscillating 13CO2 efflux from the stem. Carbohydrates might have been transported in the phloem to

  7. Novel E-ring camptothecin keto analogues (S38809 and S39625) are stable, potent, and selective topoisomerase I inhibitors without being substrates of drug efflux transporters.

    PubMed

    Takagi, Kazutaka; Dexheimer, Thomas S; Redon, Christophe; Sordet, Olivier; Agama, Keli; Lavielle, Gilbert; Pierré, Alain; Bates, Susan E; Pommier, Yves

    2007-12-01

    Camptothecin (CPT) analogues are powerful anticancer agents but are chemically unstable due to their alpha-hydroxylactone six-membered E-ring structure, which is essential for trapping topoisomerase I (Top1)-DNA cleavage complexes. To stabilize the E-ring, CPT keto analogues with a five-membered E-ring lacking the oxygen of the lactone ring (S38809 and S39625) have been synthesized. S39625 has been selected for advanced preclinical development based on its promising activity in tumor models. Here, we show that both keto analogues are active against purified Top1 and selective against Top1 in yeast and human cancer cells. The keto analogues show improved cytotoxicity toward colon, breast, and prostate cancer cells and leukemia cells compared with CPT. The drug-induced Top1-DNA cleavage complexes induced by the keto analogues show remarkable persistence both with purified Top1 and in cells following 1-h drug treatments. Moreover, we find that S39625 is not a substrate for either the ABCB1 (multidrug resistance-1/P-glycoprotein) or ABCG2 (mitoxantrone resistance/breast cancer resistance protein) drug efflux transporters, which sets S39625 apart from the clinically used CPT analogues topotecan or SN-38 (active metabolite of irinotecan). Finally, we show that nanomolar concentrations of S38809 or S39625 induce intense and persistent histone gamma-H2AX. The chemical stability of the keto analogues and the ability of S39625 to produce high levels of persistent Top1-DNA cleavage complex and its potent antiproliferative activity against human cancer cell lines make S39625 a promising new anticancer drug candidate. Histone gamma-H2AX could be used as a biomarker for the upcoming clinical trials of S39625.

  8. The Efflux Pump Inhibitor Reserpine Selects Multidrug-Resistant Streptococcus pneumoniae Strains That Overexpress the ABC Transporters PatA and PatB▿ †

    PubMed Central

    Garvey, Mark I.; Piddock, Laura J. V.

    2008-01-01

    One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer. PMID:18362193

  9. Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking.

    PubMed

    Petris, M J; Mercer, J F; Culvenor, J G; Lockhart, P; Gleeson, P A; Camakaris, J

    1996-11-15

    The Menkes P-type ATPase (MNK), encoded by the Menkes gene (MNK; ATP7A), is a transmembrane copper-translocating pump which is defective in the human disorder of copper metabolism, Menkes disease. Recent evidence that the MNK P-type ATPase has a role in copper efflux has come from studies using copper-resistant variants of cultured Chinese hamster ovary (CHO) cells. These variants have MNK gene amplification and consequently overexpress MNK, the extents of which correlate with the degree of elevated copper efflux. Here, we report on the localization of MNK in these copper-resistant CHO cells when cultured in different levels of copper. Immunofluorescence studies demonstrated that MNK is predominantly localized to the Golgi apparatus of cells in basal medium. In elevated copper conditions there was a rapid trafficking of MNK from the Golgi to the plasma membrane. This shift in steady-state distribution of MNK was reversible and not dependent on new protein synthesis. In media containing basal copper, MNK accumulated in cytoplasmic vesicles after treatment of cells with a variety of agents that inhibit endosomal recycling. We suggest that MNK continuously recycles between the Golgi and the plasma membrane and elevated copper shifts the steady-state distribution from the Golgi to the plasma membrane. These data reveal a novel system of regulated protein trafficking which ultimately leads to the efflux of an essential yet potentially toxic ligand, where the ligand itself appears directly and specifically to stimulate the trafficking of its own transporter.

  10. Transport to Rhebpress activity.

    PubMed

    Garrido, Amanda; Brandt, Marta; Djouder, Nabil

    2016-01-01

    The small GTPases from the rat sarcoma (Ras) superfamily are a heterogeneous group of proteins of about 21 kDa that act as molecular switches, modulating cell signaling pathways and controlling diverse cellular processes. They are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). Ras homolog enriched in brain (Rheb) is a member of the Ras GTPase superfamily and a key activator of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). We recently determined that microspherule protein 1 (MCRS1) maintains Rheb at lysosomal surfaces in an amino acid-dependent manner. MCRS1 depletion promotes the formation of the GDP-bound form of Rheb, which is then delocalized from the lysosomal platform and transported to endocytic recycling vesicles, leading to mTORC1 inactivation. During this delocalization process, Rheb-GDP remains farnesylated and associated with cellular endomembranes. These findings provide new insights into the regulation of small GTPases, whose activity depends on both their GTP/GDP switch state and their capacity to move between different cellular membrane-bound compartments. Dynamic spatial transport between compartments makes it possible to alter the proximity of small GTPases to their activatory sites depending on the prevailing physiological and cellular conditions.

  11. 13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

    PubMed Central

    2011-01-01

    Background Synthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. Methods RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux. Results Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells. Conclusion 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway. PMID:22129452

  12. VvBOR1, the grapevine ortholog of AtBOR1, encodes an efflux boron transporter that is differentially expressed throughout reproductive development of Vitis vinifera L.

    PubMed

    Pérez-Castro, Ramón; Kasai, Koji; Gainza-Cortés, Felipe; Ruiz-Lara, Simón; Casaretto, José A; Peña-Cortés, Hugo; Tapia, Jaime; Fujiwara, Toru; González, Enrique

    2012-02-01

    Boron (B) is an essential micronutrient for normal development of roots, shoots and reproductive tissues in plants. Due to its role in the structure of rhamnogalacturonan II, a polysaccharide required for pollen tube growth, B deficiency has been associated with the occurrence of parthenocarpic seedless grapes in some varieties of Vitis vinifera L. Despite that, it is unclear how B is mobilized and accumulated in reproductive tissues. Here we describe the characterization of an efflux B transporter, VvBOR1, homolog to AtBOR1, which is involved in B xylem loading in Arabidopsis thaliana roots. VvBOR1-green fluorescent protein (GFP) fusion protein expressed in A. thaliana localizes in the proximal plasma membrane domain in root pericycle cells, and VvBOR1 overexpression restores the wild-type phenotype in A. thaliana bor1-3 mutant plants exposed to B deficiency. Complementation of a mutant yeast strain indicates that VvBOR1 corresponds to a B efflux transporter. Transcriptional analyses during grapevine reproductive development show that the VvBOR1 gene is preferentially expressed in flowers at anthesis and a direct correlation between the expression pattern and B content in grapes was established, suggesting the involvement of this transporter in B accumulation in grapevine berries.

  13. 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

  14. 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

  15. Centrifuge-induced hypergravity and glutamate efflux by reversal of high-affinity, sodium-dependent transporters from rat brain synaptosomes.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Himmelreich, N.

    Glutamate uptake by high affinity sodium-dependent glutamate transporters is essential for termination of the synaptic transmission. Glutamate transporters may also contribute to an increase in extracellular glutamate. Glutamate efflux can occur by reversal of the sodium-dependent glutamate transporters during ATP depletion and dissipation of the sodium gradient across the cell membrane. Depolarization-induced calcium independent release of neurotransmitter from synaptosomal cytosolic pool is Na+-dependent and due to reverse of the neurotransmitter transporters also. We used monovalent organic cations N-methyl-D-glucamine (NMDG) to replace extracellular sodium, suggesting that the reducing of Na+ elucidate further the mechanism underlying Ca2+-independent glutamate release. A reduction in extracellular sodium would facilitate reversal of sodium-dependent transporters with extrusion of glutamate. We have compared the basal release of glutamate in Ca2+-free Na+-supplemented and NMDG-supplemented medium in control and after exposure of animals to long-arm centrifuge-induced hypergravity (ten G, during one hour). Replacement of sodium by NMDG enhanced basal level of neurotransmitter. The value of basal level increased to 110± 4% and 140± 2% in the medium with NMDG in comparison with Na+ under the control and hypergravity conditions, respectively. It is likely to reflect the enhancement of the neurotransmitter level in cytosolic pool. Thermodynamic considerations show that the extracellular level of a amino acid neurotransmitter, such as glutamate, that can be generated by transporter reversal are directly proportional to the intracellular concentration of the intracellular concentration of amino acid. KCl-stimulated glutamate release from cytosolic pool increased not statistically after hypergravity loading. We examined the effects of transporter inhibitors DL-threo-beta-benzyloxyaspartate ( DL-TBOA) on the release to elucidate whether reverse transport via the

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

    PubMed

    Suzuki, Toyofumi; Fukami, Toshiro; Tomono, Kazuo

    2015-03-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    1999-01-01

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

  20. 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

  1. Roles of inner blood-retinal barrier organic anion transporter 3 in the vitreous/retina-to-blood efflux transport of p-aminohippuric acid, benzylpenicillin, and 6-mercaptopurine.

    PubMed

    Hosoya, Ken-ichi; Makihara, Akihide; Tsujikawa, Yuki; Yoneyama, Daisuke; Mori, Shinobu; Terasaki, Tetsuya; Akanuma, Shin-ichi; Tomi, Masatoshi; Tachikawa, Masanori

    2009-04-01

    The purpose of the present study was to characterize rat organic anion transporter (Oat) 3 (Oat3, Slc22a8) in the efflux transport at the inner blood-retinal barrier (BRB). Reverse transcription-polymerase chain reaction analysis showed that rat (r) Oat3 mRNA is expressed in retinal vascular endothelial cells (RVECs), but not rOat1 and rOat2 mRNA. The expression of Oat3 in the retina and human cultured retinal endothelial cells was further confirmed by Western blot analysis. Immunohistochemical staining in RVECs showed that rOat3 is colocalized with glucose transporter 1, but not P-glycoprotein, suggesting that rOat3 is possibly located at the abluminal membrane of the RVEC. The contribution of rOat3 to the efflux of [(3)H]p-aminohippuric acid ([(3)H]PAH), [(3)H]benzylpenicillin ([(3)H]PCG), and [(14)C]6-mercaptopurine ([(14)C]6-MP), substrates of rOat3, from the vitreous humor/retina to the circulating blood across the inner BRB was evaluated using the microdialysis method. [(3)H]PAH, [(3)H]PCG, [(14)C]6-MP, and [(14)C] or [(3)H]d-mannitol, a bulk flow marker, were biexponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]PAH, [(3)H]PCG, and [(14)C]6-MP during the terminal phase was approximately 2-fold greater than that of d-mannitol. This efflux transport was reduced in the retinal presence of probenecid, PAH, and PCG, whereas it was not inhibited by digoxin. In conclusion, rOat3 is expressed at the inner BRB and involved in the vitreous humor/retina-to-blood transport of PAH, PCG, and 6-MP. This transport system is one mechanism to limit the retinal distribution of PAH, PCG, and 6-MP.

  2. The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development

    PubMed Central

    Sakhtah, Hassan; Koyama, Leslie; Zhang, Yihan; Morales, Diana K.; Fields, Blanche L.; Price-Whelan, Alexa; Hogan, Deborah A.; Shepard, Kenneth; Dietrich, Lars E. P.

    2016-01-01

    Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based self-resistance, controlled by a simple circuit, in a Gram-negative pathogen. PMID:27274079

  3. The Pseudomonas aeruginosa efflux pump MexGHI-OpmD transports a natural phenazine that controls gene expression and biofilm development.

    PubMed

    Sakhtah, Hassan; Koyama, Leslie; Zhang, Yihan; Morales, Diana K; Fields, Blanche L; Price-Whelan, Alexa; Hogan, Deborah A; Shepard, Kenneth; Dietrich, Lars E P

    2016-06-21

    Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHI-OpmD in the opportunistic pathogen Pseudomonas aeruginosa Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHI-OpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based self-resistance, controlled by a simple circuit, in a Gram-negative pathogen.

  4. 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.

  5. 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.

  6. Dopamine Transporter Activity Is Modulated by α-Synuclein.

    PubMed

    Butler, Brittany; Saha, Kaustuv; Rana, Tanu; Becker, Jonas P; Sambo, Danielle; Davari, Paran; Goodwin, J Shawn; Khoshbouei, Habibeh

    2015-12-04

    The duration and strength of the dopaminergic signal are regulated by the dopamine transporter (DAT). Drug addiction and neurodegenerative and neuropsychiatric diseases have all been associated with altered DAT activity. The membrane localization and the activity of DAT are regulated by a number of intracellular proteins. α-Synuclein, a protein partner of DAT, is implicated in neurodegenerative disease and drug addiction. Little is known about the regulatory mechanisms of the interaction between DAT and α-synuclein, the cellular location of this interaction, and the functional consequences of this interaction on the basal, amphetamine-induced DAT-mediated dopamine efflux, and membrane microdomain distribution of the transporter. Here, we found that the majority of DAT·α-synuclein protein complexes are found at the plasma membrane of dopaminergic neurons or mammalian cells and that the amphetamine-mediated increase in DAT activity enhances the association of these proteins at the plasma membrane. Further examination of the interaction of DAT and α-synuclein revealed a transient interaction between these two proteins at the plasma membrane. Additionally, we found DAT-induced membrane depolarization enhances plasma membrane localization of α-synuclein, which in turn increases dopamine efflux and enhances DAT localization in cholesterol-rich membrane microdomains.

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

    PubMed

    Rowell, Peter P; Volk, Kelly A

    2004-01-01

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

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

    PubMed

    Amaral, Leonard; Spengler, Gabriella; Molnar, Joseph

    2016-11-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. Interaction of BDE-47 and its Hydroxylated Metabolite 6-OH-BDE-47 with the Human ABC Efflux Transporters P-gp and BCRP: Considerations for Human Exposure and Risk Assessment

    EPA Science Inventory

    ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp; also known as MDR1, ABCB1) and breast cancer resistance protein (BCRP; also known as ABCG2), are membrane-bound proteins that mediate the cellular efflux of xenobiotics as an important defense against chemic...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-09

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

  13. 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

  14. 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.

  15. ABCG1 is involved in vitamin E efflux.

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

    Ruhfus, B; Kinne, R K

    1996-01-01

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

  18. Laboratory Exercise on Active Transport.

    ERIC Educational Resources Information Center

    Stalheim-Smith, Ann; Fitch, Greg K.

    1985-01-01

    Describes a laboratory exercise which demonstrates qualitatively the specificity of the transport mechanism, including a consideration of the competitive inhibition, and the role of adenosine triphosphate (ATP) in active transport. The exercise, which can be completed in two to three hours by groups of four students, consistently produces reliable…

  19. Activated transport in AMTEC electrodes

    NASA Technical Reports Server (NTRS)

    Williams, R. M.; Jeffries-Nakamura, B.; Ryan, M. A.; Underwood, M. L.; O'Connor, D.; Kikkert, S.

    1992-01-01

    Transport of alkali, metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. The temperature dependence of the diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and preexponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process.

  20. 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

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

    PubMed

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

    1999-06-15

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

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

    PubMed Central

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

    2010-01-01

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

  3. Activated transport in AMTEC electrodes

    SciTech Connect

    Williams, R.M.; Jeffries-Nakamura, B.; Ryan, M.A.; Underwood, M.L.; O`Connor, D.; Kikkert, S.

    1992-07-01

    Transport of alkali metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant, reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. Little can be learned about the detailed mass transport process from the ac impedance or current voltage curves of an electrode at one set of operating parameters, because the transport process includes a number of important physical parameters that are not all uniquely determined by one experiment. The temperature dependence of diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and pre-exponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process. 17 refs.

  4. Activated transport in AMTEC electrodes

    SciTech Connect

    Williams, R.M.; Jeffries-Nakamura, B.; Ryan, M.A.; Underwood, M.L.; O'Connor, D.; Kikkert, S.

    1992-01-01

    Transport of alkali metal atoms through porous cathodes of alkali metal thermal-to-electric converter (AMTEC) cells is responsible for significant, reducible losses in the electrical performance of these cells. Experimental evidence for activated transport of metal atoms at grain surfaces and boundaries within some AMTEC electrodes has been derived from temperature dependent studies as well as from analysis of the detailed frequency dependence of ac impedance results for other electrodes, including thin, mature molybdenum electrodes which exhibit transport dominated by free molecular flow of sodium gas at low frequencies or dc conditions. Activated surface transport will almost always exist in parallel with free molecular flow transport, and the process of alkali atom adsorption/desorption from the electrode surface will invariably be part of the transport process, and possibly a dominant part in some cases. Little can be learned about the detailed mass transport process from the ac impedance or current voltage curves of an electrode at one set of operating parameters, because the transport process includes a number of important physical parameters that are not all uniquely determined by one experiment. The temperature dependence of diffusion coefficient of the alkali metal through the electrode in several cases provides an activation energy and pre-exponential, but at least two activated processes may be operative, and the activation parameters should be expected to depend on the alkali metal activity gradient that the electrode experiences. In the case of Pt/W/Mn electrodes operated for 2500 hours, limiting currents varied with electrode thickness, and the activation parameters could be assigned primarily to the surface/grain boundary diffusion process. 17 refs.

  5. CHX14 is a plasma membrane K-efflux transporter that regulates K+ redistribution in "Arabidopsis thaliana"

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Potassium (K(+)) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. "CHX14" expression was induced by elevated K(+) and histochemical analysis...

  6. Ceramide 1-Phosphate Increases P-Glycoprotein Transport Activity at the Blood-Brain Barrier via Prostaglandin E2 Signaling.

    PubMed

    Mesev, Emily V; Miller, David S; Cannon, Ronald E

    2017-04-01

    P-glycoprotein, an ATP-driven efflux pump, regulates permeability of the blood-brain barrier (BBB). Sphingolipids, endogenous to brain tissue, influence inflammatory responses and cell survival in vitro. Our laboratory has previously shown that sphingolipid signaling by sphingosine 1-phosphate decreases basal P-glycoprotein transport activity. Here, we investigated the potential for another sphingolipid, ceramide 1-phosphate (C1P), to modulate efflux pumps at the BBB. Using confocal microscopy and measuring luminal accumulation of fluorescent substrates, we assessed the transport activity of several efflux pumps in isolated rat brain capillaries. C1P treatment induced P-glycoprotein transport activity in brain capillaries rapidly and reversibly. In contrast, C1P did not affect transport activity of two other major efflux transporters, multidrug resistance protein 2 and breast cancer resistance protein. C1P induced P-glycoprotein transport activity without changing transporter protein expression. Inhibition of the key signaling components in the cyclooxygenase-2 (COX-2)/prostaglandin E2 signaling cascade (phospholipase A2, COX-2, multidrug resistance protein 4, and G-protein-coupled prostaglandin E2 receptors 1 and 2), abolished P-glycoprotein induction by C1P. We show that COX-2 and prostaglandin E2 are required for C1P-mediated increases in P-glycoprotein activity independent of transporter protein expression. This work describes how C1P activates a signaling cascade to dynamically regulate P-glycoprotein transport at the BBB and offers potential clinical targets to modulate neuroprotection and drug delivery to the CNS.

  7. Ceramide 1-Phosphate Increases P-Glycoprotein Transport Activity at the Blood-Brain Barrier via Prostaglandin E2 Signaling

    PubMed Central

    Mesev, Emily V.; Miller, David S.

    2017-01-01

    P-glycoprotein, an ATP-driven efflux pump, regulates permeability of the blood-brain barrier (BBB). Sphingolipids, endogenous to brain tissue, influence inflammatory responses and cell survival in vitro. Our laboratory has previously shown that sphingolipid signaling by sphingosine 1-phosphate decreases basal P-glycoprotein transport activity. Here, we investigated the potential for another sphingolipid, ceramide 1-phosphate (C1P), to modulate efflux pumps at the BBB. Using confocal microscopy and measuring luminal accumulation of fluorescent substrates, we assessed the transport activity of several efflux pumps in isolated rat brain capillaries. C1P treatment induced P-glycoprotein transport activity in brain capillaries rapidly and reversibly. In contrast, C1P did not affect transport activity of two other major efflux transporters, multidrug resistance protein 2 and breast cancer resistance protein. C1P induced P-glycoprotein transport activity without changing transporter protein expression. Inhibition of the key signaling components in the cyclooxygenase-2 (COX-2)/prostaglandin E2 signaling cascade (phospholipase A2, COX-2, multidrug resistance protein 4, and G-protein–coupled prostaglandin E2 receptors 1 and 2), abolished P-glycoprotein induction by C1P. We show that COX-2 and prostaglandin E2 are required for C1P-mediated increases in P-glycoprotein activity independent of transporter protein expression. This work describes how C1P activates a signaling cascade to dynamically regulate P-glycoprotein transport at the BBB and offers potential clinical targets to modulate neuroprotection and drug delivery to the CNS. PMID:28119480

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

    Liao, Julie; Schurr, Michael J.

    2013-01-01

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

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

    PubMed

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

    2017-03-02

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

  11. Calcium Efflux from Barnacle Muscle Fibers

    PubMed Central

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

    1974-01-01

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

  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. Functional, structural and phylogenetic analysis of domains underlying the Al-sensitivity of the aluminium-activated malate/anion transporter, TaALMT1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    TaALMT1 (Triticum aestivum Aluminum Activated Malate Transporter) is the founding member of a novel gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small subgroup of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (...

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

    PubMed

    Rey, Ana

    2015-05-01

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

  15. [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.

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

    PubMed

    Hermawan, Adam; Wagner, Ernst; Roidl, Andreas

    2016-03-01

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

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

    PubMed

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

    2017-04-01

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

  18. Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein.

    PubMed

    Matsushima, Soichiro; Maeda, Kazuya; Kondo, Chihiro; Hirano, Masaru; Sasaki, Makoto; Suzuki, Hiroshi; Sugiyama, Yuichi

    2005-09-01

    Until recently, it was generally believed that the transport of various organic anions across the bile canalicular membrane was mainly mediated by multidrug resistance-associated protein 2 (MRP2/ABCC2). However, a number of new reports have shown that some organic anions are also substrates of multidrug resistance 1 (MDR1/ABCB1) and/or breast cancer resistance protein (BCRP/ABCG2), implying MDR1 and BCRP could also be involved in the biliary excretion of organic anions in humans. In the present study, we constructed new double-transfected Madin-Darby canine kidney II (MDCKII) cells expressing organic anion-transporting polypeptide 1B1 (OATP1B1)/MDR1 and OATP1B1/BCRP, and we investigated the transcellular transport of four kinds of organic anions, estradiol-17beta-d-glucuronide (EG), estrone-3-sulfate (ES), pravastatin (PRA), and cerivastatin (CER), to identify which efflux transporters mediate the biliary excretion of compounds using double-transfected cells. We observed the vectorial transport of EG and ES in all the double transfectants. MRP2 showed the highest efflux clearance of EG among these efflux transporters, whereas BCRP-mediated clearance of ES was the highest in these double transfectants. In addition, two kinds of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, CER and PRA, were also substrates of all these efflux transporters. The rank order of the efflux clearance of PRA mediated by each transporter was the same as that of EG, whereas the contribution of MDR1 to the efflux of CER was relatively greater than for PRA. This experimental system is very useful for identifying which transporters are involved in the biliary excretion of organic anions that cannot easily penetrate the plasma membrane.

  19. ArsP: a methylarsenite efflux permease

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    1990-01-01

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

  1. P-glycoprotein mediated efflux limits the transport of the novel anti-Parkinson's disease candidate drug FLZ across the physiological and PD pathological in vitro BBB models.

    PubMed

    Liu, Qian; Hou, Jinfeng; Chen, Xiaoguang; Liu, Gengtao; Zhang, Dan; Sun, Hua; Zhang, Jinlan

    2014-01-01

    FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.

  2. Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment

    EPA Science Inventory

    Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

  3. 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

  4. Efflux Transporters at the Blood-Brain Barrier Limit Delivery and Efficacy of Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib (PD-0332991) in an Orthotopic Brain Tumor Model

    PubMed Central

    Parrish, Karen E.; Pokorny, Jenny; Mittapalli, Rajendar K.; Bakken, Katrina; Sarkaria, Jann N.

    2015-01-01

    6-Acetyl-8-cyclopentyl-5-methyl-2-([5-(piperazin-1-yl)pyridin-2-yl]amino)pyrido(2,3-d)pyrimidin-7(8H)-one [palbociclib (PD-0332991)] is a cyclin-dependent kinase 4/6 inhibitor approved for the treatment of metastatic breast cancer and is currently undergoing clinical trials for many solid tumors. Glioblastoma (GBM) is the most common primary brain tumor in adults and has limited treatment options. The cyclin-dependent kinase 4/6 pathway is commonly dysregulated in GBM and is a promising target in treating this devastating disease. The blood-brain barrier (BBB) limits the delivery of drugs to invasive regions of GBM, where the efflux transporters P-glycoprotein and breast cancer resistance protein can prevent treatments from reaching the tumor. The purpose of this study was to examine the mechanisms limiting the effectiveness of palbociclib therapy in an orthotopic xenograft model. The in vitro intracellular accumulation results demonstrated that palbociclib is a substrate for both P-glycoprotein and breast cancer resistance protein. In vivo studies in transgenic mice confirmed that efflux transport is responsible for the limited brain distribution of palbociclib. There was an ∼115-fold increase in brain exposure at steady state in the transporter deficient mice when compared with wild-type mice, and the efflux inhibitor elacridar significantly increased palbociclib brain distribution. Efficacy studies demonstrated that palbociclib is an effective therapy when GBM22 tumor cells are implanted in the flank, but ineffective in an orthotopic (intracranial) model. Moreover, doses designed to mimic brain exposure were ineffective in treating flank tumors. These results demonstrate that efflux transport in the BBB is involved in limiting the brain distribution of palbociclib and this has critical implications in determining effective dosing regimens of palbociclib therapy in the treatment of brain tumors. PMID:26354993

  5. Drug interaction between sunitinib and cimetidine and contribution of the efflux transporter ATP-binding cassette C2 to biliary excretion of sunitinib in rats.

    PubMed

    Arakawa-Todo, Maki; Ueyama, Jun; Nomura, Hiroshi; Abe, Fumie; Tsukiyama, Ikuto; Matsuura, Katsuhiko; Hasegawa, Takaaki

    2013-08-01

    The present study investigated the effect of the H2 antagonist cimetidine on the pharmacokinetics of a multi-targeted receptor tyrosine kinase (RTK) inhibitor, sunitinib, in Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic mutant rats (EHBR) lacking the efflux transporter, ATP-binding cassette C2 protein (ABCC2). Rats received an intraperitoneal injection of cimetidine (10 mg/kg) once a day for three days. On day 4, sunitinib (3 mg/kg) was administered intravenously 30 min after the final injection of cimetidine or saline to SD rats. Disappearance of sunitinib from plasma was significantly delayed by cimetidine. The pharmacokinetic parameter of sunitinib, systemic clearance (CLSYS), was significantly reduced and the half-life was significantly prolonged, with no change in the volume of distribution at steady-state (VSS). When the effect of cimetidine on the biliary excretion of sunitinib at steady-state condition was investigated in SD rats, cimetidine had no effect on some transporter-mediated biliary excretion of sunitinib. Furthermore, the contribution of ABCC2 to the biliary excretion of sunitinib was also examined in SD rats and EHBR. The biliary clearance of sunitinib was significantly lower in EHBR, but the biliary excretion rate of EHBR was not different from that of SD rats, and the contribution of biliary excretion to systemic elimination was small, suggesting that sunitinib is mainly eliminated by cytochrome P450 3A4 (CYP3A4)-mediated metabolism and is not excreted into the bile via ABCC2. These findings indicate that co-administration of cimetidine alters the pharmacokinetics of sunitinib probably due to inhibition of CYP3A4, suggesting the possibility that cimetidine should be used carefully for patients with cancer being treated with sunitinib therapy.

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

    PubMed

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

    1986-09-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    1996-08-01

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

  9. Activation of ion transport systems during cell volume regulation

    SciTech Connect

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

    1987-01-01

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

  10. Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance.

    PubMed

    Blair, Jessica M A; Richmond, Grace E; Piddock, Laura J V

    2014-01-01

    Gram-negative bacteria express a plethora of efflux pumps that are capable of transporting structurally varied molecules, including antibiotics, out of the bacterial cell. This efflux lowers the intracellular antibiotic concentration, allowing bacteria to survive at higher antibiotic concentrations. Overexpression of some efflux pumps can cause clinically relevant levels of antibiotic resistance in Gram-negative pathogens. This review discusses the role of efflux in resistance of clinical isolates of Gram-negative bacteria, the regulatory mechanisms that control efflux pump expression, the recent advances in our understanding of efflux pump structure and how inhibition of efflux is a promising future strategy for tackling multidrug resistance in Gram-negative pathogens.

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

    PubMed Central

    Blair, Jessica M. A.

    2016-01-01

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

  12. 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.

  13. An electrically tight in vitro blood-brain barrier model displays net brain-to-blood efflux of substrates for the ABC transporters, P-gp, Bcrp and Mrp-1.

    PubMed

    Helms, Hans Christian; Hersom, Maria; Kuhlmann, Louise Borella; Badolo, Lasina; Nielsen, Carsten Uhd; Brodin, Birger

    2014-09-01

    Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95 ± 0.1 · 10(-6) cm·s(-1) and high transendothelial electrical resistances of 1,177 ± 101 Ω·cm(2). Bidirectional transport studies with (3)H-digoxin, (3)H-estrone-3-sulphate and (3)H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5 ± 0.2 for digoxin, 4.4 ± 0.5 for estrone-3-sulphate and 2.4 ± 0.1 for etoposide were observed. These were reduced to 1.1 ± 0.08, 1.4 ± 0.2 and 1.5 ± 0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar + reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport.

  14. Active and passive transport of drugs in the human placenta.

    PubMed

    Włoch, Stanisław; Pałasz, Artur; Kamiński, Marcin

    2009-10-01

    The human placenta, characterized by the processes of passive transport and facilitated diffusion, contains numerous active transport proteins, usually located in the microvilli of the syncytiotrophoblast or in the endothelium of the capillaries of the villi. These proteins use either the energy from ATP hydrolysis or other mechanisms resulting, among others, from the formation of the maternofetal ion gradient, which facilitates the transfer of various endogenous substances or xenobiotics across the body membranes. The proteins either trigger the efflux of these substances from the fetal tissues via the placenta into the maternal bloodstream, or conversely they accumulate them in the fetal tissues. Both the placenta and the fetus are equipped with independent systems of enzymes of 1st and 2nd phase of substrate metabolism, such as CYP450, glucuronyltransferase or sulphatase. An active therapy with a wide range of drugs, often at high toxicity levels, either shortly before or during pregnancy, has naturally posed a question concerning the degree of impermeability of the placental barrier and how effectively it can be crossed, including any possible negative embryotoxic or teratogenic consequences. Such hazards seem to be quite real, as many drugs are substrates for ABC transporters. Also the placenta itself, including its structure, is subject to vast transformations during pregnancy which may be observed as the thinning of the barrier separating the maternal blood from the fetal one, from 20-30 microm in the first trimester of gestation down to 2-4 microm in the third trimester of gestation.

  15. Effects of rhinacanthin-C on function and expression of drug efflux transporters in Caco-2 cells.

    PubMed

    Wongwanakul, Ratjika; Vardhanabhuti, Nontima; Siripong, Pongpun; Jianmongkol, Suree

    2013-09-01

    Rhinacanthin-C is a bioactive naphthoquinone ester found in Rhinacanthus nasutus Kurz (Acanthaceae). This compound has potential therapeutic value as an anticancer and antiviral agent. The purposes of this study were to determine the effects of this compound on the function and the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2), using the in vitro model of Caco-2 cells. The activities of P-gp and MRP2 were determined by following the intracellular accumulation of calcein and 5(6)-carboxy-2',7'-dichlorofluorescein in the uptake assays with fluorescence spectroscopy. The expression of P-gp after prolonged exposure was evaluated by flow cytometry with the use of a fluorescein isothiocyanate-conjugated anti-human P-gp antibody. Our results showed that the inhibitory effect of rhinacanthin-C was more potent toward P-gp than MRP2, and was reversible. Short-term exposure of Caco-2 cells with rhinacanthin-C (100 μM) resulted in increase in P-gp expression without any significant change in its function. Extended exposure of Caco-2 cells to the naphthoquinone at the highest non-cytotoxic concentration (0.625 μM) for 7 days had no effect on the expression and the function of P-gp. These findings suggested that rhinacanthin-C might raise the problem of herb-drug interaction when co-administered with other P-gp substrates.

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

    PubMed

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

    2011-06-01

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

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

    PubMed

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

    2010-10-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  20. 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.

  1. Common folds and transport mechanisms of secondary active transporters.

    PubMed

    Shi, Yigong

    2013-01-01

    Secondary active transporters exploit the electrochemical potential of solutes to shuttle specific substrate molecules across biological membranes, usually against their concentration gradient. Transporters of different functional families with little sequence similarity have repeatedly been found to exhibit similar folds, exemplified by the MFS, LeuT, and NhaA folds. Observations of multiple conformational states of the same transporter, represented by the LeuT superfamily members Mhp1, AdiC, vSGLT, and LeuT, led to proposals that structural changes are associated with substrate binding and transport. Despite recent biochemical and structural advances, our understanding of substrate recognition and energy coupling is rather preliminary. This review focuses on the common folds and shared transport mechanisms of secondary active transporters. Available structural information generally supports the alternating access model for substrate transport, with variations and extensions made by emerging structural, biochemical, and computational evidence.

  2. Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes

    PubMed Central

    Bachour-El Azzi, Pamela; Sharanek, Ahmad; Burban, Audrey; Li, Ruoya; Guével, Rémy Le; Abdel-Razzak, Ziad; Stieger, Bruno; Guguen-Guillouzo, Christiane; Guillouzo, André

    2015-01-01

    The role of hepatobiliary transporters in drug-induced liver injury remains poorly understood. Various in vivo and in vitro biological approaches are currently used for studying hepatic transporters; however, appropriate localization and functional activity of these transporters are essential for normal biliary flow and drug transport. Human hepatocytes (HHs) are considered as the most suitable in vitro cell model but erratic availability and inter-donor functional variations limit their use. In this work, we aimed to compare localization of influx and efflux transporters and their functional activity in differentiated human HepaRG hepatocytes with fresh HHs in conventional (CCHH) and sandwich (SCHH) cultures. All tested influx and efflux transporters were correctly localized to canalicular [bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), multidrug resistance protein 1 (MDR1), and MDR3] or basolateral [Na+-taurocholate co-transporting polypeptide (NTCP) and MRP3] membrane domains and were functional in all models. Contrary to other transporters, NTCP and BSEP were less abundant and active in HepaRG cells, cellular uptake of taurocholate was 2.2- and 1.4-fold and bile excretion index 2.8- and 2.6-fold lower, than in SCHHs and CCHHs, respectively. However, when taurocholate canalicular efflux was evaluated in standard and divalent cation-free conditions in buffers or cell lysates, the difference between the three models did not exceed 9.3%. Interestingly, cell imaging showed higher bile canaliculi contraction/relaxation activity in HepaRG hepatocytes and larger bile canaliculi networks in SCHHs. Altogether, our results bring new insights in mechanisms involved in bile acids accumulation and excretion in HHs and suggest that HepaRG cells represent a suitable model for studying hepatobiliary transporters and drug-induced cholestasis. PMID:25690737

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

    PubMed

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

    2015-04-01

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

  4. Multidrug efflux pumps in Staphylococcus aureus and their clinical implications.

    PubMed

    Jang, Soojin

    2016-01-01

    Antibiotic resistance is rapidly spreading among bacteria such as Staphylococcus aureus, an opportunistic bacterial pathogen that causes a variety of diseases in humans. For the last two decades, bacterial multidrug efflux pumps have drawn attention due to their potential association with clinical multidrug resistance. Numerous researchers have demonstrated efflux-mediated resistance in vitro and in vivo and found novel multidrug transporters using advanced genomic information about bacteria. This article aims to provide a concise summary of multidrug efflux pumps and their important clinical implications, focusing on recent findings concerning S. aureus efflux pumps.

  5. Targeting efflux pumps to overcome antifungal drug resistance.

    PubMed

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

    2016-08-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

    Jung, Hyungjin; Best, Makenzie; Akkus, Ozan

    2015-07-01

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

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

    PubMed

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

    2016-10-20

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

  9. Action of Steroidal Diamines on Active Transport and Permeability Properties of Escherichia coli

    PubMed Central

    Silver, Simon; Levine, Elaine

    1968-01-01

    The steroidal diamine irehdiamine A (IDA) is a potent inhibitor of bacteriophage growth and macromolecular synthesis in Escherichia coli. By using radioactive 42K and 14C-thiomethylgalactoside (TMG), rapid effects of IDA and related steroids, both on the influx of potassium and TMG via their respective transport systems and on the efflux (leakage) of radioactivity from the treated cells, have been measured. IDA affects both the influx and efflux of 42K at concentrations of steroid as low as 2 × 10−5m. Because of the increased leakage, it is not possible to tell whether there is a direct effect reducing the rate of active transport of potassium. The primary diamine, IDA, and its bis-secondary, bis-tertiary, and bis-quaternary diamine analogues are decreasingly effective in altering cell permeability properties in the order 1° > 2° > 3° > 4°. The effects of IDA on potassium transport are mirrored by similar effects on the transport of TMG. Therefore, the action of IDA is on the cell membrane and not directly on one or another transport system. The effects of IDA on cell permeability can reasonably explain the inhibitory actions of the drugs on bacteriophage growth and cellular metabolism. PMID:4877122

  10. Compartmental models for apical efflux by P-glycoprotein. Part 1. Evaluation of model complexity

    PubMed Central

    Nagar, Swati; Tucker, Jalia; Weiskircher, Erica A.; Bhoopathy, Siddhartha; Hidalgo, Ismael J.; Korzekwa, Ken

    2013-01-01

    Purpose With the goal of quantifying P-gp transport kinetics, Part 1 of these manuscripts evaluates different compartmental models and Part 2 applies these models to kinetic data. Methods Models were developed to simulate the effect of apical efflux transporters on intracellular concentrations of six drugs. The effect of experimental variability on model predictions was evaluated. Several models were evaluated, and characteristics including membrane configuration, lipid content, and apical surface area (asa) were varied. Results Passive permeabilities from MDCK-MDR1 cells in the presence of cyclosporine gave lower model errors than from MDCK control cells. Consistent with the results in Part 2, model configuration had little impact on calculated model errors. The 5-compartment model was the simplest model that reproduced experimental lag times. Lipid content and asa had minimal effect on model errors, predicted lag times, and intracellular concentrations. Including endogenous basolateral uptake activity can decrease model errors. Models with and without explicit membrane barriers differed markedly in their predicted intracellular concentrations for basolateral drug exposure. Single point data resulted in clearances similar to time course data. Conclusions Compartmental models are useful to evaluate the impact of efflux transporters on intracellular concentrations. Whereas a 3-compartment model may be sufficient to predict the impact of transporters that efflux drugs from the cell, a 5-compartment model with explicit membranes may be required to predict intracellular concentrations when efflux occurs from the membrane. More complex models including additional compartments may be unnecessary. PMID:24019023

  11. Political activity for physical activity: health advocacy for active transport

    PubMed Central

    2011-01-01

    Effective health advocacy is a priority for efforts to increase population participation in physical activity. Local councils are an important audience for this advocacy. The aim of the current study was to describe features of advocacy for active transport via submissions to city council annual plans in New Zealand, and the impact of an information sheet to encourage the health sector to be involved in this process. Written submissions to city council's annual consultation process were requested for 16 city councils over the period of three years (2007/08, 2008/09, and 2009/10). Submissions were reviewed and categories of responses were created. An advocacy information sheet encouraging health sector participation and summarising some of the evidence-base related to physical activity, active transport and health was released just prior to the 2009/10 submission time. Over the period of the study, city councils received 47,392 submissions, 17% of which were related to active transport. Most submissions came from city residents, with a small proportion (2%) from the health sector. The largest category of submissions was in support of pedestrian and cycling infrastructure, design and maintenance of facilities and additional features to support use of these transport modes. Health arguments featured prominently in justifications for active transport initiatives, including concerns about injury risk, obesity, physical inactivity, personal safety and facilities for people with disabilities. There was evidence that the information sheet was utilised by some health sector submitters (12.5%), providing tentative support for initiatives of this nature. In conclusion, the study provides novel information about the current nature of health advocacy for active transport and informs future advocacy efforts about areas for emphasis, such as health benefits of active transport, and potential alliances with other sectors such as environmental sustainability, transport and urban

  12. 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

  13. 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.

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

    SciTech Connect

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

    2016-10-21

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

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

    DOE PAGES

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

    2016-10-21

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

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

    PubMed

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

    2015-11-02

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

  17. Modified host cells with efflux pumps

    DOEpatents

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

    2016-08-30

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

  18. Energetics of active transport processes.

    PubMed

    Essig, A; Caplan, S R

    1968-12-01

    Discussions of active transport usually assume stoichiometry between the rate of transport J(+) and the metabolic rate J(r). However, the observation of a linear relationship between J(+) and J(r) does not imply a stoichiometric relationship, i.e., complete coupling. Since coupling may possibly be incomplete, we examine systems of an arbitrary degree of coupling q, regarding stoichiometry as a limiting case. We consider a sodium pump, with J(+) and J(r) linear functions of the electrochemical potential difference, -X(+), and the chemical affinity of the metabolic driving reaction, A. The affinity is well defined even for various complex reaction pathways. Incorporation of a series barrier and a parallel leak does not affect the linearity of the composite observable system. The affinity of some region of the metabolic chain may be maintained constant, either by large pools of reactants or by regulation. If so, this affinity can be evaluated by two independent methods. Sodium transport is conveniently characterized by the open-circuit potential (Deltapsi)(I=0) and the natural limits, level flow (J(+))(X+=0), and static head X(0) (+) = (X(+))(J+=0). With high degrees of coupling -X(0) (+)/F approaches the electromotive force E(Na) (Ussing); -X(0) (+)/F cannot be identified with ((RT/F) ln f)(X+=0), where f is the flux ratio. The efficiency eta = -J(+)X(+)/J(r)A is of significance only when appreciable energy is being converted from one form to another. When either J(+) or -X(+) is small eta is low; the significant parameters are then the efficacies epsilon(J+) = J(+)/J(r)A and epsilon(X+) = -X(+)/J(r)A, respectively maximal at level flow and static head. Leak increases both J(+) and epsilon(J+) for isotonic saline reabsorption, but diminishes -X(0) (+) and epsilon(Xfemale symbol). Electrical resistance reflects both passive parameters and metabolism. Various fundamental relations are preserved despite coupling of passive ion and water flows.

  19. Regulation of hepatic drug transporter activity and expression by organochlorine pesticides.

    PubMed

    Bucher, Simon; Le Vee, Marc; Jouan, Elodie; Fardel, Olivier

    2014-03-01

    Organochlorine (OC) pesticides constitute a major class of persistent and toxic organic pollutants, known to modulate drug-detoxifying enzymes. In the present study, OCs were demonstrated to also alter the activity and expression of human hepatic drug transporters. Activity of the sinusoidal influx transporter OCT1 (organic cation transporter 1) was thus inhibited by endosulfan, chlordane, heptachlor, lindane, and dieldrine, but not by dichlorodiphenyltrichloroethane isomers, whereas those of the canalicular efflux pumps MRP2 (multidrug resistance-associated protein 2) and BCRP (breast cancer resistance protein) were blocked by endosulfan, chlordane, heptachlor, and chlordecone; this latter OC additionally inhibited the multidrug resistance gene 1 (MDR1)/P-glycoprotein (P-gp) activity. OCs, except endosulfan, were next found to induce MDR1/P-gp and MRP2 mRNA expressions in hepatoma HepaRG cells; some of them also upregulated BCRP. By contrast, expression of sinusoidal transporters was not impaired (organic anion-transporting polypeptide (OATP) 1B1 and OATP2B1) or was downregulated (sodium taurocholate co-transporting polypeptide (NTCP) and OCT1). Such regulations of drug transporter activity and expression, depending on the respective nature of OCs and transporters, may contribute to the toxicity of OC pesticides.

  20. 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.

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

    PubMed

    Shao, Fei; Ford, David A

    2013-08-01

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

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

    PubMed Central

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

    2015-01-01

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

  3. 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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-12-01

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

  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. Red blood cell cation transports in uraemic anaemia: evidence for an increased K/Cl co-transport activity. Effects of dialysis and erythropoietin treatment.

    PubMed

    De Franceschi, L; Olivieri, O; Girelli, D; Lupo, A; Bernich, P; Corrocher, R

    1995-10-01

    This study examines the role of uraemia and the effect of different dialysis treatments on red cell cation transport. We evaluated the main cation transport systems in erythrocytes of non-dialysed end-stage renal disease (ESRD) subjects, of patients undergoing haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD), as well as the changes induced by human recombinant erythropoietin (r-HuEPO) administration. In uraemic undialysed and dialysed patients, we observed an increase in K/Cl co-transport activity and in shrinkage-induced amiloride-sensitive (HMA-sensitive) Na efflux (Na/H exchange) and a decrease in Na/K pump and Na/K/Cl co-transport activity, while Na/Li exchange was increased only in dialysed patients. In uraemic erythrocytes, we showed for the first time an increased K/Cl co-transport activity, which was cell age independent. Generally, the different method of dialysis (CAPD or HD) did not modify the cation transport abnormalities observed. During the treatment with r-HuEPO, all the systems, with the exception of the Na/K pump and Na/K/Cl co-transport, increased their activities following the increase of circulating young red cells. The changes produced under r-HuEPO administration were transient and cation transports returned to the baseline values within 100 days of treatment, indicating a primary and prominent pathogenetic role of uraemia in modulating the red cell membrane cation transport activities.

  8. Regulation of Human Hepatic Drug Transporter Activity and Expression by Diesel Exhaust Particle Extract

    PubMed Central

    Le Vee, Marc; Jouan, Elodie; Stieger, Bruno; Lecureur, Valérie; Fardel, Olivier

    2015-01-01

    Diesel exhaust particles (DEPs) are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe) may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC) uptake transporters organic anion-transporting polypeptides (OATP) 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC) efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT) 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP), whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP). Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a reference activator of the aryl hydrocarbon receptor (AhR) pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute to their

  9. 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.

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

    PubMed

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

    2011-05-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1998-04-01

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

  12. 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

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

    PubMed Central

    Nowak, Jennifer; Schneiders, Thamarai; Seifert, Harald

    2015-01-01

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

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

    PubMed

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

    2017-02-01

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

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

    PubMed

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

    2015-11-01

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

  16. eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells

    PubMed Central

    Yeh, Johannes T.-H.; Nam, Kwangho; Yeh, Joshua T.-H.; Perrimon, Norbert

    2017-01-01

    The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter. PMID:28176814

  17. 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

  18. 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.

  19. Abcb and Abcc transporter homologs are expressed and active in larvae and adults of zebra mussel and induced by chemical stress.

    PubMed

    Navarro, Anna; Weißbach, Susann; Faria, Melissa; Barata, Carlos; Piña, Benjamin; Luckenbach, Till

    2012-10-15

    Multixenobiotic resistance (MXR) of aquatic invertebrates has so far been associated with cellular efflux activity mediated by P-glycoprotein (ABCB1) and MRP (multidrug resistance protein; ABCC) type ABC (ATP binding cassette) transporters. Expression and activity of an abcb1/Abcb1 homolog has been shown in eggs and larvae of the zebra mussel Dreissena polymorpha. Here we report identification of a partial cDNA sequence of an abcc/Abcc homolog from zebra mussel that is transcribed and active as a cellular efflux transporter in embryos and gill tissue of adult mussels. Transcript expression levels were comparatively low in eggs and sharply increased after fertilization, then maintaining high expression levels in 1 and 2 dpf (days post fertilization) larvae. MK571, a known inhibitor of mammalian ABCC transporters, blocks efflux of calcein-am in larvae and gill tissue as indicated by elevated calcein fluorescence; this indicates the presence of active Abcc protein in cells of the larvae and gills. Dacthal and mercury used as chemical stressors both induced expression of abcb1 and abcc mRNAs in larvae; accordingly, assays with calcein-am and ABCB1 inhibitor reversin 205 and ABCC inhibitor MK571 indicated enhanced Abcb1 and Abcc efflux activities. Responses to chemicals were different in gills, where abcb1 transcript abundances were enhanced in dacthal and mercury treatments, whereas abcc mRNA was only increased with mercury. Abcb1 and Abcc activities did not in all cases show increases that were according to respective mRNA levels; thus, Abcc activity was significantly higher with dacthal, whereas Abcb1 activity was unchanged with mercury. Our data indicate that abcb1/Abcb1 and abcc/Abcc transporters are expressed and active in larvae and adult stages of zebra mussel. Expression of both genes is induced as cellular stress response, but regulation appears to differ in larvae and tissue of adult stages.

  20. Sodium efflux in plant roots: what do we really know?

    PubMed

    Britto, D T; Kronzucker, H J

    2015-08-15

    The efflux of sodium (Na(+)) ions across the plasma membrane of plant root cells into the external medium is surprisingly poorly understood. Nevertheless, Na(+) efflux is widely regarded as a major mechanism by which plants restrain the rise of Na(+) concentrations in the cytosolic compartments of root cells and, thus, achieve a degree of tolerance to saline environments. In this review, several key ideas and bodies of evidence concerning root Na(+) efflux are summarized with a critical eye. Findings from decades past are brought to bear on current thinking, and pivotal studies are discussed, both "purely physiological", and also with regard to the SOS1 protein, the only major Na(+) efflux transporter that has, to date, been genetically characterized. We find that the current model of rapid transmembrane sodium cycling (RTSC), across the plasma membrane of root cells, is not adequately supported by evidence from the majority of efflux studies. An alternative hypothesis cannot be ruled out, that most Na(+) tracer efflux from the root in the salinity range does not proceed across the plasma membrane, but through the apoplast. Support for this idea comes from studies showing that Na(+) efflux, when measured with tracers, is rarely affected by the presence of inhibitors or the ionic composition in saline rooting media. We conclude that the actual efflux of Na(+) across the plasma membrane of root cells may be much more modest than what is often reported in studies using tracers, and may predominantly occur in the root tips, where SOS1 expression has been localized.

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

    PubMed

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

    2017-01-23

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

  2. The transport properties of activated carbon fibers

    SciTech Connect

    di Vittorio, S.L. . Dept. of Materials Science and Engineering); Dresselhaus, M.S. . Dept. of Electrical Engineering and Computer Science Massachusetts Inst. of Tech., Cambridge, MA . Dept. of Physics); Endo, M. . Dept. of Electrical Engineering); Issi, J-P.; Piraux, L.

    1990-07-01

    The transport properties of activated isotropic pitch-based carbon fibers with surface area 1000 m{sup 2}/g have been investigated. We report preliminary results on the electrical conductivity, the magnetoresistance, the thermal conductivity and the thermopower of these fibers as a function of temperature. Comparisons are made to transport properties of other disordered carbons. 19 refs., 4 figs.

  3. The Transport Properties of Activated Carbon Fibers

    DOE R&D Accomplishments Database

    di Vittorio, S. L.; Dresselhaus, M. S.; Endo, M.; Issi, J-P.; Piraux, L.

    1990-07-01

    The transport properties of activated isotropic pitch-based carbon fibers with surface area 1000 m{sup 2}/g have been investigated. We report preliminary results on the electrical conductivity, the magnetoresistance, the thermal conductivity and the thermopower of these fibers as a function of temperature. Comparisons are made to transport properties of other disordered carbons.

  4. 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

  5. Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins

    PubMed Central

    Tseng, Pei-Chi; Lee, Tzong-Shyuan; Lee, Wen-Jane; Chang, Pey-Jium; Chiang, An-Na

    2016-01-01

    Metabolic syndrome (MetS) is a complicated health problem that encompasses a variety of metabolic disorders. In this study, we analyzed the relationship between the major biochemical parameters associated with MetS and circulating levels of microRNA (miR)-33, miR-103, and miR-155. We found that miRNA-33 levels were positively correlated with levels of fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, LDL-cholesterol, and triacylglycerol, but negatively correlated with HDL-cholesterol levels. In the cellular study, miR-33 levels were increased in macrophages treated with high glucose and cholesterol-lowering drugs atorvastatin and pitavastatin. miR-33 has been reported to play an essential role in cholesterol homeostasis through ATP-binding cassette transporter A1 (ABCA1) regulation and reverse cholesterol transport. However, the molecular mechanism underlying the linkage between miR-33 and statin treatment remains unclear. In the present study, we investigated whether atorvastatin and pitavastatin exert their functions through the modulation of miR-33 and ABCA1-mediated cholesterol efflux from macrophages. The results showed that treatment of the statins up-regulated miR-33 expression, but down-regulated ABCA1 mRNA levels in RAW264.7 cells and bone marrow-derived macrophages. Statin-mediated ABCA1 regulation occurs at the post-transcriptional level through targeting of the 3′-UTR of the ABCA1 transcript by miR-33. Additionally, we found significant down-regulation of ABCA1 protein expression in macrophages treated with statins. Finally, we showed that high glucose and statin treatment significantly suppressed cholesterol efflux from macrophages. These findings have highlighted the complexity of statins, which may exert detrimental effects on metabolic abnormalities through regulation of miR-33 target genes. PMID:27139226

  6. 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

  7. Endothelium-dependent relaxation of the pig aorta: relationship to stimulation of 86Rb efflux from isolated endothelial cells.

    PubMed Central

    Gordon, J. L.; Martin, W.

    1983-01-01

    Bradykinin, adenosine triphosphate (ATP) and acetylcholine each relaxed histamine-contracted strips of pig aorta in a dose-dependent manner. These relaxations were abolished when the endothelium was removed. Relaxation induced by ATP was mimicked by adenosine diphosphate (ADP) but adenosine monophosphate (AMP) and adenosine were about 120 times less potent. Relaxation induced by acetylcholine was antagonized by atropine in a competitive manner, and carbachol induced the same degree of relaxation as acetylcholine, but was about 10 times less potent. The calcium ionophore, A23187, also induced a dose-dependent relaxation of pig aortic strips provided the endothelium was present, suggesting that a rise in the level of ionized calcium within the endothelial cells is one means by which vascular smooth muscle relaxation can be triggered. Bradykinin, ATP, ADP, AMP, adenosine and A23187 each induced a dose-dependent increase in 86Rb efflux from preloaded pig aortic endothelial cells. The dose-response curves for stimulation of 86Rb efflux and for endothelium-dependent relaxation were similar for each individual compound. ADP was equipotent with ATP, but AMP and adenosine were about 120 times less potent. Neither acetylcholine nor carbachol, in concentrations that induce endothelium-dependent relaxation, had any effect on 86Rb efflux from isolated aortic endothelial cells. Lanthanum, which blocks calcium influx, abolished the increases in 86Rb efflux induced by bradykinin and ATP, and the calcium ionophore A23187 was the most effective stimulant of 86Rb efflux, suggesting that the potassium transport induced by these agents is calcium-activated. It is concluded that endothelial responses to bradykinin and ATP can be assessed by monitoring 86Rb efflux, which probably reflects a calcium-activated efflux of potassium associated with the endothelium-dependent vascular relaxation induced by these agents. This pathway is apparently not involved in endothelial responses to

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

    PubMed

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

    2011-07-29

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

  9. The role of the polymorphic efflux transporter P-glycoprotein on the brain accumulation of d-methylphenidate and d-amphetamine.

    PubMed

    Zhu, Hao-Jie; Wang, Jun-Sheng; DeVane, C Lindsay; Williard, Robin L; Donovan, Jennifer L; Middaugh, Lawrence D; Gibson, Brian B; Patrick, Kennerly S; Markowitz, John S

    2006-07-01

    The psychostimulant medications methylphenidate (MPH) and amphetamine (AMP), available in various ratios or enantiopure formulations of their respective active dextrorotary isomers, constitute the majority of agents used in the treatment of attention-deficit/hyperactivity disorder (ADHD). Substantial interindividual variability occurs in their pharmacokinetics and tolerability. Little is known regarding the potential role of drug transporters such as P-glycoprotein (P-gp) in psychostimulant pharmacokinetics and response. Therefore, experiments were carried out in P-gp knockout (KO) mice versus wild-type (WT) mice after intraperitoneal dosing (2.5 mg/kg) of d-MPH or (3.0 mg/kg) of d-AMP. After the administration of each psychostimulant, locomotor activity was assessed at 30-min intervals for 2 h. Total brain-to-plasma drug concentration ratios were determined at 10-, 30-, and 80-min postdosing time-points. The results showed no statistically supported genotypic difference in d-AMP-induced locomotor activity stimulation or in brain-to-plasma ratio of d-AMP. As for d-MPH, the P-gp KO mice had 33% higher brain concentrations (p < 0.05) and 67.5% higher brain-to-plasma ratios (p < 0.01) than WT controls at the 10-min postdosing timepoint. However, in spite of elevated brain concentrations, d-MPH-induced locomotor activity increase was attenuated for P-gp compared with that for WT mice. These data indicate that P-gp has no apparent effect on the pharmacokinetics and pharmacodynamics of d-AMP. In addition, d-MPH is a relatively weak P-gp substrate, and its entry into the brain may be limited by P-gp. Furthermore, the mechanism by which d-MPH-induced locomotor activity was attenuated in P-gp KO mice remains to be elucidated.

  10. Structural comparison of bacterial multidrug efflux pumps of the major facilitator superfamily

    PubMed Central

    Ranaweera, Indrika; Shrestha, Ugina; Ranjana, K.C.; Kakarla, Prathusha; Willmon, T. Mark; Hernandez, Alberto J.; Mukherjee, Mun Mun; Barr, Sharla R.; Varela, Manuel F.

    2016-01-01

    The biological membrane is an efficient barrier against water-soluble substances. Solute transporters circumvent this membrane barrier by transporting water-soluble solutes across the membrane to the other sides. These transport proteins are thus required for all living organisms. Microorganisms, such as bacteria, effectively exploit solute transporters to acquire useful nutrients for growth or to expel substances that are inhibitory to their growth. Overall, there are distinct types of related solute transporters that are grouped into families or superfamilies. Of these various transporters, the major facilitator superfamily (MFS) represents a very large and constantly growing group and are driven by solute- and ion-gradients, making them passive and secondary active transporters, respectively. Members of the major facilitator superfamily transport an extreme variety of structurally different substrates such as antimicrobial agents, amino acids, sugars, intermediary metabolites, ions, and other small molecules. Importantly, bacteria, especially pathogenic ones, have evolved multidrug efflux pumps which belong to the major facilitator superfamily. Furthermore, members of this important superfamily share similar primary sequences in the form of highly conserved sequence motifs that confer useful functional properties during transport. The transporters of the superfamily also share similarities in secondary structures, such as possessing 12- or 14-membrane spanning α-helices and the more recently described 3-helix structure repeat element, known as the MFS fold. The three-dimensional structures of bacterial multidrug efflux pumps have been determined for only a few members of the superfamily, all drug pumps of which are surprisingly from Escherichia coli. This review briefly summarizes the structural properties of the bacterial multidrug efflux pumps of the major facilitator superfamily in a comparative manner and provides future directions for study. PMID:27065631

  11. Structural comparison of bacterial multidrug efflux pumps of the major facilitator superfamily.

    PubMed

    Ranaweera, Indrika; Shrestha, Ugina; Ranjana, K C; Kakarla, Prathusha; Willmon, T Mark; Hernandez, Alberto J; Mukherjee, Mun Mun; Barr, Sharla R; Varela, Manuel F

    The biological membrane is an efficient barrier against water-soluble substances. Solute transporters circumvent this membrane barrier by transporting water-soluble solutes across the membrane to the other sides. These transport proteins are thus required for all living organisms. Microorganisms, such as bacteria, effectively exploit solute transporters to acquire useful nutrients for growth or to expel substances that are inhibitory to their growth. Overall, there are distinct types of related solute transporters that are grouped into families or superfamilies. Of these various transporters, the major facilitator superfamily (MFS) represents a very large and constantly growing group and are driven by solute- and ion-gradients, making them passive and secondary active transporters, respectively. Members of the major facilitator superfamily transport an extreme variety of structurally different substrates such as antimicrobial agents, amino acids, sugars, intermediary metabolites, ions, and other small molecules. Importantly, bacteria, especially pathogenic ones, have evolved multidrug efflux pumps which belong to the major facilitator superfamily. Furthermore, members of this important superfamily share similar primary sequences in the form of highly conserved sequence motifs that confer useful functional properties during transport. The transporters of the superfamily also share similarities in secondary structures, such as possessing 12- or 14-membrane spanning α-helices and the more recently described 3-helix structure repeat element, known as the MFS fold. The three-dimensional structures of bacterial multidrug efflux pumps have been determined for only a few members of the superfamily, all drug pumps of which are surprisingly from Escherichia coli. This review briefly summarizes the structural properties of the bacterial multidrug efflux pumps of the major facilitator superfamily in a comparative manner and provides future directions for study.

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

    PubMed

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

    2013-01-01

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

  13. The Ferroportin Metal Efflux Proteins Function in Iron and Cobalt Homeostasis in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin is the sole iron efflux transporter in animals, and there are two closely related orthologs in Arabidopsis, FPN1 and FPN2. FPN1 localizes to the pl...

  14. Ratchet transport powered by chiral active particles

    PubMed Central

    Ai, Bao-quan

    2016-01-01

    We numerically investigate the ratchet transport of mixtures of active and passive particles in a transversal asymmetric channel. A big passive particle is immersed in a ‘sea’ of active particles. Due to the chirality of active particles, the longitudinal directed transport is induced by the transversal asymmetry. For the active particles, the chirality completely determines the direction of the ratchet transport, the counterclockwise and clockwise particles move to the opposite directions and can be separated. However, for the passive particle, the transport behavior becomes complicated, the direction is determined by competitions among the chirality, the self-propulsion speed, and the packing fraction. Interestingly, within certain parameters, the passive particle moves to the left, while active particles move to the right. In addition, there exist optimal parameters (the chirality, the height of the barrier, the self-propulsion speed and the packing fraction) at which the rectified efficiency takes its maximal value. Our findings could be used for the experimental pursuit of the ratchet transport powered by chiral active particles. PMID:26795952

  15. Extensive sphingolipid depletion does not affect lipid raft integrity or lipid raft localization and efflux function of the ABC transporter MRP1

    PubMed Central

    Klappe, Karin; Dijkhuis, Anne-Jan; Hummel, Ina; vanDam, Annie; Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex; Permentier, Hjalmar; Kok, Jan W.

    2013-01-01

    We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C16 species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1. PMID:20604746

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

    PubMed Central

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

    2016-01-01

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

  17. Matrix metalloproteinase 8 degrades apolipoprotein A-I and reduces its cholesterol efflux capacity.

    PubMed

    Salminen, Aino; Åström, Pirjo; Metso, Jari; Soliymani, Rabah; Salo, Tuula; Jauhiainen, Matti; Pussinen, Pirkko J; Sorsa, Timo

    2015-04-01

    Various cell types in atherosclerotic lesions express matrix metalloproteinase (MMP)-8. We investigated whether MMP-8 affects the structure and antiatherogenic function of apolipoprotein (apo) A-I, the main protein component of HDL particles. Furthermore, we studied serum lipid profiles and cholesterol efflux capacity in MMP-8-deficient mouse model. Incubation of apoA-I (28 kDa) with activated MMP-8 yielded 22 kDa and 25 kDa apoA-I fragments. Mass spectrometric analyses revealed that apoA-I was cleaved at its carboxyl-terminal part. Treatment of apoA-I and HDL with MMP-8 resulted in significant reduction (up to 84%, P < 0.001) in their ability to facilitate cholesterol efflux from cholesterol-loaded THP-1 macrophages. The cleavage of apoA-I by MMP-8 and the reduction in its cholesterol efflux capacity was inhibited by doxycycline. MMP-8-deficient mice had significantly lower serum triglyceride (TG) levels (P = 0.003) and larger HDL particles compared with wild-type (WT) mice. However, no differences were observed in the apoA-I levels or serum cholesterol efflux capacities between the mouse groups. Proteolytic modification of apoA-I by MMP-8 may impair the first steps of reverse cholesterol transport, leading to increased accumulation of cholesterol in the vessel walls. Eventually, inhibition of MMPs by doxycycline may reduce the risk for atherosclerotic vascular diseases.

  18. Stochastic steps in secondary active sugar transport.

    PubMed

    Adelman, Joshua L; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D F; Choe, Seungho; Abramson, Jeff; Rosenberg, John M; Wright, Ernest M; Grabe, Michael

    2016-07-05

    Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state.

  19. Stochastic steps in secondary active sugar transport

    PubMed Central

    Adelman, Joshua L.; Ghezzi, Chiara; Bisignano, Paola; Loo, Donald D. F.; Choe, Seungho; Abramson, Jeff; Rosenberg, John M.; Wright, Ernest M.; Grabe, Michael

    2016-01-01

    Secondary active transporters, such as those that adopt the leucine-transporter fold, are found in all domains of life, and they have the unique capability of harnessing the energy stored in ion gradients to accumulate small molecules essential for life as well as expel toxic and harmful compounds. How these proteins couple ion binding and transport to the concomitant flow of substrates is a fundamental structural and biophysical question that is beginning to be answered at the atomistic level with the advent of high-resolution structures of transporters in different structural states. Nonetheless, the dynamic character of the transporters, such as ion/substrate binding order and how binding triggers conformational change, is not revealed from static structures, yet it is critical to understanding their function. Here, we report a series of molecular simulations carried out on the sugar transporter vSGLT that lend insight into how substrate and ions are released from the inward-facing state of the transporter. Our simulations reveal that the order of release is stochastic. Functional experiments were designed to test this prediction on the human homolog, hSGLT1, and we also found that cytoplasmic release is not ordered, but we confirmed that substrate and ion binding from the extracellular space is ordered. Our findings unify conflicting published results concerning cytoplasmic release of ions and substrate and hint at the possibility that other transporters in the superfamily may lack coordination between ions and substrate in the inward-facing state. PMID:27325773

  20. 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.

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

    PubMed

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

    2007-04-01

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

  2. Differences in trans-stimulated chloroquine efflux kinetics are linked to PfCRT in Plasmodium falciparum

    PubMed Central

    Sanchez, Cecilia P.; Rohrbach, Petra; McLean, Jeremy E.; Fidock, David A.; Stein, Wilfred D.; Lanzer, Michael

    2010-01-01

    Summary The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum has remained controversial. Currently discussed models include a carrier or a channel for chloroquine, the former actively expelling the drug, the latter facilitating its passive diffusion, out of the parasite’s food vacuole, where chloroquine accumulates and inhibits haem detoxification. Here we have challenged both models using an established trans-stimulation efflux protocol. While carriers may demonstrate trans-stimulation, channels do not. Our data reveal that extracellular chloroquine stimulates chloroquine efflux in the presence and absence of metabolic energy in both chloroquine-sensitive and -resistant parasites, resulting in a hyperbolic increase in the apparent initial efflux rates as the concentration of external chloroquine increases. In the absence of metabolic energy, the apparent initial efflux rates were comparable in both parasites. Significant differences were only observed in the presence of metabolic energy, where consistently higher apparent initial efflux rates were found in chloroquine-resistant parasites. As trans-stimulation is characteristic of a carrier, and not a channel, we interpret our data in favour of a carrier for chloroquine being present in both chloroquine-sensitive and -resistant parasites, however, with different transport modalities. PMID:17493125

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

    PubMed

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

    2012-11-01

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

  4. Two promoter rearrangements in a drug efflux transporter gene are responsible for the appearance and spread of multidrug resistance phenotype MDR2 in Botrytis cinerea isolates in French and German vineyards.

    PubMed

    Mernke, D; Dahm, S; Walker, A-S; Lalève, A; Fillinger, S; Leroch, M; Hahn, M

    2011-10-01

    In French and German vineyards, Botrytis cinerea isolates with multiple fungicide resistance phenotypes have been observed with increasing frequencies. Multidrug resistance (MDR) results from mutations that lead to constitutive overexpression of genes encoding drug efflux transporters. In MDR2 and MDR3 strains, overexpression of the major facilitator superfamily transporter gene mfsM2 has been found to result from a rearrangement in the mfsM2 promoter (type A), caused by insertion of a retroelement (RE)-derived sequence. Here, we report the discovery of another, similar RE-induced rearrangement of the mfsM2 promoter (type B) in a subpopulation of French MDR2 isolates. MDR2 isolates harboring either type A or type B mutations in mfsM2 show the same resistance phenotypes and similar levels of mfsM2 overexpression. RE sequences similar to those in mfsM2 were found in low copy numbers in other but not all B. cinerea strains analyzed, including non-MDR2 strains. Population genetic analyses support the hypothesis that the two rearrangement mutations have only occurred once, and are responsible for the appearance and subsequent spread of all known MDR2 and MDR3 strains in French and German wine-growing regions.

  5. An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon

    PubMed Central

    Spivey, Vicky L; Whalan, Rachael H; Hirst, Elizabeth M A; Smerdon, Stephen J; Buxton, Roger S

    2013-01-01

    The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence. PMID:23915284

  6. Interaction of ABC transport proteins with toxic metals at the level of gene and transport activity in the PLHC-1 fish cell line.

    PubMed

    Della Torre, Camilla; Zaja, Roko; Loncar, Jovica; Smital, Tvrtko; Focardi, Silvano; Corsi, Ilaria

    2012-06-25

    The aim of this study was to investigate the interaction of four toxic metals with ABC transport proteins in piscine cell line PLHC-1. Cells were exposed for 24 h to 0.01-1 μM of CdCl(2), HgCl(2), As(2)O(3), or K(2)Cr(2)O(7) and the expression of a series of ABC genes (abcb1, abcc1-4) was determined using qRT-PCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC transporters. P-glycoprotein (P-gp) activity was measured in PLHC-1/dox (P-gp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. After 24 h exposure, abcc2-4 genes were dose-dependently up-regulated by all metals, while abcb1 and abcc1 were less affected. Up-regulation of abcc2 was more pronounced, with up to 8-fold increase in expression. Abcc3 and abcc4 were moderately inducible by HgCl(2) with 3.3-fold and 2.2-fold, respectively. All metals caused a significant inhibition of both P-gp (2.9- to 4-fold vs. controls) and MRP (1.3- to 1.8-fold) transport activities. Modulation of ABC genes and transport activities was further investigated in PLHC-1/wt cells exposed to 1 μM HgCl(2) for 72 h and in Hg resistant cells selected by long term cultivation of PLHC-1/wt cells in increasing concentrations of HgCl(2). Exposure to HgCl(2) for 72 h induced MRP genes expression and efflux activity. The long term cultivation of PLHC-1/wt cells in HgCl(2), did not cause prolonged up-regulation of the tested abc genes but resulted in higher MRP transport activities as determined by the increased sensitivity of these cells to MK571 (MRP specific inhibitor). Results of the present study indicated specific interaction of metals with selected ABC transport proteins. Modulation of ABC transporters takes place at both transcriptional and functional level. An active involvement of efflux pumps in Hg clearance in fish is suggested.

  7. Development of novel active transport membrande devices

    SciTech Connect

    Laciak, D.V.

    1994-11-01

    Air Products has undertaken a research program to fabricate and evaluate gas separation membranes based upon promising ``active-transport`` (AT) materials recently developed in our laboratories. Active Transport materials are ionic polymers and molten salts which undergo reversible interaction or reaction with ammonia and carbon dioxide. The materials are useful for separating these gases from mixtures with hydrogen. Moreover, AT membranes have the unique property of possessing high permeability towards ammnonia and carbon dioxide but low permeability towards hydrogen and can thus be used to permeate these components from a gas stream while retaining hydrogen at high pressure.

  8. Enhanced Corneal Absorption of Erythromycin by Modulating P-Glycoprotein and MRP Mediated Efflux with Corticosteroids

    PubMed Central

    Hariharan, Sudharshan; Gunda, Sriram; Mishra, Gyan P.; Pal, Dhananjay; Mitra, Ashim K.

    2015-01-01

    Purpose The objectives were (i) to test in vivo functional activity of MRP2 on rabbit corneal epithelium and (ii) to evaluate modulation of P-gp and MRP2 mediated efflux of erythromycin when co-administered with corticosteroids. Methods Cultured rabbit primary corneal epithelial cells (rPCECs) was employed as an in vitro model for rabbit cornea. Cellular accumulation and bi-directional transport studies were conducted across Madin-Darby Canine Kidney (MDCK) cells overexpressing MDR1 and MRP2 proteins to delineate transporter specific interaction of steroids. Ocular pharmacokinetic studies were conducted in rabbits following a single-dose infusion of erythromycin in the presence of specific inhibitors and steroids. Results Bi-directional transport of erythromycin across MDCK-MDR1 and MDCK-MRP2 cells showed significant difference between BL-AP and AP-BL permeability, suggesting that erythromycin is a substrate for P-gp and MRP2. Cellular accumulation of erythromycin in rPCEC was inhibited by steroids in a dose dependent manner. MK571, a specific MRP inhibitor, modulated the aqueous humor concentration of erythromycin in vivo. Even, steroids inhibited P-gp and MRP2 mediated efflux with maximum increase in ka, AUC0−∞, Cmax and Clast values of erythromycin, observed with 6α-methyl prednisolone. Conclusion MRP2 is functionally active along with P-gp in effluxing drug molecules out of corneal epithelium. Steroids were able to significantly inhibit both P-gp and MRP2 mediated efflux of erythromycin. PMID:18958406

  9. Health Impacts of Active Transportation in Europe

    PubMed Central

    Rojas-Rueda, David; de Nazelle, Audrey; Andersen, Zorana J.; Braun-Fahrländer, Charlotte; Bruha, Jan; Bruhova-Foltynova, Hana; Desqueyroux, Hélène; Praznoczy, Corinne; Ragettli, Martina S.; Tainio, Marko; Nieuwenhuijsen, Mark J.

    2016-01-01

    Policies that stimulate active transportation (walking and bicycling) have been related to heath benefits. This study aims to assess the potential health risks and benefits of promoting active transportation for commuting populations (age groups 16–64) in six European cities. We conducted a health impact assessment using two scenarios: increased cycling and increased walking. The primary outcome measure was all-cause mortality related to changes in physical activity level, exposure to fine particulate matter air pollution with a diameter <2.5 μm, as well as traffic fatalities in the cities of Barcelona, Basel, Copenhagen, Paris, Prague, and Warsaw. All scenarios produced health benefits in the six cities. An increase in bicycle trips to 35% of all trips (as in Copenhagen) produced the highest benefits among the different scenarios analysed in Warsaw 113 (76–163) annual deaths avoided, Prague 61 (29–104), Barcelona 37 (24–56), Paris 37 (18–64) and Basel 5 (3–9). An increase in walking trips to 50% of all trips (as in Paris) resulted in 19 (3–42) deaths avoided annually in Warsaw, 11(3–21) in Prague, 6 (4–9) in Basel, 3 (2–6) in Copenhagen and 3 (2–4) in Barcelona. The scenarios would also reduce carbon dioxide emissions in the six cities by 1,139 to 26,423 (metric tonnes per year). Policies to promote active transportation may produce health benefits, but these depend of the existing characteristics of the cities. Increased collaboration between health practitioners, transport specialists and urban planners will help to introduce the health perspective in transport policies and promote active transportation. PMID:26930213

  10. Regulators of Slc4 bicarbonate transporter activity

    PubMed Central

    Thornell, Ian M.; Bevensee, Mark O.

    2015-01-01

    The Slc4 family of transporters is comprised of anion exchangers (AE1-4), Na+-coupled bicarbonate transporters (NCBTs) including electrogenic Na/bicarbonate cotransporters (NBCe1 and NBCe2), electroneutral Na/bicarbonate cotransporters (NBCn1 and NBCn2), and the electroneutral Na-driven Cl-bicarbonate exchanger (NDCBE), as well as a borate transporter (BTR1). These transporters regulate intracellular pH (pHi) and contribute to steady-state pHi, but are also involved in other physiological processes including CO2 carriage by red blood cells and solute secretion/reabsorption across epithelia. Acid-base transporters function as either acid extruders or acid loaders, with the Slc4 proteins moving HCO−3 either into or out of cells. According to results from both molecular and functional studies, multiple Slc4 proteins and/or associated splice variants with similar expected effects on pHi are often found in the same tissue or cell. Such apparent redundancy is likely to be physiologically important. In addition to regulating pHi, a HCO−3 transporter contributes to a cell's ability to fine tune the intracellular regulation of the cotransported/exchanged ion(s) (e.g., Na+ or Cl−). In addition, functionally similar transporters or splice variants with different regulatory profiles will optimize pH physiology and solute transport under various conditions or within subcellular domains. Such optimization will depend on activated signaling pathways and transporter expression profiles. In this review, we will summarize and discuss both well-known and more recently identified regulators of the Slc4 proteins. Some of these regulators include traditional second messengers, lipids, binding proteins, autoregulatory domains, and less conventional regulators. The material presented will provide insight into the diversity and physiological significance of multiple members within the Slc4 gene family. PMID:26124722

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

    PubMed

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

    2014-10-01

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

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

    PubMed

    Iyer, Ramkumar; Erwin, Alice L

    2015-01-01

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

  13. 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

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

    PubMed

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

    2017-01-01

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

  15. Fluid transport by active elastic membranes

    NASA Astrophysics Data System (ADS)

    Evans, Arthur A.; Lauga, Eric

    2011-09-01

    A flexible membrane deforming its shape in time can self-propel in a viscous fluid. Alternatively, if the membrane is anchored, its deformation will lead to fluid transport. Past work in this area focused on situations where the deformation kinematics of the membrane were prescribed. Here we consider models where the deformation of the membrane is not prescribed, but instead the membrane is internally forced. Both the time-varying membrane shape and the resulting fluid motion result then from a balance between prescribed internal active stresses, internal passive resistance, and external viscous stresses. We introduce two specific models for such active internal forcing: one where a distribution of active bending moments is prescribed, and one where active inclusions exert normal stresses on the membrane by pumping fluid through it. In each case, we asymptotically calculate the membrane shape and the fluid transport velocities for small forcing amplitudes, and recover our results using scaling analysis.

  16. Astrocytic GABA transporter activity modulates excitatory neurotransmission

    PubMed Central

    Boddum, Kim; Jensen, Thomas P.; Magloire, Vincent; Kristiansen, Uffe; Rusakov, Dmitri A.; Pavlov, Ivan; Walker, Matthew C.

    2016-01-01

    Astrocytes are ideally placed to detect and respond to network activity. They express ionotropic and metabotropic receptors, and can release gliotransmitters. Astrocytes also express transporters that regulate the extracellular concentration of neurotransmitters. Here we report a previously unrecognized role for the astrocytic GABA transporter, GAT-3. GAT-3 activity results in a rise in astrocytic Na+ concentrations and a consequent increase in astrocytic Ca2+ through Na+/Ca2+ exchange. This leads to the release of ATP/adenosine by astrocytes, which then diffusely inhibits neuronal glutamate release via activation of presynaptic adenosine receptors. Through this mechanism, increases in astrocytic GAT-3 activity due to GABA released from interneurons contribute to 'diffuse' heterosynaptic depression. This provides a mechanism for homeostatic regulation of excitatory transmission in the hippocampus. PMID:27886179

  17. 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

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

    PubMed Central

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

    2016-01-01

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

  19. Induction of influx and efflux transporters and cytochrome P450 3A4 in primary human hepatocytes by rifampin, rifabutin, and rifapentine.

    PubMed

    Williamson, Beth; Dooley, Kelly E; Zhang, Yuan; Back, David J; Owen, Andrew

    2013-12-01

    Rifampin is a potent inducer of cytochrome P450 (CYP) enzymes and transporters. Drug-drug interactions during tuberculosis treatment are common. Induction by rifapentine and rifabutin is understudied. Rifampin and rifabutin significantly induced CYP3A4 (80-fold and 20-fold, respectively) in primary human hepatocytes. The induction was concentration dependent. Rifapentine induced CYP3A4 in hepatocytes from 3 of 6 donors. Data were also generated for ABCB1, ABCC1, ABCC2, organic anion-transporting polypeptide 1B1 (OATP1B1), and OATP1B3. This work serves as a basis for further study of the extent to which rifamycins induce key metabolism and transporter genes.

  20. Induction of Influx and Efflux Transporters and Cytochrome P450 3A4 in Primary Human Hepatocytes by Rifampin, Rifabutin, and Rifapentine

    PubMed Central

    Williamson, Beth; Dooley, Kelly E.; Zhang, Yuan; Back, David J.

    2013-01-01

    Rifampin is a potent inducer of cytochrome P450 (CYP) enzymes and transporters. Drug-drug interactions during tuberculosis treatment are common. Induction by rifapentine and rifabutin is understudied. Rifampin and rifabutin significantly induced CYP3A4 (80-fold and 20-fold, respectively) in primary human hepatocytes. The induction was concentration dependent. Rifapentine induced CYP3A4 in hepatocytes from 3 of 6 donors. Data were also generated for ABCB1, ABCC1, ABCC2, organic anion-transporting polypeptide 1B1 (OATP1B1), and OATP1B3. This work serves as a basis for further study of the extent to which rifamycins induce key metabolism and transporter genes. PMID:24060875

  1. 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

  2. Functional characterization of mouse organic anion transporting peptide 1a4 in the uptake and efflux of drugs across the blood-brain barrier.

    PubMed

    Ose, Atsushi; Kusuhara, Hiroyuki; Endo, Chihiro; Tohyama, Kimio; Miyajima, Mari; Kitamura, Satoshi; Sugiyama, Yuichi

    2010-01-01

    This study investigated the role of a multispecific organic anion transporter, Oatp1a4/Slco1a4, in drug transport across the blood-brain barrier. In vitro transport studies using human embryonic kidney 293 cells expressing mouse Oatp1a4 identified the following compounds as Oatp1a4 substrates: pitavastatin (K(m) = 8.3 microM), rosuvastatin (K(m) = 12 microM), pravastatin, taurocholate (K(m) = 40 microM), digoxin, ochratoxin A, and [d-penicillamine(2,5)]-enkephalin. Double immunohistochemical staining of Oatp1a4 with P-glycoprotein (P-gp) or glial fibrillary acidic protein demonstrated that Oatp1a4 signals colocalized with P-gp signals partly but not with glial fibrillary acidic protein, suggesting that Oatp1a4 is expressed in both the luminal and the abluminal membranes of mouse brain capillary endothelial cells. The brain-to-blood transport of pitavastatin, rosuvastatin, pravastatin, and taurocholate after microinjection into the cerebral cortex was significantly decreased in Oatp1a4(-/-) mice compared with that in wild-type mice. The blood-to-brain transport of pitavastatin, rosuvastatin, taurocholate, and ochratoxin A, determined by in situ brain perfusion, was significantly lower in Oatp1a4(-/-) mice than in wild-type mice, whereas transport of pravastatin and [D-penicillamine(2,5)]-enkephalin was unchanged. The blood-to-brain transport of digoxin was significantly lower in Oatp1a4(-/-) mice than in wild-type mice only when P-gp was inhibited by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918). Taken together, these results show that Oatp1a4 can mediate the brain-to-blood and blood-to-brain transport of its substrate drugs across the blood-brain barrier. The brain-to-plasma ratio of taurocholate, pitavastatin, and rosuvastatin was close to the capillary volume in wild-type mice, and it was not affected by Oatp1a4 dysfunction. Whether Oatp1a4 can deliver drugs from the blood

  3. Molecular Dynamics Computer Simulations of Multidrug RND Efflux Pumps.

    PubMed

    Ruggerone, Paolo; Vargiu, Attilio V; Collu, Francesca; Fischer, Nadine; Kandt, Christian

    2013-01-01

    Over-expression of multidrug efflux pumps of the Resistance Nodulation Division (RND) protein super family counts among the main causes for microbial resistance against pharmaceuticals. Understanding the molecular basis of this process is one of the major challenges of modern biomedical research, involving a broad range of experimental and computational techniques. Here we review the current state of RND transporter investigation employing molecular dynamics simulations providing conformational samples of transporter components to obtain insights into the functional mechanism underlying efflux pump-mediated antibiotics resistance in Escherichia coli and Pseudomonas aeruginosa.

  4. Molecular Dynamics Computer Simulations of Multidrug RND Efflux Pumps

    PubMed Central

    Ruggerone, Paolo; Vargiu, Attilio V.; Collu, Francesca; Fischer, Nadine; Kandt, Christian

    2013-01-01

    Over-expression of multidrug efflux pumps of the Resistance Nodulation Division (RND) protein super family counts among the main causes for microbial resistance against pharmaceuticals. Understanding the molecular basis of this process is one of the major challenges of modern biomedical research, involving a broad range of experimental and computational techniques. Here we review the current state of RND transporter investigation employing molecular dynamics simulations providing conformational samples of transporter components to obtain insights into the functional mechanism underlying efflux pump-mediated antibiotics resistance in Escherichia coli and Pseudomonas aeruginosa. PMID:24688701

  5. 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

  6. Prolonged Oral Administration of a Pan-Retinoic Acid Receptor Antagonist Inhibits Spermatogenesis in Mice With a Rapid Recovery and Changes in the Expression of Influx and Efflux Transporters.

    PubMed

    Chung, Sanny S W; Wang, Xiangyuan; Wolgemuth, Debra J

    2016-04-01

    We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

  7. Control of Angiogenesis by AIBP-mediated Cholesterol Efflux

    PubMed Central

    Fang, Longhou; Choi, Soo-Ho; Baek, Ji Sun; Liu, Chao; Almazan, Felicidad; Ulrich, Florian; Wiesner, Philipp; Taleb, Adam; Deer, Elena; Pattison, Jennifer; Torres-Vázquez, Jesús; Li, Andrew C.; Miller, Yury I.

    2013-01-01

    Cholesterol is a structural component of the cell, indispensable for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell death. To prevent cholesterol overload, ATP-binding cassette (ABC) transporters mediate cholesterol efflux from the cells to apolipoprotein A-I (ApoA-I) and to the ApoA-I-containing high-density lipoprotein (HDL)1-3. Maintaining efficient cholesterol efflux is essential for normal cellular function4-6. However, the role of cholesterol efflux in angiogenesis and the identity of its local regulators are poorly understood. Here we show that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Remarkably, Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis. PMID:23719382

  8. ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity

    PubMed Central

    Gauthier, Charlotte; Ozvegy-Laczka, Csilla; Szakacs, Gergely; Sarkadi, Balazs; Di Pietro, Attilio

    2013-01-01

    ABCG2 is a key human ATP-binding cassette (ABC) transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated glutathione (GSH) efflux and, upon depletion of intracellular GSH, induce “collateral sensitivity” leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and 3H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded. PMID:24312054

  9. Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter

    PubMed Central

    Steinkellner, Thomas; Yang, Jae-Won; Montgomery, Therese R.; Chen, Wei-Qiang; Winkler, Marie-Therese; Sucic, Sonja; Lubec, Gert; Freissmuth, Michael; Elgersma, Ype; Sitte, Harald H.; Kudlacek, Oliver

    2012-01-01

    The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP+) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKIIT305D), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP+ efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome. PMID:22778257

  10. 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.

  11. Group X Secretory Phospholipase A2 Negatively Regulates ABCA1 and ABCG1 expression and cholesterol efflux in macrophages

    PubMed Central

    Shridas, Preetha; Bailey, William M; Gizard, Florence; Oslund, Rob C; Gelb, Michael H; Bruemmer, Dennis; Webb, Nancy R

    2010-01-01

    Objectives Group X secretory phospholipase A2 (GX sPLA2) potently hydrolyzes plasma membranes to generate lysophospholipids and free fatty acids and has been implicated in inflammatory diseases including atherosclerosis. Here we identify a novel role for GX sPLA2 in modulating ABCA1 and ABCG1 expression and hence macrophage cholesterol efflux. Methods and Results Overexpression or exogenous addition of GX sPLA2 significantly reduced ABCA1 and ABCG1 expression in J774 macrophage-like cells, whereas GX sPLA2 deficiency in mouse peritoneal macrophages (MPMs) was associated with enhanced expression. Altered ABC transporter expression led to reduced cholesterol efflux in GX sPLA2 overexpressing J774 cells, and increased efflux in GX sPLA2-deficient MPMs. Gene regulation was dependent on GX sPLA2 catalytic activity, mimicked by arachidonic acid, abrogated when LXRα/β expression was suppressed, and partially reversed by the LXR agonist T0901317. Reporter assays indicated that GX sPLA2 suppresses the ability of LXR to trans-activate its promoters through a mechanism involving the C-terminal portion of LXR spanning the ligand binding domain. Conclusions GX sPLA2 modulates gene expression in macrophages by generating lipolytic products that suppress LXR activation. GX sPLA2 may play a previously unrecognized role in atherosclerotic lipid accumulation by negatively regulating genes critical for cellular cholesterol efflux. PMID:20844270

  12. Ca(2+)/calmodulin-dependent protein kinase IIα (αCaMKII) controls the activity of the dopamine transporter: implications for Angelman syndrome.

    PubMed

    Steinkellner, Thomas; Yang, Jae-Won; Montgomery, Therese R; Chen, Wei-Qiang; Winkler, Marie-Therese; Sucic, Sonja; Lubec, Gert; Freissmuth, Michael; Elgersma, Ype; Sitte, Harald H; Kudlacek, Oliver

    2012-08-24

    The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.

  13. Bursts of Active Transport in Living Cells

    NASA Astrophysics Data System (ADS)

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-01

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  14. Bursts of active transport in living cells.

    PubMed

    Wang, Bo; Kuo, James; Granick, Steve

    2013-11-15

    We show, using a large new data set, that the temporally resolved speed of active cargo transport in living cells follows a scaling law over several decades of time and length. The statistical regularities display a time-averaged shape that we interpret to reflect stress buildup, followed by rapid release. The scaling power law agrees quantitatively with those reported in inanimate systems (jammed colloids and granular media, and magnetic Barkhausen noise), suggesting a common origin in pushing through a crowded environment in a weak force regime. The implied regulation of the speed of active cellular transport due to environmental obstruction results in bursts of speed and acceleration. These findings extend the classical notion of molecular crowding.

  15. Exogenously produced CO2 doubles the CO2 efflux from three north temperate lakes

    NASA Astrophysics Data System (ADS)

    Wilkinson, Grace M.; Buelo, Cal D.; Cole, Jonathan J.; Pace, Michael L.

    2016-03-01

    It is well established that lakes are typically sources of CO2 to the atmosphere. However, it remains unclear what portion of CO2 efflux is from endogenously processed organic carbon or from exogenously produced CO2 transported into lakes. We estimated high-frequency CO2 and O2 efflux from three north temperate lakes in summer to determine the proportion of the total CO2 efflux that was exogenously produced. Two of the lakes were amended with nutrients to experimentally enhance endogenous CO2 uptake. In the unfertilized lake, 50% of CO2 efflux was from exogenous sources and hydrology had a large influence on efflux. In the fertilized lakes, endogenous CO2 efflux was negative (into the lake) yet exogenous CO2 made the lakes net sources of CO2 to the atmosphere. Shifts in hydrologic regimes and nutrient loading have the potential to change whether small lakes act primarily as reactors or vents in the watershed.

  16. Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine

    PubMed Central

    Nakano, Takanari; Inoue, Ikuo; Takenaka, Yasuhiro; Ono, Hiraku; Katayama, Shigehiro; Awata, Takuya; Murakoshi, Takayuki

    2016-01-01

    Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen 3H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given 3H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given 3H-cholesterol in mice, we found that lumen-to-BBM 3H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux

  17. Alpinetin enhances cholesterol efflux and inhibits lipid accumulation in oxidized low-density lipoprotein-loaded human macrophages.

    PubMed

    Jiang, Zhengming; Sang, Haiqiang; Fu, Xin; Liang, Ying; Li, Ling

    2015-01-01

    Alpinetin is a natural flavonoid abundantly present in the ginger family. Here, we investigated the effect of alpinetin on cholesterol efflux and lipid accumulation in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 macrophages and human peripheral blood monocyte-derived macrophages (HMDMs). After exposing THP-1 macrophages to alpinetin, cholesterol efflux was determined by liquid scintillator. The mRNA and protein levels of peroxisome proliferator-activated receptor gamma (PPAR-γ), liver X receptor alpha (LXR-α), ATP-binding cassette transporter A1 (ABCA1), and ABCG1 and scavenger receptor class B member 1 were determined by reverse-transcriptase PCR (RT-PCR) and Western blot analysis, respectively. Alpinetin promoted apolipoprotein A-I- and high-density-lipoprotein-mediated cholesterol efflux and elevated PPAR-γ and LXR-α mRNA and protein expression in a dose-dependent fashion in ox-LDL-treated THP-1 macrophages and HMDMs. Small interfering RNA-mediated silencing of PPAR-γ or LXR-α dose dependently reversed alpinetin-increased cholesterol efflux in THP-1 macrophages, indicating the involvement of PPAR-γ and LXR-α in alpinetin-promoted cholesterol efflux. Alpinetin inhibited ox-LDL-induced lipid accumulation and enhanced the expression of ABCA1 and ABCG1 mRNA and protein, which was reversed by specific knockdown of PPAR-γ or LXR-α. Taken together, our results reveal that alpinetin exhibits positive effects on cholesterol efflux and inhibits ox-LDL-induced lipid accumulation, which might be through PPAR-γ/LXR-α/ABCA1/ABCG1 pathway.

  18. Susceptibility of juvenile and adult blood–brain barrier to endothelin-1: regulation of P-glycoprotein and breast cancer resistance protein expression and transport activity

    PubMed Central

    2012-01-01

    Background P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play a critical role in keeping neurotoxic substances from entering the brain. We and others have previously reported an impact of inflammation on the regulation of adult blood–brain barrier (BBB) efflux transporters. However, studies in children have not been done. From the pediatric clinical perspective, it is important to understand how the central nervous system (CNS) and BBB drug efflux transporters differ in childhood from those of adults under normal and inflammatory conditions. Therefore, we examined and compared the regulation of P-gp and BCRP expression and transport activity in young and adult BBB and investigated the molecular mechanisms underlying inflammatory responses. Methods Rats at postnatal day (P) P21 and P84, corresponding to the juvenile and adult stages of human brain maturation, respectively, were treated with endothelin-1 (ET-1) given by the intracerebroventricular (icv) route. Twenty-four hours later, we measured P-gp and BCRP protein expression in isolated brain capillary by immunoblotting as well as by transport activity in vivo by measuring the unbound drug partitioning coefficient of the brain (Kp,uu,brain) of known efflux transporter substrates administered intravenously. Glial activation was measured by immunohistochemistry. The release of cytokines/chemokines (interleukins-1α, 1-β (IL-1β), -6 (IL-6), -10 (IL-10), monocyte chemoattractant protein (MCP-1/CCL2), fractalkine and tissue inhibitor of metalloproteinases-1 (TIMP-1)) were simultaneously measured in brain and serum samples using the Agilent Technology cytokine microarray. Results We found that juvenile and adult BBBs exhibited similar P-gp and BCRP transport activities in the normal physiological conditions. However, long-term exposure of the juvenile brain to low-dose of ET-1 did not change BBB P-gp transport activity but tended to decrease BCRP transport activity in the juvenile brain, while a

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

    PubMed

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

    2011-01-01

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

  20. Calcium- and potassium-permeable plasma membrane transporters are activated by copper in Arabidopsis root tips: linking copper transport with cytosolic hydroxyl radical production.

    PubMed

    Rodrigo-Moreno, Ana; Andrés-Colás, Nuria; Poschenrieder, Charlotte; Gunsé, Benet; Peñarrubia, Lola; Shabala, Sergey

    2013-04-01

    Transition metals such as copper can interact with ascorbate or hydrogen peroxide to form highly reactive hydroxyl radicals (OH(•) ), with numerous implications to membrane transport activity and cell metabolism. So far, such interaction was described for extracellular (apoplastic) space but not cytosol. Here, a range of advanced electrophysiological and imaging techniques were applied to Arabidopsis thaliana plants differing in their copper-transport activity: Col-0, high-affinity copper transporter COPT1-overexpressing (C1(OE) ) seedlings, and T-DNA COPT1 insertion mutant (copt1). Low Cu concentrations (10 µm) stimulated a dose-dependent Gd(3+) and verapamil sensitive net Ca(2+) influx in the root apex but not in mature zone. C1(OE) also showed a fivefold higher Cu-induced K(+) efflux at the root tip level compared with Col-0, and a reduction in basal peroxide accumulation at the root tip after copper exposure. Copper caused membrane disruptions of the root apex in C1(OE) seedlings but not in copt1 plants; this damage was prevented by pretreatment with Gd(3+) . Our results suggest that copper transport into cytosol in root apex results in hydroxyl radical generation at the cytosolic side, with a consequent regulation of plasma membrane OH(•) -sensitive Ca(2+) and K(+) transport systems.

  1. A gene (tmpA) for an efflux protein of the transporter family III from Brevibacterium linens OC2, an antibacterial substance-producing strain.

    PubMed

    Boucabeille, C; Simonet, J M; Henckes, G

    1999-06-01

    A gene (tmpA) encoding a putative transmembrane protein has been cloned from B. linens OC2, an antibacterial substance-producing strain. The deduced TmpA protein sequence shares similarities to members of the transporter family III exploiting the transmembrane proton gradient to provide export of toxic compounds such as antiseptics or antibiotics. Northern blot analysis indicated that tmpA gene is expressed. Length of RNA messenger and overlapping of ORFs upstream tmpA gene suggested that it might belong to an operon. The tmpA gene is unusual among B. linens species since it was not detected among eight B. linens collection strains and 40 B. linens industrial strains.

  2. 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.

  3. Synaptic GABA release prevents GABA transporter type-1 reversal during excessive network activity

    PubMed Central

    Savtchenko, Leonid; Megalogeni, Maria; Rusakov, Dmitri A.; Walker, Matthew C.; Pavlov, Ivan

    2015-01-01

    GABA transporters control extracellular GABA, which regulates the key aspects of neuronal and network behaviour. A prevailing view is that modest neuronal depolarization results in GABA transporter type-1 (GAT-1) reversal causing non-vesicular GABA release into the extracellular space during intense network activity. This has important implications for GABA uptake-targeting therapies. Here we combined a realistic kinetic model of GAT-1 with experimental measurements of tonic GABAA receptor currents in ex vivo hippocampal slices to examine GAT-1 operation under varying network conditions. Our simulations predict that synaptic GABA release during network activity robustly prevents GAT-1 reversal. We test this in the 0 Mg2+ model of epileptiform discharges using slices from healthy and chronically epileptic rats and find that epileptiform activity is associated with increased synaptic GABA release and is not accompanied by GAT-1 reversal. We conclude that sustained efflux of GABA through GAT-1 is unlikely to occur during physiological or pathological network activity. PMID:25798861

  4. Functions and transport of silicon in plants.

    PubMed

    Ma, J F; Yamaji, N

    2008-10-01

    Silicon exerts beneficial effects on plant growth and production by alleviating both biotic and abiotic stresses including diseases, pests, lodging, drought, and nutrient imbalance. Recently, two genes (Lsi1 and Lsi2) encoding Si transporters have been identified from rice. Lsi1 (low silicon 1) belongs to a Nod26-like major intrinsic protein subfamily in aquaporin, while Lsi2 encodes a putative anion transporter. Lsi1 is localized on the distal side of both exodermis and endodermis in rice roots, while Lsi2 is localized on the proximal side of the same cells. Lsi1 shows influx transport activity for Si, while Lsi2 shows efflux transport activity. Therefore, Lsi1 is responsible for transport of Si from the external solution to the root cells, whereas Lsi2 is an efflux transporter responsible for the transport of Si from the root cells to the apoplast. Coupling of Lsi1 with Lsi2 is required for efficient uptake of Si in rice.

  5. Active learning in transportation engineering education

    NASA Astrophysics Data System (ADS)

    Weir, Jennifer Anne

    The objectives of this research were (1) to develop experimental active-based-learning curricula for undergraduate courses in transportation engineering and (2) to assess the effectiveness of an active-learning-based traffic engineering curriculum through an educational experiment. The researcher developed a new highway design course as a pilot study to test selected active-learning techniques before employing them in the traffic engineering curriculum. Active-learning techniques, including multiple-choice questions, short problems completed by individual students or small groups, and group discussions, were used as active interludes within lectures. The researcher also collected and analyzed student performance and attitude data from control and experimental classes to evaluate the relative effectiveness of the traditional lecture (control) approach and the active-learning (experimental) approach. The results indicate that the active-learning approach adopted for the experimental class did have a positive impact on student performance as measured by exam scores. The students in the experimental class also indicated slightly more positive attitudes at the end of the course than the control class, although the difference was not significant. The author recommends that active interludes similar to those in the experimental curricula be used in other courses in civil engineering.

  6. The cation efflux transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes.

    PubMed

    Wenzlau, Janet M; Juhl, Kirstine; Yu, Liping; Moua, Ong; Sarkar, Suparna A; Gottlieb, Peter; Rewers, Marian; Eisenbarth, George S; Jensen, Jan; Davidson, Howard W; Hutton, John C

    2007-10-23

    Type 1 diabetes (T1D) results from progressive loss of pancreatic islet mass through autoimmunity targeted at a diverse, yet limited, series of molecules that are expressed in the pancreatic beta cell. Identification of these molecular targets provides insight into the pathogenic process, diagnostic assays, and potential therapeutic agents. Autoantigen candidates were identified from microarray expression profiling of human and rodent pancreas and islet cells and screened with radioimmunoprecipitation assays using new-onset T1D and prediabetic sera. A high-ranking candidate, the zinc transporter ZnT8 (Slc30A8), was targeted by autoantibodies in 60-80% of new-onset T1D compared with <2% of controls and <3% type 2 diabetic and in up to 30% of patients with other autoimmune disorders with a T1D association. ZnT8 antibodies (ZnTA) were found in 26% of T1D subjects classified as autoantibody-negative on the basis of existing markers [glutamate decarboxylase (GADA), protein tyrosine phosphatase IA2 (IA2A), antibodies to insulin (IAA), and islet cytoplasmic autoantibodies (ICA)]. Individuals followed from birth to T1D showed ZnT8A as early as 2 years of age and increasing levels and prevalence persisting to disease onset. ZnT8A generally emerged later than GADA and IAA in prediabetes, although not in a strict order. The combined measurement of ZnT8A, GADA, IA2A, and IAA raised autoimmunity detection rates to 98% at disease onset, a level that approaches that needed to detect prediabetes in a general pediatric population. The combination of bioinformatics and molecular engineering used here will potentially generate other diabetes autoimmunity markers and is also broadly applicable to other autoimmune disorders.

  7. Ascorbic acid efflux and re-uptake in endothelial cells: maintenance of intracellular ascorbate.

    PubMed

    May, James M; Qu, Zhi-chao

    2009-05-01

    Entry of vitamin C or ascorbate into most tissues requires its movement across the endothelial cell barrier of vessels. If trans-cellular ascorbate movement occurs, then it should be evident as ascorbate efflux from endothelial cells. Cultured EA.926 endothelial cells that had been loaded to about 3.5 mM intracellular ascorbate lost 70-80% of ascorbate to the medium over several hours at 37 degrees C via a non-saturable process that was insensitive to anion transport inhibitors and thiol reagents. Oxidation of this extracellular ascorbate by ascorbate oxidase or ferricyanide enhanced apparent ascorbate efflux, suggesting that efflux of the vitamin was countered in part by its re-uptake on ascorbate transporters. Although basal ascorbate efflux was not calcium-dependent, increased entry of calcium into the cells enhanced ascorbate release. These results support the hypothesis that ascorbate efflux reflects trans-endothelial cell ascorbate movement out of the blood vessel.

  8. Modulation of drug efflux by aloe materials: An In Vitro investigation across rat intestinal tissue

    PubMed Central

    Carien, Beneke; Alvaro, Viljoen; Josias, Hamman

    2013-01-01

    Background: Clinically, significant herb-drug interactions have been previously documented and can be pharmacodynamic and/or pharmacokinetic in nature. Pharmacokinetic interactions have been attributed to induction or inhibition of either metabolic enzymes or efflux transporters. Objective: The effect of gel and whole leaf materials from 3 different aloe species namely Aloe ferox, Aloe marlothii, and Aloe vera as well as polysaccharides precipitated from the A. vera materials on the bi-directional transport of cimetidine across rat intestinal tissue was investigated. Materials and Methods: Cimetidine transport studies were performed across excised rat intestinal tissue mounted in Sweetana-Grass diffusion chambers in both the apical-to-basolateral and basolateral-to-apical directions. Results: While A. vera gel and whole leaf materials did not inhibit the efflux of cimetidine, the polysaccharides precipitated from them did show a reduction of cimetidine efflux. On the other hand, both A. ferox and A. marlothii gel and whole leaf materials exhibited an inhibition effect on cimetidine efflux. Conclusions: This study identified a modulation effect of efflux transporters by certain aloe materials. This may cause herb-drug pharmacokinetic interactions when drugs that are substrates for these efflux transporters are taken simultaneously with aloe materials. On the other hand, these aloe materials may be used for drug absorption enhancement for drugs with low bioavailability due to extensive efflux. PMID:24143044

  9. Single-molecule detection with active transport

    NASA Astrophysics Data System (ADS)

    Ball, David Allan

    A glass capillary is used near the focal region of a custom-built confocal microscope to investigate the use of active transport for single-molecule detection in solution, with both one and two-photon laser excitation. The capillary tip has a diameter of several microns and is carefully aligned nearby to the sub-micron laser beam waist, collinear to the optical axis, so that a negative pressure-difference causes molecules to be drawn into the capillary, along the laser beam axis. The flow of solution, which is characterized by fluorescence correlation spectroscopy (FCS), can increase the single-molecule detection rate for slowly diffusing proteins by over a factor of 100, while the mean rate of photons during each burst is similar to that for random diffusional transport. Also, the flow is along the longest axis of the ellipsoidally-shaped confocal volume, which results in more collected photons per molecule than that for transverse flow at the same speed. When transport is dominated by flow, FCS can no longer distinguish molecules with differing translational diffusion, and hence a fluorescence fluctuation spectroscopy method based on differences in fluorescence brightness is investigated as a means for assaying different solution components, for applications in pharmaceutical drug discovery. Multi-channel fluctuation spectroscopy techniques can also be used for assays with the flow system and hence this dissertation also reports the characterization of a prototype 4-channel single-photon detector with a two-wavelength polarization-resolved optical set-up.

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

    PubMed

    Adebusuyi, Abigail; Foght, Julia

    2013-01-01

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

  11. Ascorbic Acid Efflux from Human Brain Microvascular Pericytes: Role of Re-uptake

    PubMed Central

    May, James M.; Qu, Zhi-chao

    2015-01-01

    Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8–1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 h. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25–50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism. PMID:26340060

  12. Functional Cloning and Characterization of the Multidrug Efflux Pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli▿ †

    PubMed Central

    Long, Feng; Rouquette-Loughlin, Corinne; Shafer, William M.; Yu, Edward W.

    2008-01-01

    Active efflux of antimicrobial agents is one of the most important adapted strategies that bacteria use to defend against antimicrobial factors that are present in their environment. The NorM protein of Neisseria gonorrhoeae and the YdhE protein of Escherichia coli have been proposed to be multidrug efflux pumps that belong to the multidrug and toxic compound extrusion (MATE) family. In order to determine their antimicrobial export capabilities, we cloned, expressed, and purified these two efflux proteins and characterized their functions both in vivo and in vitro. E. coli strains expressing norM or ydhE showed elevated (twofold or greater) resistance to several antimicrobial agents, including fluoroquinolones, ethidium bromide, rhodamine 6G, acriflavine, crystal violet, berberine, doxorubicin, novobiocin, enoxacin, and tetraphenylphosphonium chloride. When they were expressed in E. coli, both transporters reduced the levels of ethidium bromide and norfloxacin accumulation through a mechanism requiring the proton motive force, and direct measurements of efflux confirmed that NorM behaves as an Na+-dependent transporter. The capacities of NorM and YdhE to recognize structurally divergent compounds were confirmed by steady-state fluorescence polarization assays, and the results revealed that these transporters bind to antimicrobials with dissociation constants in the micromolar region. PMID:18591276

  13. Transport in active systems crowded by obstacles

    NASA Astrophysics Data System (ADS)

    Huang, Mu-Jie; Schofield, Jeremy; Kapral, Raymond

    2017-02-01

    The reactive and diffusive dynamics of a single chemically powered Janus motor in a crowded medium of moving but passive obstacles is investigated using molecular simulation. It is found that the reaction rate of the catalytic motor reaction decreases in a crowded medium as the volume fraction of obstacles increases as a result of a reduction in the Smoluchowski diffusion-controlled reaction rate coefficient that contributes to the overall reaction rate. A continuum model is constructed and analyzed to interpret the dependence of the steady-state reaction rate observed in simulations on the volume fraction of obstacles in the system. The steady-state concentration fields of reactant and product are shown to be sensitive to the local structure of obstacles around the Janus motor. It is demonstrated that the active motor exhibits enhanced diffusive motion at long times with a diffusion constant that decreases as the volume fraction of crowding species increases. In addition, the dynamical properties of a passive tracer particle in a system containing many active Janus motors is studied to investigate how an active environment influences the transport of non-active species. The diffusivity of a passive tracer particle in an active medium is found to be enhanced in systems with forward-moving Janus motors due to the cooperative dynamics of these motors.

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

    PubMed Central

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

    2005-01-01

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

  15. Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney.

    PubMed

    Ito, Sumito; Kusuhara, Hiroyuki; Yokochi, Miyu; Toyoshima, Junko; Inoue, Katsuhisa; Yuasa, Hiroaki; Sugiyama, Yuichi

    2012-02-01

    Cimetidine, an H₂ receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 μM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 μM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (μM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 μM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 μM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

  16. Modulation of Bacterial Multidrug Resistance Efflux Pumps of the Major Facilitator Superfamily

    PubMed Central

    Kumar, Sanath; Mukherjee, Mun Mun; Varela, Manuel F.

    2013-01-01

    Bacterial infections pose a serious public health concern, especially when an infectious disease has a multidrug resistant causative agent. Such multidrug resistant bacteria can compromise the clinical utility of major chemotherapeutic antimicrobial agents. Drug and multidrug resistant bacteria harbor several distinct molecular mechanisms for resistance. Bacterial antimicrobial agent efflux pumps represent a major mechanism of clinical resistance. The major facilitator superfamily (MFS) is one of the largest groups of solute transporters to date and includes a significant number of bacterial drug and multidrug efflux pumps. We review recent work on the modulation of multidrug efflux pumps, paying special attention to those transporters belonging primarily to the MFS. PMID:25750934

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

    PubMed

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

    2010-09-01

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

  18. 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

  19. EEPD1 Is a Novel LXR Target Gene in Macrophages Which Regulates ABCA1 Abundance and Cholesterol Efflux

    PubMed Central

    Nelson, Jessica Kristine; Koenis, Duco Steven; Scheij, Saskia; Cook, Emma Clare Laura; Moeton, Martina; Santos, Ana; Lobaccaro, Jean-Marc Adolphe; Baron, Silvere

    2017-01-01

    Objective— The sterol-responsive nuclear receptors, liver X receptors α (LXRα, NR1H3) and β (LXRβ, NR1H2), are key determinants of cellular cholesterol homeostasis. LXRs are activated under conditions of high cellular sterol load and induce expression of the cholesterol efflux transporters ABCA1 and ABCG1 to promote efflux of excess cellular cholesterol. However, the full set of genes that contribute to LXR-stimulated cholesterol efflux is unknown, and their identification is the objective of this study. Approach and Results— We systematically compared the global transcriptional response of macrophages to distinct classes of LXR ligands. This allowed us to identify both common and ligand-specific transcriptional responses in macrophages. Among these, we identified endonuclease–exonuclease–phosphatase family domain containing 1 (EEPD1/KIAA1706) as a direct transcriptional target of LXRs in human and murine macrophages. EEPD1 specifically localizes to the plasma membrane owing to the presence of a myristoylation site in its N terminus. Accordingly, the first 10 amino acids of EEPD1 are sufficient to confer plasma membrane localization in the context of a chimeric protein with GFP. Functionally, we report that silencing expression of EEPD1 blunts maximal LXR-stimulated Apo AI-dependent efflux and demonstrate that this is the result of reduced abundance of ABCA1 protein in human and murine macrophages. Conclusions— In this study, we identify EEPD1 as a novel LXR-regulated gene in macrophages and propose that it promotes cellular cholesterol efflux by controlling cellular levels and activity of ABCA1. PMID:28082258

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

    PubMed

    Hricová, Kristýna; Kolář, Milan

    2014-12-01

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

  1. A general method for determining secondary active transporter substrate stoichiometry.

    PubMed

    Fitzgerald, Gabriel A; Mulligan, Christopher; Mindell, Joseph A

    2017-01-25

    The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na(+)-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.

  2. 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.

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

    PubMed Central

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

    2012-01-01

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

  4. A domain-based approach for analyzing the function of aluminum-activated malate transporters from wheat (Triticum aestivum) and Arabidopsis thaliana in Xenopus oocytes.

    PubMed

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Furuichi, Takuya; Yamamoto, Yoko

    2014-12-01

    Wheat and Arabidopsis plants respond to aluminum (Al) ions by releasing malate from their root apices via Al-activated malate transporter. Malate anions bind with the toxic Al ions and contribute to the Al tolerance of these species. The genes encoding the transporters in wheat and Arabidopsis, TaALMT1 and AtALMT1, respectively, were expressed in Xenopus laevis oocytes and characterized electrophysiologically using the two-electrode voltage clamp system. The Al-activated currents generated by malate efflux were detected for TaALMT1 but not for AtALMT1. Chimeric proteins were generated by swapping the N- and C-terminal halves of TaALMT1 and AtALMT1 (Ta::At and At::Ta). When these chimeras were characterized in oocytes, Al-activated malate efflux was detected for the Ta::At chimera but not for At::Ta, suggesting that the N-terminal half of TaALMT1 is necessary for function in oocytes. An additional chimera, Ta(48)::At, generated by swapping 17 residues from the N-terminus of AtALMT1 with the equivalent 48 residues from TaALMT1, was sufficient to support transport activity. This 48 residue region includes a helical region with a putative transmembrane domain which is absent in AtALMT1. The deletion of this domain from Ta(48)::At led to the complete loss of transport activity. Furthermore, truncations and a deletion at the C-terminal end of TaALMT1 indicated that a putative helical structure in this region was also required for transport function. This study provides insights into the structure-function relationships of Al-activated ALMT proteins by identifying specific domains on the N- and C-termini of TaALMT1 that are critical for basal transport function and Al responsiveness in oocytes.

  5. Berberine acutely activates the glucose transport activity of GLUT1.

    PubMed

    Cok, Alexandra; Plaisier, Christina; Salie, Matthew J; Oram, Daniel S; Chenge, Jude; Louters, Larry L

    2011-07-01

    Berberine, which has a long history of use in Chinese medicine, has recently been shown to have efficacy in the treatment of diabetes. While the hypoglycemic effect of berberine has been clearly documented in animal and cell line models, such as 3T3-L1 adipocytes and L6 myotube cells, the mechanism of action appears complex with data implicating activation of the insulin signaling pathway as well as activation of the exercise or AMP kinase-mediated pathway. There have been no reports of the acute affects of berberine on the transport activity of the insulin-insensitive glucose transporter, GLUT1. Therefore, we examined the acute effects of berberine on glucose uptake in L929 fibroblast cells, a cell line that express only GLUT1. Berberine- activated glucose uptake reaching maximum stimulation of five-fold at >40 μM. Significant activation (P < 0.05) was measured within 5 min reaching a maximum by 30 min. The berberine effect was not additive to the maximal stimulation by other known stimulants, azide, methylene blue or glucose deprivation, suggesting shared steps between berberine and these stimulants. Berberine significantly reduced the K(m) of glucose uptake from 6.7 ± 1.9 mM to 0.55 ± 0.08 mM, but had no effect on the V(max) of uptake. Compound C, an inhibitor of AMP kinase, did not affect berberine-stimulated glucose uptake, but inhibitors of downstream kinases partially blocked berberine stimulation. SB203580 (inhibitor of p38 MAP kinase) did not affect submaximal berberine activation, but did lower maximal berberine stimulation by 26%, while PD98059 (inhibitor of ERK kinase) completely blocked submaximal berberine activation and decreased the maximal stimulation by 55%. It appears from this study that a portion of the hypoglycemic effects of berberine can be attributed to its acute activation of the transport activity of GLUT1.

  6. Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

    PubMed

    Noor, Sina Ibne; Pouyssegur, Jacques; Deitmer, Joachim W; Becker, Holger M

    2017-01-01

    Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail.

  7. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

    PubMed

    Bao, Huan; Dalal, Kush; Wang, Victor; Rouiller, Isabelle; Duong, Franck

    2013-08-01

    The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

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

    PubMed

    Demidchik, Vadim

    2014-05-15

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

  9. Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants.

    PubMed

    Wei, Dandan; Zhang, Wen; Wang, Cuicui; Meng, Qingwei; Li, Gang; Chen, Tony H H; Yang, Xinghong

    2017-04-01

    Tomato (Solanum lycopersicum cv. 'Moneymaker') was transformed with the choline oxidase gene codA from Arthrobacter globiformis, which was modified to allow for targeting to both chloroplasts and the cytosol. Glycine betaine (GB) was accumulated in transformed plants, while no detectable GB was found in wild-type (WT) plants. Compared to WT plants, transgenic lines showed significantly higher photosynthetic rates (Pn) and antioxidant enzyme activities and lower reactive oxygen species (ROS) accumulation in the leaves when exposed to salt stress. Furthermore, compared with WT plants, K(+) efflux decreased and Na(+) efflux increased in roots of transgenic plants under salt stress; resulted in lower Na(+)/K(+) ratios in transgenic lines. The exogenous application of GB also significantly reduced NaCl-induced K(+) efflux and increased Na(+) efflux in WT plants. A qRT-PCR assay indicated that GB enhanced NaCl-induced expression of genes encoding the K(+) transporter, Na(+)/H(+) antiporter, and H(+)-ATPase. These results suggest that the enhanced salt tolerance conferred by codA in transgenic tomato plants might be due to the regulation of ion channel and transporters by GB, which would allow high potassium levels and low sodium levels to be maintained in transgenic plants under salt stress condition.

  10. 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.

  11. A Simple Laboratory Exercise Illustrating Active Transport in Yeast Cells.

    ERIC Educational Resources Information Center

    Stambuk, Boris U.

    2000-01-01

    Describes a simple laboratory activity illustrating the chemiosmotic principles of active transport in yeast cells. Demonstrates the energy coupling mechanism of active a-glucoside uptake by Saccaromyces cerevisiae cells with a colorimetric transport assay using very simple equipment. (Contains 22 references.) (Author/YDS)

  12. Biophysical and Pharmacological Characterization of Energy-Dependent Efflux of Sb in Laboratory-Selected Resistant Strains of Leishmania (Viannia) Subgenus

    PubMed Central

    dos Reis, Priscila G.; do Monte-Neto, Rubens L.; Melo, Maria N.; Frézard, Frédéric

    2017-01-01

    The growing resistance of leishmaniasis to first-line drugs like antimonials in some regions limits the control of this parasitic disease. The precise mechanisms involved in Leishmania antimony resistance are still subject to debate. The reduction of intracellular SbIII accumulation is a common change observed in both laboratory-selected and field isolated resistant Leishmania strains, but the exact transport pathways involved in antimony resistance have not yet been elucidated. In order to functionally characterize the antimony transport routes responsible for resistance, we performed systematic transport studies of SbIII in wild-type and resistant strains of L. (Viannia) guyanensis and L. (V.) braziliensis. Those include influx and efflux assays and the influence of ABC transporters and metabolism inhibitors: prochlorperazine, probenecid, verapamil, BSO, and sodium azide. The mRNA levels of genes associated with antimony resistance (MRPA, GSH1, ODC, AQP1, ABCI4, and ARM58) were also investigated in addition to intracellular thiol levels. A strong reduction of Sb influx was observed in L. guyanensis resistant mutant (LgSbR), but not in L. braziliensis (LbSbR). Both mutants showed increased energy-dependent efflux of SbIII, when compared to their respective parental strains. In LgSbR, BSO and prochlorperazine inhibited antimony efflux and resistance was associated with increased MRPA and GSH1 mRNA levels, while in LbSbR antimony efflux was inhibited by probenicid and prochlorperazine in absence of resistance-associated gene modulation. Intracellular thiol levels were increased in both Sb-resistant mutants. An energy-dependent SbIII efflux pathway sensitive to prochlorperazine was clearly evidenced in both Sb-resistant mutants. In conclusion, the present study allowed the biophysical and pharmacological characterization of energy-dependent Sb efflux pathway apparently independent of MRPA, ABCI4, and ARM58 upregulation, in Leishmania (Vianna) mutant selected in vitro

  13. Transportation as a "Related Service": Issues that Involve Transition Activities.

    ERIC Educational Resources Information Center

    Missouri Univ., Columbia. Missouri LINC.

    The paper discusses transportation as a related service for students with disabilities expecially as related to school-to-work transition activities. First, the legislative and legal basis for providing transportation services is discussed in the form of answers to frequently asked questions: why provide transportation? what is the basis for…

  14. Chill activation of compatible solute transporters in Corynebacterium glutamicum at the level of transport activity.

    PubMed

    Ozcan, Nuran; Krämer, Reinhard; Morbach, Susanne

    2005-07-01

    The gram-positive soil bacterium Corynebacterium glutamicum harbors four osmoregulated secondary uptake systems for compatible solutes, BetP, EctP, LcoP, and ProP. When reconstituted in proteoliposomes, BetP was shown to sense hyperosmotic conditions via the increase in luminal K(+) and to respond by instant activation. To study further putative ways of stimulus perception and signal transduction, we have investigated the responses of EctP, LcoP, and BetP, all belonging to the betaine-carnitine-choline transporter family, to chill stress at the level of activity. When fully activated by hyperosmotic stress, they showed the expected increase of activity at increasing temperature. In the absence of osmotic stress, EctP was not activated by chill and LcoP to only a very low extent, whereas BetP was significantly stimulated at low temperature. BetP was maximally activated at 10 degrees C, reaching the same transport rate as that observed under hyperosmotic conditions at this temperature. A role of cytoplasmic K(+) in chill-dependent activation of BetP was ruled out, since (i) the cytoplasmic K(+) concentration did not change significantly at lower temperatures and (ii) a mutant BetP lacking the C-terminal 25 amino acids, which was previously shown to have lost the ability to be activated by luminal K(+), was fully competent in chill sensing. When heterologously expressed in Escherichia coli, BetP did not respond to chill stress. This may indicate that the membrane in which BetP is inserted plays an important role in chill activation and thus in signal transduction by BetP, different from the previously established K(+)-mediated process.

  15. Air pollution exposure: An activity pattern approach for active transportation

    NASA Astrophysics Data System (ADS)

    Adams, Matthew D.; Yiannakoulias, Nikolaos; Kanaroglou, Pavlos S.

    2016-09-01

    In this paper, we demonstrate the calculation of personal air pollution exposure during trips made by active transportation using activity patterns without personal monitors. We calculate exposure as the inhaled dose of particulate matter 2.5 μg or smaller. Two modes of active transportation are compared, and they include cycling and walking. Ambient conditions are calculated by combining mobile and stationary monitoring data in an artificial neural network space-time model. The model uses a land use regression framework and has a prediction accuracy of R2 = 0.78. Exposure is calculated at 10 m or shorter intervals during the trips using inhalation rates associated with both modes. The trips are children's routes between home and school. The average dose during morning cycling trips was 2.17 μg, during morning walking trips was 3.19 μg, during afternoon cycling trips was 2.19 μg and during afternoon walking trips was 3.23 μg. The cycling trip dose was significantly lower than the walking trip dose. The air pollution exposure during walking or cycling trips could not be strongly predicted by either the school or household ambient conditions, either individually or in combination. Multiple linear regression models regressing both the household and school ambient conditions against the dose were only able to account for, at most, six percent of the variance in the exposure. This paper demonstrates that incorporating activity patterns when calculating exposure can improve the estimate of exposure compared to its calculation from ambient conditions.

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

    PubMed

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

    2005-03-25

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

  17. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

    PubMed Central

    Mealman, Tiffany D.; McEvoy, Megan M.; Blackburn, Ninian J.

    2014-01-01

    Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. Here, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm. PMID:25313055

  18. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

    DOE PAGES

    Chacon, Kelly N.; Mealman, Tiffany D.; McEvoy, Megan M.; ...

    2014-10-13

    Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. In this paper, we use selenomethionine (SeM) active site labelsmore » in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Finally, our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.« less

  19. Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins

    SciTech Connect

    Chacon, Kelly N.; Mealman, Tiffany D.; McEvoy, Megan M.; Blackburn, Ninian J.

    2014-10-13

    Copper is an essential nutrient for all aerobic organisms but is toxic in excess. At the host–pathogen interface, macrophages respond to bacterial infection by copper-dependent killing mechanisms, whereas the invading bacteria are thought to counter with an up-regulation of copper transporters and efflux pumps. The tripartite efflux pump CusCBA and its metallochaperone CusF are vital to the detoxification of copper and silver ions in the periplasm of Escherichia coli. However, the mechanism of efflux by this complex, which requires the activation of the inner membrane pump CusA, is poorly understood. In this paper, we use selenomethionine (SeM) active site labels in a series of biological X-ray absorption studies at the selenium, copper, and silver edges to establish a “switch” role for the membrane fusion protein CusB. We determine that metal-bound CusB is required for activation of cuprous ion transfer from CusF directly to a site in the CusA antiporter, showing for the first time (to our knowledge) the in vitro activation of the Cus efflux pump. This metal-binding site of CusA is unlike that observed in the crystal structures of the CusA protein and is composed of one oxygen and two sulfur ligands. Finally, our results suggest that metal transfer occurs between CusF and apo-CusB, and that, when metal-loaded, CusB plays a role in the regulation of metal ion transfer from CusF to CusA in the periplasm.

  20. Efflux Pump Inhibitor Phenylalanine-Arginine Β-Naphthylamide Effect on the Minimum Inhibitory Concentration of Imipenem in Acinetobacter baumannii Strains Isolated From Hospitalized Patients in Shahid Motahari Burn Hospital, Tehran, Iran

    PubMed Central

    Gholami, Mehrdad; Hashemi, Ali; Hakemi-Vala, Mojdeh; Goudarzi, Hossein; Hallajzadeh, Masoumeh

    2015-01-01

    Background: Acinetobacter baumannii has emerged as a highly troublesome pathogen and a leading cause of mortality and morbidity among hospitalized burn patients. Objectives: The aims of this study were to determine the frequency of the AdeABC genes and the role of the efflux pump (s) in the imipenem resistance of A. baumannii strains isolated from burn patients. Materials and Methods: This study was conducted on 60 A. baumannii isolates collected from 240 wound samples of burn patients admitted to the Burn Unit of Shahid Motahari Burn hospital, Tehran, Iran. Antibiotic susceptibility tests were performed using the Kirby-Bauer disc diffusion and broth microdilution according to the clinical and laboratory standards institute (CLSI) guidelines. The activity of the efflux pump was evaluated using the efflux pump inhibitor, the phenylalanine-arginine Β-naphthylamide (PAΒN). The AdeABC genes were detected by polymerase chain reaction (PCR) and sequencing. Results: In this study, 100% of the isolates were resistant to cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, cefepime, piperacillin, meropenem, co-trimoxazole, and piperacillin/tazobactam; 56 (94%) to gentamicin; 50 (81%) to amikacin; 58 (97%) to imipenem; and 45 (76%) to tetracycline. Additionally,all the isolates were susceptible to colistin. The susceptibility of the strains to imipenem was highly increased in the presence of the efflux pump inhibitor such that for 58 (96.6%) of the isolates, the PAΒN reduced the minimum inhibitory concentrations (MIC) by 4- to 64-fold. The adeA and adeB genes were detected in 60 (100%) of the isolates, and the adeC gene was present in 51 (85%). Conclusions: The efflux pump may play a role in antibiotic resistance in A. baumannii isolates. The ability of A. baumannii isolates to acquire drug resistance by the efflux pump mechanism is a concern. Thus, new strategies are required in order to eliminate the efflux transport activity from resistant A. baumannii isolates causing

  1. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model.

    PubMed

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay (®) (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  2. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model

    PubMed Central

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

    Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette) transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay ® (Solvo Biotechnology, Hungary) was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2) trans-membrane estradiol-17-β-glucuronide (E17βG) transport activity, when activated by adenosine-tri-phosphate (ATP) during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology opens new and

  3. Transporting Radioactive Waste: An Engineering Activity. Grades 5-12.

    ERIC Educational Resources Information Center

    HAZWRAP, The Hazardous Waste Remedial Actions Program.

    This brochure contains an engineering activity for upper elementary, middle school, and high school students that examines the transportation of radioactive waste. The activity is designed to inform students about the existence of radioactive waste and its transportation to disposal sites. Students experiment with methods to contain the waste and…

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

    PubMed

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

    2016-11-01

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

  5. MCT Expression and Lactate Influx/Efflux in Tanycytes Involved in Glia-Neuron Metabolic Interaction

    PubMed Central

    Cortés-Campos, Christian; Elizondo, Roberto; Llanos, Paula; Uranga, Romina María; Nualart, Francisco; García, María Angeles

    2011-01-01

    Metabolic interaction via lactate between glial cells and neurons has been proposed as one of the mechanisms involved in hypothalamic glucosensing. We have postulated that hypothalamic glial cells, also known as tanycytes, produce lactate by glycolytic metabolism of glucose. Transfer of lactate to neighboring neurons stimulates ATP synthesis and thus contributes to their activation. Because destruction of third ventricle (III-V) tanycytes is sufficient to alter blood glucose levels and food intake in rats, it is hypothesized that tanycytes are involved in the hypothalamic glucose sensing mechanism. Here, we demonstrate the presence and function of monocarboxylate transporters (MCTs) in tanycytes. Specifically, MCT1 and MCT4 expression as well as their distribution were analyzed in Sprague Dawley rat brain, and we demonstrate that both transporters are expressed in tanycytes. Using primary tanycyte cultures, kinetic analyses and sensitivity to inhibitors were undertaken to confirm that MCT1 and MCT4 were functional for lactate influx. Additionally, physiological concentrations of glucose induced lactate efflux in cultured tanycytes, which was inhibited by classical MCT inhibitors. Because the expression of both MCT1 and MCT4 has been linked to lactate efflux, we propose that tanycytes participate in glucose sensing based on a metabolic interaction with neurons of the arcuate nucleus, which are stimulated by lactate released from MCT1 and MCT4-expressing tanycytes. PMID:21297988

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

    PubMed

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

    2014-06-06

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

  7. 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.

  8. Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells.

    PubMed

    Jumnongprakhon, Pichaya; Sivasinprasasn, Sivanan; Govitrapong, Piyarat; Tocharus, Chainarong; Tocharus, Jiraporn

    2017-02-20

    Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood-brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH.

  9. Heat transport in active harmonic chains

    SciTech Connect

    Zheng, Mei C.; Ellis, Fred M.; Kottos, Tsampikos; Fleischmann, Ragnar; Geisel, Theo; Prosen, Tomaz

    2011-08-15

    We show that a harmonic lattice model with amplifying and attenuating elements, when coupled to two thermal baths, exhibits unique heat transport properties. Some of these novel features include anomalous nonequilibrium steady-state heat currents, negative differential thermal conductance, as well as nonreciprocal heat transport. We find that when these elements are arranged in a PT-symmetric manner, the domain of existence of the nonequilibrium steady state is maximized. We propose an electronic experimental setup based on resistive-inductive-capacitive (RLC) transmission lines, where our predictions can be tested.

  10. Hydrogen peroxide efflux from muscle mitochondria underestimates matrix superoxide production: a correction using glutathione depletion

    PubMed Central

    TREBERG, Jason R.; QUINLAN, Casey L.; BRAND, Martin D.

    2010-01-01

    Summary The production of H2O2 by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H2O2. However, matrix enzymes such as the glutathione peroxidases can consume H2O2 and compete with efflux of H2O2, causing an underestimate of superoxide production. To assess this underestimate we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% by pretreatment with 1-chloro-2,4-dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H2O2, consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H2O2 formation from matrix-directed superoxide up to two-fold from several sites of production, defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2 to 2.5 nmol H2O2 • min−1 • mg protein−1. Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H2O2 efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H2O2 efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: [CDNB pretreated rate = control rate + (1.43*(control rate))/(0.55+control rate)]. These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria. PMID:20491900

  11. Transport and biological activities of bile acids.

    PubMed

    Zwicker, Brittnee L; Agellon, Luis B

    2013-07-01

    Bile acids have emerged as important biological molecules that support the solubilization of various lipids and lipid-soluble compounds in the gut, and the regulation of gene expression and cellular function. Bile acids are synthesized from cholesterol in the liver and eventually released into the small intestine. The majority of bile acids are recovered in the distal end of the small intestine and then returned to the liver for reuse. The components of the mechanism responsible for the recycling of bile acids within the enterohepatic circulation have been identified whereas the mechanism for intracellular transport is less understood. Recently, the ileal lipid binding protein (ILBP; human gene symbol FABP6) was shown to be needed for the efficient transport of bile acids from the apical side to the basolateral side of enterocytes in the distal intestine. This review presents an overview of the transport of bile acids between the liver and the gut as well as within hepatocytes and enterocytes. A variety of pathologies is associated with the malfunction of the bile acid transport system.

  12. Broad Specificity Efflux pumps and Their Role in Multidrug Resistance of Gram Negative Bacteria

    PubMed Central

    Nikaido, Hiroshi; Pagès, Jean-Marie

    2013-01-01

    Antibiotic resistance mechanisms reported in Gram-negative bacteria are producing a worldwide health problem. The continuous dissemination of «multi-drug resistant» (MDR) bacteria drastically reduces the efficacy of our antibiotic “arsenal” and consequently increases the frequency of therapeutic failure. In MDR bacteria, the over-expression of efflux pumps that expel structurally-unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data indicate an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological level, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may open the way to rationally develop an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms. PMID:21707670

  13. HDL-apolipoprotein A-I exchange is independently associated with cholesterol efflux capacity

    PubMed Central

    Borja, Mark S.; Ng, Kit F.; Irwin, Angela; Hong, Jaekyoung; Wu, Xing; Isquith, Daniel; Zhao, Xue-Qiao; Prazen, Bryan; Gildengorin, Virginia; Oda, Michael N.; Vaisar, Tomáš

    2015-01-01

    HDL is the primary mediator of cholesterol mobilization from the periphery to the liver via reverse cholesterol transport (RCT). A critical first step in this process is the uptake of cholesterol from lipid-loaded macrophages by HDL, a function of HDL inversely associated with prevalent and incident cardiovascular disease. We hypothesized that the dynamic ability of HDL to undergo remodeling and exchange of apoA-I is an important and potentially rate-limiting aspect of RCT. In this study, we investigated the relationship between HDL-apoA-I exchange (HAE) and serum HDL cholesterol (HDL-C) efflux capacity. We compared HAE to the total and ABCA1-specific cholesterol efflux capacity of 77 subjects. We found that HAE was highly correlated with both total (r = 0.69, P < 0.0001) and ABCA1-specific (r = 0.47, P < 0.0001) efflux, and this relationship remained significant after adjustment for HDL-C or apoA-I. Multivariate models of sterol efflux capacity indicated that HAE accounted for approximately 25% of the model variance for both total and ABCA1-specific efflux. We conclude that the ability of HDL to exchange apoA-I and remodel, as measured by HAE, is a significant contributor to serum HDL efflux capacity, independent of HDL-C and apoA-I, indicating that HDL dynamics are an important factor in cholesterol efflux capacity and likely RCT. PMID:26254308

  14. Broad-specificity efflux pumps and their role in multidrug resistance of Gram-negative bacteria.

    PubMed

    Nikaido, Hiroshi; Pagès, Jean-Marie

    2012-03-01

    Antibiotic resistance mechanisms reported in Gram-negative bacteria are causing a worldwide health problem. The continuous dissemination of 'multidrug-resistant' (MDR) bacteria drastically reduces the efficacy of our antibiotic 'arsenal' and consequently increases the frequency of therapeutic failure. In MDR bacteria, the overexpression of efflux pumps that expel structurally unrelated drugs contributes to the reduced susceptibility by decreasing the intracellular concentration of antibiotics. During the last decade, several clinical data have indicated an increasing involvement of efflux pumps in the emergence and dissemination of resistant Gram-negative bacteria. It is necessary to clearly define the molecular, functional and genetic bases of the efflux pump in order to understand the translocation of antibiotic molecules through the efflux transporter. The recent investigation on the efflux pump AcrB at its structural and physiological levels, including the identification of drug affinity sites and kinetic parameters for various antibiotics, may pave the way towards the rational development of an improved new generation of antibacterial agents as well as efflux inhibitors in order to efficiently combat efflux-based resistance mechanisms.

  15. Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (Helianthus tuberosus L.) tubers.

    PubMed

    Szarka, András; Horemans, Nele; Passarella, Salvatore; Tarcsay, Akos; Orsi, Ferenc; Salgó, András; Bánhegyi, Gábor

    2008-10-01

    Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism.

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed Central

    1989-01-01

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

  19. Regulation of ABC Transporters at the Blood-Brain Barrier

    PubMed Central

    Miller, David S.

    2015-01-01

    ATP Binding Cassette (ABC) transporters at the blood-brain barrier function as ATP-driven xenobiotic efflux pumps and limit delivery of small molecule drugs to the brain. Here I review recent progress in understanding the regulation of the expression and transport activity of these transporters and comment on how this new information might aid in improving drug delivery to the brain. PMID:25670036

  20. Absorption, Conjugation and Efflux of the Flavonoids, Kaempferol and Galangin, Using the Intestinal CACO-2/TC7 Cell Model.

    PubMed

    Barrington, Robert; Williamson, Gary; Bennett, Richard N; Davis, Barry D; Brodbelt, Jennifer S; Kroon, Paul A

    2009-01-01

    Flavonoids are biologically active compounds in food with potential health effects. We have used the Caco-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of 5 kaempferol conjugates: 3-, 7- and 4'-glucuronide, a sulphate and a glucurono-sulphate; and 4 galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC-MS, and HPLC with post column metal complexation/tandem MS. Transport studies showed that the flavonols were conjugated inside the cells then transported across the monolayer or effluxed back to the apical side. Sulphated conjugates were preferentially effluxed back to the apical side, whereas glucuronides were mostly transported to the basolateral side. For kaempferol, a small amount of the unconjugated aglycone permeated in both directions, indicating some passive diffusion. When kaempferol-3-glucuronide and quercetin7-sulphate were applied to either side of the cells, no permeation in either direction was observed, indicating that conjugates cannot re-cross the cell monolayer. Formation of apical kaempferol-7- and 4'-glucuronides was readily saturated, whereas formation of other conjugates at the apical side and all at the basolateral side increased with increasing concentration of kaempferol, implying different transporters are responsible at the apical and basolateral sides. The results highlight the important but complex metabolic changes occurring in flavonoids during absorption.

  1. Absorption, Conjugation and Efflux of the Flavonoids, Kaempferol and Galangin, Using the Intestinal CACO-2/TC7 Cell Model

    PubMed Central

    Barrington, Robert; Williamson, Gary; Bennett, Richard N; Davis, Barry D; Brodbelt, Jennifer S; Kroon, Paul A

    2009-01-01

    Flavonoids are biologically active compounds in food with potential health effects. We have used the Caco-2 cell monolayer model to study the absorption and metabolism of two flavonols, a class of flavonoids, specifically kaempferol and galangin. Metabolism experiments allowed identification of 5 kaempferol conjugates: 3-, 7- and 4′-glucuronide, a sulphate and a glucurono-sulphate; and 4 galangin conjugates: 3-, 5- and 7-glucuronides, and a sulphate, using specific enzyme hydrolysis, HPLC-MS, and HPLC with post column metal complexation/tandem MS. Transport studies showed that the flavonols were conjugated inside the cells then transported across the monolayer or effluxed back to the apical side. Sulphated conjugates were preferentially effluxed back to the apical side, whereas glucuronides were mostly transported to the basolateral side. For kaempferol, a small amount of the unconjugated aglycone permeated in both directions, indicating some passive diffusion. When kaempferol-3-glucuronide and quercetin7-sulphate were applied to either side of the cells, no permeation in either direction was observed, indicating that conjugates cannot re-cross the cell monolayer. Formation of apical kaempferol-7- and 4′-glucuronides was readily saturated, whereas formation of other conjugates at the apical side and all at the basolateral side increased with increasing concentration of kaempferol, implying different transporters are responsible at the apical and basolateral sides. The results highlight the important but complex metabolic changes occurring in flavonoids during absorption. PMID:20046888

  2. An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux

    PubMed Central

    Gillespie, Mark A; Gold, Elizabeth S; Ramsey, Stephen A; Podolsky, Irina; Aderem, Alan; Ranish, Jeffrey A

    2015-01-01

    LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment-quantitative mass spectrometry (PE-QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand- and binding-dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll-like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro-inflammatory and anti-inflammatory pathways that results in repression of macrophage cholesterol efflux. PMID:25755249

  3. An LXR-NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux.

    PubMed

    Gillespie, Mark A; Gold, Elizabeth S; Ramsey, Stephen A; Podolsky, Irina; Aderem, Alan; Ranish, Jeffrey A

    2015-05-05

    LXR-cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment-quantitative mass spectrometry (PE-QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand- and binding-dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll-like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3-LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro-inflammatory and anti-inflammatory pathways that results in repression of macrophage cholesterol efflux.

  4. Olive oil polyphenols enhance the expression of cholesterol efflux related genes in vivo in humans. A randomized controlled trial.

    PubMed

    Farràs, Marta; Valls, Rosa M; Fernández-Castillejo, Sara; Giralt, Montserrat; Solà, Rosa; Subirana, Isaac; Motilva, María-José; Konstantinidou, Valentini; Covas, María-Isabel; Fitó, Montserrat

    2013-07-01

    Both oleic acid and polyphenols have been shown to increase high-density lipoprotein (HDL) cholesterol and to protect HDL from oxidation, a phenomenon associated with a low cholesterol efflux from cells. Our goal was to determine whether polyphenols from olive oil could exert an in vivo nutrigenomic effect on genes related to cholesterol efflux in humans. In a randomized, controlled, cross-over trial, 13 pre/hypertensive patients were assigned 30 ml of two similar olive oils with high (961 mg/kg) and moderate (289 mg/kg) polyphenol content. We found an increase in ATP binding cassette transporter-A1, scavenger receptor class B type 1, peroxisome proliferator-activated receptor (PPAR)BP, PPARα, PPARγ, PPARδ and CD36 gene expression in white blood cells at postprandial after high polyphenol olive oil when compared with moderate polyphenol olive oil intervention (P<.017), with COX-1 reaching borderline significance (P=.024). Linear regression analyses showed that changes in gene expression were related to a decrease in oxidized low-density lipoproteins and with an increase in oxygen radical absorbance capacity and olive oil polyphenols (P<.05). Our results indicate a significant role of olive oil polyphenols in the up-regulation of genes involved in the cholesterol efflux from cells to HDL in vivo in humans. These results are in agreement with previous ones concerning the fact that benefits associated with polyphenol-rich olive oil consumption on cardiovascular risk could be mediated through an in vivo nutrigenomic effect in humans.

  5. Study of active cooling for supersonic transports

    NASA Technical Reports Server (NTRS)

    Brewer, G. D.; Morris, R. E.

    1975-01-01

    The potential benefits of using the fuel heat sink of hydrogen fueled supersonic transports for cooling large portions of the aircraft wing and fuselage are examined. The heat transfer would be accomplished by using an intermediate fluid such as an ethylene glycol-water solution. Some of the advantages of the system are: (1) reduced costs by using aluminum in place of titanium, (2) reduced cabin heat loads, and (3) more favorable environmental conditions for the aircraft systems. A liquid hydrogen fueled, Mach 2.7 supersonic transport aircraft design was used for the reference uncooled vehicle. The cooled aircraft designs were analyzed to determine their heat sink capability, the extent and location of feasible cooled surfaces, and the coolant passage size and spacing.

  6. Modeling of active transmembrane transport in a mixture theory framework.

    PubMed

    Ateshian, Gerard A; Morrison, Barclay; Hung, Clark T

    2010-05-01

    This study formulates governing equations for active transport across semi-permeable membranes within the framework of the theory of mixtures. In mixture theory, which models the interactions of any number of fluid and solid constituents, a supply term appears in the conservation of linear momentum to describe momentum exchanges among the constituents. In past applications, this momentum supply was used to model frictional interactions only, thereby describing passive transport processes. In this study, it is shown that active transport processes, which impart momentum to solutes or solvent, may also be incorporated in this term. By projecting the equation of conservation of linear momentum along the normal to the membrane, a jump condition is formulated for the mechano-electrochemical potential of fluid constituents which is generally applicable to nonequilibrium processes involving active transport. The resulting relations are simple and easy to use, and address an important need in the membrane transport literature.

  7. [Model of active peristaltic transport in biosystems].

    PubMed

    Klochkov, B N; Romanov, A S

    2013-01-01

    A nonlinear distributed mathematical model of soft vessel with the nonmonotonous static characteristic is proposed and considered. The model describes space-time dynamics of vessel clearance change. Wave phenomena in vessels of different nature and the possibility of peristaltic fluid pumping are discussed and analyzed. The model is rather common in character and represents a description of the whole class of transport phenomena. Lymphatic vessels are particularly considered.

  8. Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB.

    PubMed

    Tikhonova, Elena B; Devroy, Vishakha K; Lau, Sze Yi; Zgurskaya, Helen I

    2007-02-01

    Periplasmic membrane fusion proteins (MFPs) are essential components of the type I protein secretion systems and drug efflux pumps in Gram-negative bacteria. Previous studies suggested that MFPs connect the inner and outer membrane components of the transport systems and by this means co-ordinate the transfer of substrates across the two membranes. In this study, we purified and reconstituted the macrolide transporter MacAB from Escherichia coli. Here, MacA is a periplasmic MFP and MacB is an ABC-type transporter. Similar to other MFP-dependent transporters from E. coli, the in vivo function of MacAB requires the outer membrane channel TolC. The purified MacB displayed a basal ATPase activity in detergent micelles. This activity conformed to Michaelis-Menten kinetics but was unresponsive to substrates or accessory proteins. Upon reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45-fold higher than the activity of MacB alone. Both the N- and C-terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the presence of macrolides. Our results suggest that MacA is a functional subunit of the MacB transporter.

  9. The sodium-bicarbonate cotransporter NBCe1 supports glutamine efflux via SNAT3 (SLC38A3) co-expressed in Xenopus oocytes.

    PubMed

    Wendel, Christina; Becker, Holger M; Deitmer, Joachim W

    2008-02-01

    The glutamine transporter SNAT3 contributes to the glutamine fluxes in liver, kidney, and brain. We heterologously co-expressed SNAT3 with the electrogenic sodium-bicarbonate cotransporter NBCe1 in Xenopus laevis oocytes and measured cytosolic pH and membrane current in voltage clamp. Because of the increased buffer capacity contributed by the NBCe1 (Becker and Deitmer in J Biol Chem 279:28057-28062, 2004), we hypothesized that this may enhance the proton-coupled glutamine transport via SNAT3 in the presence of CO2/HCO3-. Addition and removal of glutamine activated not only SNAT3 but also NBCe1, as indicated by the increased membrane current. The NBCe1 current during glutamine removal was more than 50% larger than during glutamine addition, suggesting that NBCe1 enhances glutamine efflux rather than glutamine uptake. This was confirmed by radio-labeled glutamine flux measurements; influx of glutamine was significantly decreased, whereas efflux of glutamine was increased when SNAT3 was co-expressed with NBCe1. A model is presented that attempts to explain the role of intracellular pH, bicarbonate transport, and buffering capacity mediated by NBCe1 for uptake and efflux of glutamine via SNAT3.

  10. In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers

    PubMed Central

    Findlay, Heather E.; Harris, Nicola J.; Booth, Paula J.

    2016-01-01

    Nature encapsulates reactions within membrane-bound compartments, affording sequential and spatial control over biochemical reactions. Droplet Interface Bilayers are evolving into a valuable platform to mimic this key biological feature in artificial systems. A major issue is manipulating flow across synthetic bilayers. Droplet Interface Bilayers must be functionalised, with seminal work using membrane-inserting toxins, ion channels and pumps illustrating the potential. Specific transport of biomolecules, and notably transport against a concentration gradient, across these bilayers has yet to be demonstrated. Here, we successfully incorporate the archetypal Major Facilitator Superfamily transporter, lactose permease, into Droplet Interface Bilayers and demonstrate both passive and active, uphill transport. This paves the way for controllable transport of sugars, metabolites and other essential biomolecular substrates of this ubiquitous transporter superfamily in DIB networks. Furthermore, cell-free synthesis of lactose permease during DIB formation also results in active transport across the interface bilayer. This adds a specific disaccharide transporter to the small list of integral membrane proteins that can be synthesised via in vitro transcription/translation for applications of DIB-based artificial cell systems. The introduction of a means to promote specific transport of molecules across Droplet Interface Bilayers against a concentration gradient gives a new facet to droplet networks. PMID:27996025

  11. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2010-01-27

    Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl- efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl- efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees C. The effect of NAC on Cl- transport was measured by Cl- efflux measurements and by X-ray microanalysis. Cl- efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl- efflux with nearly 80% in CFBE cells. The intracellular Cl- concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl- efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.

  12. Computer simulations suggest direct and stable tip to tip interaction between the outer membrane channel TolC and the isolated docking domain of the multidrug RND efflux transporter AcrB.

    PubMed

    Schmidt, Thomas H; Raunest, Martin; Fischer, Nadine; Reith, Dirk; Kandt, Christian

    2016-07-01

    One way by which bacteria achieve antibiotics resistance is preventing drug access to its target molecule for example through an overproduction of multi-drug efflux pumps of the resistance nodulation division (RND) protein super family of which AcrAB-TolC in Escherichia coli is a prominent example. Although representing one of the best studied efflux systems, the question of how AcrB and TolC interact is still unclear as the available experimental data suggest that either both proteins interact in a tip to tip manner or do not interact at all but are instead connected by a hexamer of AcrA molecules. Addressing the question of TolC-AcrB interaction, we performed a series of 100 ns - 1 µs-molecular dynamics simulations of membrane-embedded TolC in presence of the isolated AcrB docking domain (AcrB(DD)). In 5/6 simulations we observe direct TolC-AcrB(DD) interaction that is only stable on the simulated time scale when both proteins engage in a tip to tip manner. At the same time we find TolC opening and closing freely on extracellular side while remaining closed at the inner periplasmic bottleneck region, suggesting that either the simulated time is too short or additional components are required to unlock TolC.

  13. Amphetamine activates Rho GTPase signaling to mediate dopamine transporter internalization and acute behavioral effects of amphetamine

    PubMed Central

    Wheeler, David S.; Underhill, Suzanne M.; Stolz, Donna B.; Murdoch, Geoffrey H.; Thiels, Edda; Romero, Guillermo; Amara, Susan G.

    2015-01-01

    Acute amphetamine (AMPH) exposure elevates extracellular dopamine through a variety of mechanisms that include inhibition of dopamine reuptake, depletion of vesicular stores, and facilitation of dopamine efflux across the plasma membrane. Recent work has shown that the DAT substrate AMPH, unlike cocaine and other nontransported blockers, can also stimulate endocytosis of the plasma membrane dopamine transporter (DAT). Here, we show that when AMPH enters the cytoplasm it rapidly stimulates DAT internalization through a dynamin-dependent, clathrin-independent process. This effect, which can be observed in transfected cells, cultured dopamine neurons, and midbrain slices, is mediated by activation of the small GTPase RhoA. Inhibition of RhoA activity with C3 exotoxin or a dominant-negative RhoA blocks AMPH-induced DAT internalization. These actions depend on AMPH entry into the cell and are blocked by the DAT inhibitor cocaine. AMPH also stimulates cAMP accumulation and PKA-dependent inactivation of RhoA, thus providing a mechanism whereby PKA- and RhoA-dependent signaling pathways can interact to regulate the timing and robustness of AMPH’s effects on DAT internalization. Consistent with this model, the activation of D1/D5 receptors that couple to PKA in dopamine neurons antagonizes RhoA activation, DAT internalization, and hyperlocomotion observed in mice after AMPH treatment. These observations support the existence of an unanticipated intracellular target that mediates the effects of AMPH on RhoA and cAMP signaling and suggest new pathways to target to disrupt AMPH action. PMID:26553986

  14. 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.

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

    PubMed Central

    Speer, B S; Salyers, A A

    1990-01-01

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

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

    PubMed

    Bhatt, Anish; Rohatgi, Anand

    2016-01-01

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

  17. Molecular cloning and characterization of a multidrug efflux pump, SmfY, from Serratia marcescens.

    PubMed

    Shahcheraghi, Fereshteh; Minato, Yusuke; Chen, Jing; Mizushima, Tohru; Ogawa, Wakano; Kuroda, Teruo; Tsuchiya, Tomofusa

    2007-04-01

    We cloned a gene smfY for multidrug efflux pump from chromosomal DNA of Serratia marcescens using drug-hypersensitive Escherichia coli KAM32 as the host, and characterized the pump. E. coli KAM32/pESM42 carrying the smfY showed significantly increased MICs of various drugs including DAPI, norfloxacin, benzalkonium chloride, acriflavine and ethidium bromide, compared with the control. We also detected energy-dependent ethidium and acriflavine efflux due to the SmfY. Sequence analysis revealed that the SmfY was a multidrug efflux pump of the MF (Major Facilitator) superfamily transporters. This is the first report of a multidrug efflux pump belonging to the MF superfamily in S. marcescens.

  18. The role played by drug efflux pumps in bacterial multidrug resistance.

    PubMed

    Chitsaz, Mohsen; Brown, Melissa H

    2017-02-28

    Antimicrobial resistance is a current major challenge in chemotherapy and infection control. The ability of bacterial and eukaryotic cells to recognize and pump toxic compounds from within the cell to the environment before they reach their targets is one of the important mechanisms contributing to this phenomenon. Drug efflux pumps are membrane transport proteins that require energy to export substrates and can be selective for a specific drug or poly-specific that can export multiple structurally diverse drug compounds. These proteins can be classified into seven groups based on protein sequence homology, energy source and overall structure. Extensive studies on efflux proteins have resulted in a wealth of knowledge that has made possible in-depth understanding of the structures and mechanisms of action, substrate profiles, regulation and possible inhibition of many clinically important efflux pumps. This review focuses on describing known families of drug efflux pumps using examples that are well characterized structurally and/or biochemically.

  19. 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

  20. Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

    PubMed

    Fournier, Natalie; Tardivel, Sylviane; Benoist, Jean-François; Vedie, Benoît; Rousseau-Ralliard, Delphine; Nowak, Maxime; Allaoui, Fatima; Paul, Jean-Louis

    2016-04-01

    A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages.