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Sample records for active efflux transport

  1. SPTLC1 binds ABCA1 to negatively regulate trafficking and cholesterol efflux activity of the transporter.

    PubMed

    Tamehiro, Norimasa; Zhou, Suiping; Okuhira, Keiichiro; Benita, Yair; Brown, Cari E; Zhuang, Debbie Z; Latz, Eicke; Hornemann, Thorsten; von Eckardstein, Arnold; Xavier, Ramnik J; Freeman, Mason W; Fitzgerald, Michael L

    2008-06-10

    ABCA1 transport of cholesterol and phospholipids to nascent HDL particles plays a central role in lipoprotein metabolism and macrophage cholesterol homeostasis. ABCA1 activity is regulated both at the transcriptional level and at the post-translational level. To explore mechanisms involved in the post-translational regulation of the transporter, we have used affinity purification and mass spectrometry to identify proteins that bind ABCA1 and influence its activity. Previously, we demonstrated that an interaction between beta1-syntrophin stimulated ABCA1 activity, at least in part, be slowing the degradation of the transporter. This work demonstrates that one subunit of the serine palmitoyltransferase enzyme, SPTLC1, but not subunit 2 (SPTLC2), is copurified with ABCA1 and negatively regulates its function. In human THP-I macrophages and in mouse liver, the ABCA1-SPTLC1 complex was detected by co-immunoprecipitation, demonstrating that the interaction occurs in cellular settings where ABCA1 activity is critical for HDL genesis. Pharmacologic inhibition of SPTLC1 with myriocin, which resulted in the disruption of the SPTLC1-ABCA1 complex, and siRNA knockdown of SPTLC1 expression both stimulated ABCA1 efflux by nearly 60% ( p < 0.05). In contrast, dominant-negative mutants of SPTLC1 inhibited ABCA1 efflux, indicating that a reduced level of sphingomyelin synthesis could not explain the effect of myriocin on ABCA1 activity. In 293 cells, the SPTLC1 inhibition of ABCA1 activity led to the blockade of the exit of ABCA1 from the endoplasmic reticulum. In contrast, myriocin treatment of macrophages increased the level of cell surface ABCA1. In composite, these results indicate that the physical interaction of ABCA1 and SPTLC1 results in reduction of ABCA1 activity and that inhibition of this interaction produces enhanced cholesterol efflux. PMID:18484747

  2. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals.

    PubMed

    Pearson, J P; Van Delden, C; Iglewski, B H

    1999-02-01

    Many gram-negative bacteria communicate by N-acyl homoserine lactone signals called autoinducers (AIs). In Pseudomonas aeruginosa, cell-to-cell signaling controls expression of extracellular virulence factors, the type II secretion apparatus, a stationary-phase sigma factor (sigmas), and biofilm differentiation. The fact that a similar signal, N-(3-oxohexanoyl) homoserine lactone, freely diffuses through Vibrio fischeri and Escherichia coli cells has led to the assumption that all AIs are freely diffusible. In this work, transport of the two P. aeruginosa AIs, N-(3-oxododecanoyl) homoserine lactone (3OC12-HSL) (formerly called PAI-1) and N-butyryl homoserine lactone (C4-HSL) (formerly called PAI-2), was studied by using tritium-labeled signals. When [3H]C4-HSL was added to cell suspensions of P. aeruginosa, the cellular concentration reached a steady state in less than 30 s and was nearly equal to the external concentration, as expected for a freely diffusible compound. In contrast, [3H]3OC12-HSL required about 5 min to reach a steady state, and the cellular concentration was 3 times higher than the external level. Addition of inhibitors of the cytoplasmic membrane proton gradient, such as azide, led to a strong increase in cellular accumulation of [3H]3OC12-HSL, suggesting the involvement of active efflux. A defined mutant lacking the mexA-mexB-oprM-encoded active-efflux pump accumulated [3H]3OC12-HSL to levels similar to those in the azide-treated wild-type cells. Efflux experiments confirmed these observations. Our results show that in contrast to the case for C4-HSL, P. aeruginosa cells are not freely permeable to 3OC12-HSL. Instead, the mexA-mexB-oprM-encoded efflux pump is involved in active efflux of 3OC12-HSL. Apparently the length and/or degree of substitution of the N-acyl side chain determines whether an AI is freely diffusible or is subject to active efflux by P. aeruginosa.

  3. P-glycoprotein in sheep liver and small intestine: gene expression and transport efflux activity.

    PubMed

    Ballent, M; Wilkens, M R; Maté, L; Muscher, A S; Virkel, G; Sallovitz, J; Schröder, B; Lanusse, C; Lifschitz, A

    2013-12-01

    The role of the transporter P-glycoprotein (P-gp) in the disposition kinetics of different drugs therapeutically used in veterinary medicine has been demonstrated. Considering the anatomo-physiological features of the ruminant species, the constitutive expression of P-gp (ABCB1) along the sheep gastrointestinal tract was studied. Additionally, the effect of repeated dexamethasone (DEX) administrations on the ABCB1 gene expression in the liver and small intestine was also assessed. The ABCB1 mRNA expression was determined by real-time quantitative PCR. P-gp activity was evaluated in diffusion chambers to determine the efflux of rhodamine 123 (Rho 123) in the ileum from experimental sheep. The constitutive ABCB1 expression was 65-fold higher in the liver than in the intestine (ileum). The highest ABCB1 mRNA expression along the small intestine was observed in the ileum (between 6- and 120-fold higher). The treatment with DEX did not elicit a significant effect on the P-gp gene expression levels in any of the investigated gastrointestinal tissues. Consistently, no significant differences were observed in the intestinal secretion of Rho 123, between untreated control (Peff S-M = 3.99 × 10(-6)  ± 2.07 × 10(-6) ) and DEX-treated animals (Peff S-M = 6.00 × 10(-6)  ± 2.5 × 10(-6) ). The understanding of the efflux transporters expression and activity along the digestive tract may help to elucidate clinical implications emerging from drug interactions in livestock.

  4. Opioids and efflux transporters. Part 4: influence of N-substitution on P-glycoprotein substrate activity of noroxymorphone analogues.

    PubMed

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

    2014-08-01

    The efflux transporter protein P-glycoprotein (P-gp) is capable of affecting the central distribution of diverse neurotherapeutics, including opioid analgesics, through their active removal from the brain. P-gp located at the blood brain barrier has been implicated in the development of tolerance to opioids and demonstrated to be up-regulated in rats tolerant to morphine and oxycodone. We have previously examined the influence of hydrogen-bonding oxo-substitutents on the P-gp-mediated efflux of 4,5-epoxymorphinan analgesics, as well as that of N-substituted analogues of meperidine. Structure-activity relationships (SAR) governing N-substituent effects on opioid efficacy is well-established, however the influence of such structural modifications on P-gp-mediated efflux is unknown. Here, we present SAR describing P-gp recognition of a short series of N-modified 4,5-epoxymorphinans. Oxymorphone, naloxone, naltrexone, and nalmexone all failed to demonstrate P-gp substrate activity, indicating these opioid scaffolds contain structural features that preclude recognition by the transporter. These results are examined using mathematical molecular modeling and discussed in comparison to other opioid scaffolds bearing similar N-substituents. PMID:24915880

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

    PubMed

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

    2015-04-22

    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.

  6. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2008-01-01

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

  8. 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

  9. Bacterial multi-drug efflux transporters

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  11. Efflux transporters of the human placenta.

    PubMed

    Young, Amber M; Allen, Courtni E; Audus, Kenneth L

    2003-01-21

    The use of pharmaceuticals during pregnancy is often a necessity for the health of the mother. Until recently, the placenta was viewed as a passive organ through which molecules are passed indiscriminately between mother and fetus. In reality, the placenta contains a plethora of transporters, some of which appear to be specifically dedicated to removal of xenobiotics and toxic endogenous compounds. Drug efflux transporters such as P-glycoprotein (P-gp), several multidrug resistant associated proteins (MRPs) and breast cancer resistant protein (BCRP) may provide mechanisms that protect the developing fetus. Bile acid transporters may also play a role in exporting compounds back into the maternal compartment. Steroid hormones directly influence the level of expression and function in some of these transporters. Investigating the link between the hormones of pregnancy and these drug efflux transporters is one possible key in developing strategies to deliver drugs to the mother with minimal fetal risk. PMID:12535577

  12. Development of chitosan-SLN microparticles for chemotherapy: in vitro approach through efflux-transporter modulation.

    PubMed

    Dharmala, Kiran; Yoo, Jin Wook; Lee, Chi H

    2008-11-12

    Drug efflux-transporters serve as a major barrier to anticancer drugs at the target site. One strategy to enhance the therapeutic efficacy of drugs against cancer is to increase their available concentrations at the target site by suppressing or modulating efflux-transporters. This manuscript deals with the development and evaluation of the particle type drug delivery system made of stearic acid (Solid Lipid Nanoparticle - SLN) and chitosan for the delivery of Phenethyl Isothiocyanate (PEITC), a tumor-suppressive agent, through the pulmonary route. The rationale behind the particle type drug delivery system involves a prior release of the efflux-transporter inhibitors, such as tamoxifen, verapamil HCl or nifedipine, to suppress or modulate the efflux activity of ABC transporters followed by the release of the efflux-transporter substrate, PEITC. The efficacy of Chitosan-SLN Microparticles (CSM) as a carrier for PEITC was evaluated by investigating the release profiles of PEITC loaded in CSM and its cytotoxicity in the presence or absence of the efflux-transporter inhibitors. An initial burst release of the inhibitors, followed by gradual, sustained release of PEITC and subsequent increase in cytotoxicity was observed. This finding indicated that the efflux transporter inhibitors significantly affected the PEITC uptake rate by Calu-3 cells. Judging from these results, CSM can be an efficient drug delivery system for the substrates susceptible to the efflux-transporters. PMID:18723057

  13. 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

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

    PubMed

    Chatard, Morgane; Puech, Clémentine; Roche, Frederic; Perek, Nathalie

    2016-01-01

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

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

    PubMed Central

    Chatard, Morgane; Puech, Clémentine

    2016-01-01

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

  16. Induction of expression and functional activity of P-glycoprotein efflux transporter by bioactive plant natural products.

    PubMed

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

    2011-11-01

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

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

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2016-01-01

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that ‘aromatic clamps’ formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such ‘clamps’ may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins. PMID:26961153

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

    PubMed

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

    2015-12-01

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

  20. Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux.

    PubMed

    Binda, Francesca; Dipace, Concetta; Bowton, Erica; Robertson, Sabrina D; Lute, Brandon J; Fog, Jacob U; Zhang, Minjia; Sen, Namita; Colbran, Roger J; Gnegy, Margaret E; Gether, Ulrik; Javitch, Jonathan A; Erreger, Kevin; Galli, Aurelio

    2008-10-01

    The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.

  1. The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

    PubMed

    Halwachs, Sandra; Kneuer, Carsten; Gohlsch, Katrin; Müller, Marian; Ritz, Vera; Honscha, Walther

    2016-02-01

    In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter. PMID:26907376

  2. The sea urchin embryo as a model for studying efflux transporters: Roles and energy cost

    PubMed Central

    Epel, David; Cole, Bryan; Hamdoun, Amro; Thurber, Rebecca Vega

    2011-01-01

    We describe the use of the sea urchin as a model for studying efflux transporters and estimating energy cost for the cytotoxin protective system provided by these transporters. The unfertilized egg has low transport activity, which increases to a new steady state shortly after fertilization. Activity results from p-glycoprotein (p-gp) and MRP type transporters which protect the embryo from cytotoxic drugs that can disrupt cell division or induce apoptosis. The energy cost is estimated from a novel use of calcein-AM as a substrate; keeping 0.25 μM substrate levels out of the cell utilizes only 0.023% of steady state respiration. PMID:16740304

  3. [Role of HDL in Cholesterol Efflux and Reverse Cholesterol Transport].

    PubMed

    Ayaori, Makoto

    2016-01-01

    Low plasma levels of HDL-cholesterol (HDL-C) have been consistently associated with an increased risk of atherosclerotic cardiovascular diseases (CVD), and it is thus considered to be an anti-atherogenic lipoprotein. The development of novel therapies to enhance the atheroprotective properties of HDL may have the potential to further reduce the residual risk. Reverse cholesterol transport (RCT) is believed to be a primary atheroprotective property of HDL and its major protein, apolipoprotein A-I(apoA-I). HDL and apoA-I have been shown to promote the efflux of excess cholesterol from macrophage-derived foam cells via the cholesterol transporters, ATP-binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B, type I (SR-BI), and then transport it back to the liver for excretion into bile and eventually into the feces. In this regard, a validated murine assay that quantifies macrophage RCT may be a better predictor of atherosclerosis than the steady-state plasma concentration of HDL-C. Indeed, a recent clinical study demonstrated that the ability of serum HDL to mediate cholesterol efflux from macrophages was independently and negatively associated with the CVD risk even after adjustment for HDL-C levels, suggesting that HDL functionality is more important than its quantity. Therefore, the future development of HDL-targeted therapy should take both aspects into consideration to further reduce the residual risk.

  4. [Role of HDL in Cholesterol Efflux and Reverse Cholesterol Transport].

    PubMed

    Ayaori, Makoto

    2016-01-01

    Low plasma levels of HDL-cholesterol (HDL-C) have been consistently associated with an increased risk of atherosclerotic cardiovascular diseases (CVD), and it is thus considered to be an anti-atherogenic lipoprotein. The development of novel therapies to enhance the atheroprotective properties of HDL may have the potential to further reduce the residual risk. Reverse cholesterol transport (RCT) is believed to be a primary atheroprotective property of HDL and its major protein, apolipoprotein A-I(apoA-I). HDL and apoA-I have been shown to promote the efflux of excess cholesterol from macrophage-derived foam cells via the cholesterol transporters, ATP-binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B, type I (SR-BI), and then transport it back to the liver for excretion into bile and eventually into the feces. In this regard, a validated murine assay that quantifies macrophage RCT may be a better predictor of atherosclerosis than the steady-state plasma concentration of HDL-C. Indeed, a recent clinical study demonstrated that the ability of serum HDL to mediate cholesterol efflux from macrophages was independently and negatively associated with the CVD risk even after adjustment for HDL-C levels, suggesting that HDL functionality is more important than its quantity. Therefore, the future development of HDL-targeted therapy should take both aspects into consideration to further reduce the residual risk. PMID:27192798

  5. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    1996-01-01

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

  8. Silicon efflux transporters isolated from two pumpkin cultivars contrasting in Si uptake.

    PubMed

    Mitani-Ueno, Namiki; Yamaji, Naoki; Ma, Jian Feng

    2011-07-01

    The accumulation of silicon (Si) differs greatly with plant species and cultivars due to different ability of the roots to take up Si. In Si accumulating plants such as rice, barley and maize, Si uptake is mediated by the influx (Lsi1) and efflux (Lsi2) transporters. Here we report isolation and functional analysis of two Si efflux transporters (CmLsi2-1 and CmLsi2-2) from two pumpkin (Cucurbita moschata Duch.) cultivars contrasting in Si uptake. These cultivars are used for rootstocks of bloom and bloomless cucumber, respectively. Different from mutations in the Si influx transporter CmLsi1, there was no difference in the sequence of either CmLsi2 between two cultivars. Both CmLsi2-1 and CmLsi2-2 showed an efflux transport activity for Si and they were expressed in both the roots and shoots. These results confirm our previous finding that mutation in CmLsi1, but not in CmLsi2-1 and CmLsi2-2 are responsible for bloomless phenotype resulting from low Si uptake.

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

    PubMed

    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

    2016-04-21

    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.

  10. 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

  11. Why Does the Intestine Lack Basolateral Efflux Transporters for Cationic Compounds? A Provocative Hypothesis.

    PubMed

    Proctor, William R; Ming, Xin; Bourdet, David; Han, Tianxiang Kevin; Everett, Ruth S; Thakker, Dhiren R

    2016-02-01

    Transport proteins in intestinal epithelial cells facilitate absorption of nutrients/compounds that are organic anions, cations, and zwitterions. For two decades, we have studied intestinal absorption and transport of hydrophilic ionic compounds, with specific focus on transport properties of organic cations and their interactions with intestinal transporters and tight junction proteins. Our data reveal how complex interactions between a compound and transporters in intestinal apical/basolateral (BL) membranes and tight junction proteins define oral absorption, and that the BL membrane lacks an efflux transporter that can transport positively charged compounds. Based on our investigations of transport mechanisms of zwitterionic, anionic, and cationic compounds, we postulate that physicochemical properties of these ionic species, in relation to the intestinal micro pH environment, have exerted evolutionary pressure for development of transporters that can handle apical uptake/efflux of all 3 ionic species and BL efflux of anions and zwitterions, but such evolutionary pressure is lacking for development of a BL efflux transporter for cationic compounds. This review provides an overview of intestinal uptake/efflux transporters and describes our studies on intestinal transport of cationic, anionic, and zwitterionic drugs that led to hypothesize that there are no cation-selective BL efflux transporters in the intestine. PMID:26869413

  12. 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.

  13. The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.

    PubMed

    Holmes, Ann R; Keniya, Mikhail V; Ivnitski-Steele, Irena; Monk, Brian C; Lamping, Erwin; Sklar, Larry A; Cannon, Richard D

    2012-03-01

    Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains. PMID:22203607

  14. 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

  15. Danofloxacin-mesylate is a substrate for ATP-dependent efflux transporters

    PubMed Central

    Schrickx, J A; Fink-Gremmels, J

    2007-01-01

    Background and purpose: Next to its broad antimicrobial spectrum, the therapeutic advantages of the fluoroquinolone antimicrobial drug Danofloxacin-Mesylate (DM) are attributed to its rapid distribution to the major target tissues such as lungs, intestines and the mammary gland in animals. Previous analyses revealed that effective drug concentrations are achieved also in luminal compartments of these organs, suggesting that active transport proteins facilitate excretion into the luminal space. Members of the ATP-Binding Cassette (ABC) superfamily, including P-gp, BCRP and MRP2 are known to be expressed in many tissue barriers and in cell-membranes facing luminal compartments. Hence we hypothesized that DM is a substrate for one of these efflux-transporters. Experimental approach: Confluent monolayers of Caco-2 cells, grown on microporous membranes in two-chamber devices were used. DM concentrations were measured by fluorimetric assay after HPLC of the culture media. Key results: DM transport across Caco-2 cells was asymmetric, with a rate of secretion exceeding that of absorption. The P-gp inhibitors PSC833 and GF120918 and the MRP-inhibitor MK571 partially decreased the secretion of DM and increased its absorption rate. The BCRP inhibitor, Ko143, decreased secretion only at a concentration of 1 μM. When DM was applied together with ciprofloxacin, secretion as well as absorption of DM decreased. Conclusions and Implications: DM is a substrate for the efflux transporters P-gp and MRP2, whereas the specific role of BCRP in DM transport needs further evaluation. These findings provide a mechanistic basis for the understanding of the pharmacokinetics of DM in healthy and diseased individuals. PMID:17211460

  16. Telatinib reverses chemotherapeutic multidrug resistance mediated by ABCG2 efflux transporter in vitro and in vivo

    PubMed Central

    Sodani, Kamlesh; Patel, Atish; Anreddy, Nagaraju; Singh, Satyakam; Yang, Dong-Hua; Kathawala, Rishil J; Kumar, Priyank; Talele, Tanaji T; Chen, Zhe-Sheng

    2014-01-01

    Multidrug resistance (MDR) is a phenomenon where cancer cells become simultaneously resistant to anticancer drugs with different structures and mechanisms of action. MDR has been shown to be associated with overexpression of ATP-binding cassette (ABC) transporters. Here, we report that telatinib, a small molecule tyrosine kinase inhibitor, enhances the anticancer activity of ABCG2 substrate anticancer drugs by inhibiting ABCG2 efflux transporter activity. Co-incubation of ABCG2-overexpressing drug resistant cell lines with telatinib and ABCG2 substrate anticancer drugs significantly reduced cellular viability, whereas telatinib alone did not significantly affect drug sensitive and drug resistant cell lines. Telatinib at 1 μM did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines. Telatinib at 1 μM significantly enhanced the intracellular accumulation of [3H]-mitoxantrone (MX) in ABCG2-overexpressing cell lines. In addition, telatinib at 1 μM significantly reduced the rate of [3H]-MX efflux from ABCG2-overexpressing cells. Furthermore, telatinib significantly inhibited ABCG2-mediated transport of [3H]-E217βG in ABCG2 overexpressing membrane vesicles. Telatinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner, indicating that telatinib might be a substrate of ABCG2. Binding interactions of telatinib were found to be in transmembrane region of homology modeled human ABCG2. In addition, telatinib (15 mg/kg) with doxorubicin (1.8 mg/kg) significantly decreased the growth rate and tumor size of ABCG2 overexpressing tumors in a xenograft nude mouse model. These results, provided that they can be translated to humans, suggesting that telatinib, in combination with specific ABCG2 substrate drugs may be useful in treating tumors that overexpress ABCG2. PMID:24565910

  17. 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.

  18. 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.

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

    PubMed

    Ahmad, Aijaz; Khan, Amber; Manzoor, Nikhat

    2013-01-23

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

  20. 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.

  1. 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. PMID:27480280

  2. Importance of Non-Diffusive Transport for Soil CO2 Efflux in a Temperate Mountain Grassland

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

    A key focus in climate change is on the dynamics and predictions of the soil CO2 efflux (SCE) from terrestrial ecosystems. Limited knowledge of CO2 transport through the soil restricts our understanding of the various biotic and abiotic processes underlying these emissions. Diffusion is often thought to be the main transport mechanism for trace gases in soils, an assumption that is reflected in the increasing popularity of the flux-gradient approach (FGA). Based on Fick's law, the FGA calculates soil CO2 efflux from CO2 concentration profiles, given good estimates of the diffusion coefficient. The latter can be calculated via different commonly used models, and solid-state sensors allow continuous high-frequency measurements of soil CO2 concentrations with minimal disturbance to the soil conditions in a cost-effective way. Fast growing evidence of pressure pumping and advection, makes it impossible to disregard non-diffusive gas transport when evaluating diel and day-to-day dynamics of soil CO2 emissions. We have analyzed combined measurements from solid-state sensors and soil chambers to gain insight in the CO2 transport mechanisms in a grassland site in the Austrian Alps. The FGA-derived efflux underestimated the chamber efflux by 10 to 87% at our site, depending on which model was used for calculation of the diffusion coefficient. We found that the actual transport rates correlated well with irradiation and wind speed, even more when the soil moisture content was below 33%. These findings suggest that bulk soil air transport was enhanced by pressure changes induced by wind shear at the surface and by local heating of the soil surface. Considering the importance of non-diffusive transport processes is a prerequisite when using solid-state CO2 concentration measurements to estimate soil CO2 efflux at any given site.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux

    PubMed Central

    1995-01-01

    We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter

  8. 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. PMID:25035400

  9. 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.

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

    PubMed

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

    2015-06-01

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

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

    NASA Astrophysics Data System (ADS)

    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.

  12. Cytotoxic thio-malate is transported by both an aluminum-responsive malate efflux pathway in wheat and the MAE1 malate permease in Schizosaccharomyces pombe.

    PubMed

    Osawa, Hiroki; Matsumoto, Hideaki

    2006-07-01

    Aluminum (Al) tolerance in wheat (Triticum aestivum L.) is mainly achieved by malate efflux, which is regulated by the expression of the recently identified gene, presumably encoding an Al-activated malate efflux transporter (ALMT1). However, the transport mechanism is not fully understood, partly as a result of the rapid turnover of its substrate. We developed a tool to study malate transport in wheat by screening biological compounds using the well-characterized Schizosaccharomyces pombe malate transporter (SpMAE1). Expression of SpMAE1 in both S. pombe and Saccharomyces cerevisiae, which has no SpMAE1 homologue, caused hypersensitivity to thio-malic acid. This hypersensitivity was prominent at pH 3.5, but not pH 4.5, and was accompanied by an increase in thiol content, indicating that SpMAE1 mediates the uptake of thio-malic acid at a specific low pH. In wheat, root apices were able to accumulate thio-malic acid without growth reduction at pH values above 4.2. Pretreatment of root apices with thio-malic acid followed by Al treatment induced thio-malate efflux. Al-induced thio-malate efflux was much higher in Al-resistant cultivars/genotypes than in Al-sensitive ones, and was accompanied by a decrease in thiol-content. Thio-malate efflux in the Al-resistant cultivar was slightly activated by lanthanum or ytterbium ion. Thio-malic acid did not alleviate the Al-induced inhibition of root elongation in wheat. Taken together, our results suggest that thio-malate acts as an analogue for malate in malate transport systems in wheat and yeast, and that it may be a useful tool for the analysis of malate transport involved in Al-tolerance and of other organic ion transport processes.

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

    PubMed Central

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

    2015-01-01

    Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates. PMID:25972842

  14. 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.

  15. 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.

  16. Extra-renal elimination of uric acid via intestinal efflux transporter BCRP/ABCG2.

    PubMed

    Hosomi, Atsushi; Nakanishi, Takeo; Fujita, Takuya; Tamai, Ikumi

    2012-01-01

    Urinary excretion accounts for two-thirds of total elimination of uric acid and the remainder is excreted in feces. However, the mechanism of extra-renal elimination is poorly understood. In the present study, we aimed to clarify the mechanism and the extent of elimination of uric acid through liver and intestine using oxonate-treated rats and Caco-2 cells as a model of human intestinal epithelium. In oxonate-treated rats, significant amounts of externally administered and endogenous uric acid were recovered in the intestinal lumen, while biliary excretion was minimal. Accordingly, direct intestinal secretion was thought to be a substantial contributor to extra-renal elimination of uric acid. Since human efflux transporter BCRP/ABCG2 accepts uric acid as a substrate and genetic polymorphism causing a decrease of BCRP activity is known to be associated with hyperuricemia and gout, the contribution of rBcrp to intestinal secretion was examined. rBcrp was confirmed to transport uric acid in a membrane vesicle study, and intestinal regional differences of expression of rBcrp mRNA were well correlated with uric acid secretory activity into the intestinal lumen. Bcrp1 knockout mice exhibited significantly decreased intestinal secretion and an increased plasma concentration of uric acid. Furthermore, a Bcrp inhibitor, elacridar, caused a decrease of intestinal secretion of uric acid. In Caco-2 cells, uric acid showed a polarized flux from the basolateral to apical side, and this flux was almost abolished in the presence of elacridar. These results demonstrate that BCRP contributes at least in part to the intestinal excretion of uric acid as extra-renal elimination pathway in humans and rats.

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

    PubMed

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

    2009-07-01

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

  18. 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

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

    PubMed Central

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

    2016-01-01

    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. PMID:27157787

  20. Swertianlarin, isolated from Swertia mussotii Franch, increases detoxification enzymes and efflux transporters expression in rats

    PubMed Central

    Feng, Xin-Chan; Du, Xiaohuang; Chen, Sheng; Yue, Dongmei; Cheng, Ying; Zhang, Liangjun; Gao, Yu; Li, Shaoxue; Chen, Lei; Peng, Zhihong; Yang, Yong; Luo, Weizao; Wang, Rongquan; Chen, Wensheng; Chai, Jin

    2015-01-01

    Swertianlarin, isolated from Swertia mussotii Franch and Enicostemma axillare, has hepatoprotective effects against cholestasis in rat models of hepatotoxicity. However, the underlying molecular mechanism is not clear. We then treated rats with swertianlarin for 7 d and then measured serum liver injury markers, lipids, and bile salts, as well as the expression of bile acid synthesis and detoxification enzymes (e.g. Cyp7a1 and Cyp3a), membrane influx and efflux transporters (e.g. Ntcp and Mrp3), nuclear receptors (e.g. Pxr and Fxr/Shp) and transcriptional factors (e.g. Nrf2 and Hnf3β) in the liver. We found a significant induction of the expression of the basolateral efflux transporters Mrp3 and Mrp4 and canalicular transporter Mdr1 in rats treated with swertianlarin, compared with the controls (1.9-fold and 2.2-fold, P < 0.005, and 3.4-fold, P < 0.05, respectively). The expression of detoxification enzymes Cyp3a, Ugt2b, Sult2a1 and Gsta1 in rats treated with swertianlarin was significantly higher than that in controls (3.7-fold, 2.8-fold, 2.1-fold, and 1.7-fold, respectively, all P < 0.05). Expression of the synthetic enzyme, Cyp8b1, was higher in rats treated with swertianlarin than that in controls (1.8-fold at mRNA level and 3.4-flod at protein level, P < 0.05). Elevated serum levels of the conjugated bile acids, taurocholic acid and taurodeoxycholic acid, and a reduction in levels of serum ALP, unconjugated bile acid αMCA, and TG were observed (all P < 0.05). In conclusion, swertianlarin significantly up-regulates hepatic bile acid detoxification enzymes and efflux transporters in rats, which can increase the water solubility of hydrophobic bile acids and elimination of conjugated bile acids. PMID:25755705

  1. A proposed role for efflux transporters in the pathogenesis of hydrocephalus

    PubMed Central

    Krishnamurthy, Satish; Tichenor, Michael D.; Satish, Akhila G.; Lehmann, David B.

    2014-01-01

    Hydrocephalus is a common brain disorder that is treated only with surgery. The basis for surgical treatment rests on the circulation theory. However, clinical and experimental data to substantiate circulation theory have remained inconclusive. In brain tissue and in the ventricles, we see that osmotic gradients drive water diffusion in water-permeable tissue. As the osmolarity of ventricular CSF increases within the cerebral ventricles, water movement into the ventricles increases and causes hydrocephalus. Macromolecular clearance from the ventricles is a mechanism to establish the normal CSF osmolarity, and therefore ventricular volume. Efflux transporters, (p-glycoprotein), are located along the blood brain barrier and play an important role in the clearance of macromolecules (endobiotics and xenobiotics) from the brain to the blood. There is clinical and experimental data to show that macromolecules are cleared out of the brain in normal and hydrocephalic brains. This article summarizes the existing evidence to support the role of efflux transporters in the pathogenesis of hydrocephalus. The location of p-gp along the pathways of macromolecular clearance and the broad substrate specificity of this abundant transporter to a variety of different macromolecules are reviewed. Involvement of p-gp in the transport of amyloid beta in Alzheimer disease and its relation to normal pressure hydrocephalus is reviewed. Finally, individual variability of p-gp expression might explain the variability in the development of hydrocephalus following intraventricular hemorrhage. PMID:25165050

  2. A proposed role for efflux transporters in the pathogenesis of hydrocephalus.

    PubMed

    Krishnamurthy, Satish; Tichenor, Michael D; Satish, Akhila G; Lehmann, David B

    2014-08-28

    Hydrocephalus is a common brain disorder that is treated only with surgery. The basis for surgical treatment rests on the circulation theory. However, clinical and experimental data to substantiate circulation theory have remained inconclusive. In brain tissue and in the ventricles, we see that osmotic gradients drive water diffusion in water-permeable tissue. As the osmolarity of ventricular CSF increases within the cerebral ventricles, water movement into the ventricles increases and causes hydrocephalus. Macromolecular clearance from the ventricles is a mechanism to establish the normal CSF osmolarity, and therefore ventricular volume. Efflux transporters, (p-glycoprotein), are located along the blood brain barrier and play an important role in the clearance of macromolecules (endobiotics and xenobiotics) from the brain to the blood. There is clinical and experimental data to show that macromolecules are cleared out of the brain in normal and hydrocephalic brains. This article summarizes the existing evidence to support the role of efflux transporters in the pathogenesis of hydrocephalus. The location of p-gp along the pathways of macromolecular clearance and the broad substrate specificity of this abundant transporter to a variety of different macromolecules are reviewed. Involvement of p-gp in the transport of amyloid beta in Alzheimer disease and its relation to normal pressure hydrocephalus is reviewed. Finally, individual variability of p-gp expression might explain the variability in the development of hydrocephalus following intraventricular hemorrhage. PMID:25165050

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

    PubMed Central

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

    2016-01-01

    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. PMID:26916090

  4. 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.

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

    PubMed

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

    2016-04-15

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

  6. 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

  7. Stem girdling affects the quantity of CO2 transported in xylem as well as CO2 efflux from soil.

    PubMed

    Bloemen, Jasper; Agneessens, Laura; Van Meulebroek, Lieven; Aubrey, Doug P; McGuire, Mary Anne; Teskey, Robert O; Steppe, Kathy

    2014-02-01

    There is recent clear evidence that an important fraction of root-respired CO2 is transported upward in the transpiration stream in tree stems rather than fluxing to the soil. In this study, we aimed to quantify the contribution of root-respired CO2 to both soil CO2 efflux and xylem CO2 transport by manipulating the autotrophic component of belowground respiration. We compared soil CO2 efflux and the flux of root-respired CO2 transported in the transpiration stream in girdled and nongirdled 9-yr-old oak trees (Quercus robur) to assess the impact of a change in the autotrophic component of belowground respiration on both CO2 fluxes. Stem girdling decreased xylem CO2 concentration, indicating that belowground respiration contributes to the aboveground transport of internal CO2 . Girdling also decreased soil CO2 efflux. These results confirmed that root respiration contributes to xylem CO2 transport and that failure to account for this flux results in inaccurate estimates of belowground respiration when efflux-based methods are used. This research adds to the growing body of evidence that efflux-based measurements of belowground respiration underestimate autotrophic contributions.

  8. 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.

  9. AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

    PubMed Central

    Nakashima, Ryosuke; Sakurai, Keisuke; Kitagawa, Kimie; Yamasaki, Seiji; Nishino, Kunihiko

    2015-01-01

    ABSTRACT The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCE The structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for

  10. Substrate binding accelerates the conformational transitions and substrate dissociation in multidrug efflux transporter AcrB

    PubMed Central

    Wang, Beibei; Weng, Jingwei; Wang, Wenning

    2015-01-01

    The tripartite efflux pump assembly AcrAB-TolC is the major multidrug resistance transporter in E. coli. The inner membrane transporter AcrB is a homotrimer, energized by the proton movement down the transmembrane electrochemical gradient. The asymmetric crystal structures of AcrB with three monomers in distinct conformational states [access (A), binding (B) and extrusion (E)] support a functional rotating mechanism, in which each monomer of AcrB cycles among the three states in a concerted way. However, the relationship between the conformational changes during functional rotation and drug translocation has not been totally understood. Here, we explored the conformational changes of the AcrB homotrimer during the ABE to BEA transition in different substrate-binding states using targeted MD simulations. It was found that the dissociation of substrate from the distal binding pocket of B monomer is closely related to the concerted conformational changes in the translocation pathway, especially the side chain reorientation of Phe628 and Tyr327. A second substrate binding at the proximal binding pocket of A monomer evidently accelerates the conformational transitions as well as substrate dissociation in B monomer. The acceleration effect of the multi-substrate binding mode provides a molecular explanation for the positive cooperativity observed in the kinetic studies of substrate efflux and deepens our understanding of the functional rotating mechanism of AcrB. PMID:25918513

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

    PubMed

    Nagarajan, Yagnesh; Rongala, Jay; Luang, Sukanya; Singh, Abhishek; Shadiac, Nadim; Hayes, Julie; Sutton, Tim; Gilliham, Matthew; Tyerman, Stephen D; McPhee, Gordon; Voelcker, Nicolas H; Mertens, Haydyn D T; Kirby, Nigel M; Lee, Jung-Goo; Yingling, Yaroslava G; Hrmova, Maria

    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

  12. 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

  13. 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

  14. The importance of active efflux systems in the quinolone resistance of clinical isolates of Salmonella spp.

    PubMed

    Escribano, Isabel; Rodríguez, Juan Carlos; Cebrian, Laura; Royo, Gloria

    2004-11-01

    The aim of this study was to determine the importance of the active elimination of antibiotics by active efflux systems, in the decrease in fluoroquinolone sensitivity of clinical isolates of Salmonella spp. as well as the intrinsic antibiotic activity of certain active efflux system inhibitors. The effect of the active efflux system on the decrease in sensitivity to nalidixic acid, ciprofloxacin, ofloxacin and sparfloxacin was studied by investigating the variation in the in vitro activity of these compounds when assayed in association with reserpine and MC 207.110. The active efflux systems inhibited by reserpine displayed low activity in the elimination of these compounds, whereas those inhibited by MC 207.110 showed high activity in the elimination of nalidixic acid and sparfloxacin, but were less effective in the elimination of ofloxacin and ciprofloxacin. These two compounds did not exhibit intrinsic inhibitory activity against Salmonella spp. at the concentrations assayed. These mechanisms of resistance to antibiotics are complex and vary depending on the chemical composition of the antibiotics used, and perhaps the inhibitors of these systems, although they do not exhibit any intrinsic antibiotic activity, may be used as adjuvants to increase the activity of certain antibiotics. These mechanisms complement the mutations in the gyrA gene and this supports the thesis that it is necessary to lower the breakpoint established by the NCCLS for ciprofloxacin, since the strains studied have resistance mechanisms that reduce the activity of this drug and may favour the emergence of resistant mutants during treatment.

  15. Raltegravir permeability across blood-tissue barriers and the potential role of drug efflux transporters.

    PubMed

    Hoque, M Tozammel; Kis, Olena; De Rosa, María F; Bendayan, Reina

    2015-05-01

    The objectives of this study were to investigate raltegravir transport across several blood-tissue barrier models and the potential interactions with drug efflux transporters. Raltegravir uptake, accumulation, and permeability were evaluated in vitro in (i) P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), multidrug resistance-associated protein 1 (MRP1), or MRP4-overexpressing MDA-MDR1 (P-gp), HEK-ABCG2, HeLa-MRP1, or HEK-MRP4 cells, respectively; (ii) cell culture systems of the human blood-brain (hCMEC/D3), mouse blood-testicular (TM4), and human blood-intestinal (Caco-2) barriers; and (iii) rat jejunum and ileum segments using an in situ single-pass intestinal perfusion model. [(3)H]Raltegravir accumulation by MDA-MDR1 (P-gp) and HEK-ABCG2-overexpressing cells was significantly enhanced in the presence of PSC833 {6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]-7-L-valine-cyclosporine}, a P-gp inhibitor, or Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester], a BCRP inhibitor, suggesting the inhibition of a P-gp- or BCRP-mediated efflux process, respectively. Furthermore, [(3)H]raltegravir accumulation by human cerebral microvessel endothelial hCMEC/D3 and mouse Sertoli TM4 cells was significantly increased by PSC833 and Ko143. In human intestinal Caco-2 cells grown on Transwell filters, PSC833, but not Ko143, significantly decreased the [(3)H]raltegravir efflux ratios. In rat intestinal segments, [(3)H]raltegravir in situ permeability was significantly enhanced by the concurrent administration of PSC833 and Ko143. In contrast, in the transporter inhibition assays, raltegravir (10 to 500 μM) did not increase the accumulation of substrate for P-gp (rhodamine-6G), BCRP ([(3)H]mitoxantrone), or MRP1 [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)] by MDA-MDR1 (P-gp)-, HEK-ABCG2-, or HeLa-MRP1-overexpressing

  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. The quorum-sensing molecule farnesol is a modulator of drug efflux mediated by ABC multidrug transporters and synergizes with drugs in Candida albicans.

    PubMed

    Sharma, Monika; Prasad, Rajendra

    2011-10-01

    Overexpression of the CaCDR1-encoded multidrug efflux pump protein CaCdr1p (Candida drug resistance protein 1), belonging to the ATP binding cassette (ABC) superfamily of transporters, is one of the most prominent contributors of multidrug resistance (MDR) in Candida albicans. Thus, blocking or modulating the function of the drug efflux pumps represents an attractive approach in combating MDR. In the present study, we provide first evidence that the quorum-sensing molecule farnesol (FAR) is a specific modulator of efflux mediated by ABC multidrug transporters, such as CaCdr1p and CaCdr2p of C. albicans and ScPdr5p of Saccharomyces cerevisiae. Interestingly, FAR did not modulate the efflux mediated by the multidrug extrusion pump protein CaMdr1p, belonging to the major facilitator superfamily (MFS). Kinetic data revealed that FAR competitively inhibited rhodamine 6G efflux in CaCdr1p-overexpressing cells, with a simultaneous increase in an apparent K(m) without affecting the V(max) values and the ATPase activity. We also observed that when used in combination, FAR at a nontoxic concentration synergized with the drugs at their respective nonlethal concentrations, as was evident from their <0.5 fractional inhibitory concentration index (FICI) values and from the drop of 14- to 64-fold in the MIC(80) values in the wild-type strain and in azole-resistant clinical isolates of C. albicans. Our biochemical experiments revealed that the synergistic interaction of FAR with the drugs led to reactive oxygen species accumulation, which triggered early apoptosis, and that both could be partly reversed by the addition of an antioxidant. Collectively, FAR modulates drug extrusion mediated exclusively by ABC proteins and is synergistic to fluconazole (FLC), ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB). PMID:21768514

  18. Prominent Expression of Xenobiotic Efflux Transporters in Mouse Extraembryonic Fetal Membranes Compared to Placenta

    PubMed Central

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

    2008-01-01

    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 (Mdr), multidrug and toxin extrusion proteins (Mate), 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, 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) compared to placenta. Western blot 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. PMID:18566041

  19. Ferroportin and exocytoplasmic ferroxidase activity are required for brain microvascular endothelial cell iron efflux.

    PubMed

    McCarthy, Ryan C; Kosman, Daniel J

    2013-06-14

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

  20. The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis.

    PubMed

    Drdová, Edita Janková; Synek, Lukáš; Pečenková, Tamara; Hála, Michal; Kulich, Ivan; Fowler, John E; Murphy, Angus S; Zárský, Viktor

    2013-03-01

    In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.

  1. An N-Terminal Threonine Mutation Produces an Efflux-Favorable, Sodium-Primed Conformation of the Human Dopamine Transporter

    PubMed Central

    Fraser, Rheaclare; Chen, Yongyue; Guptaroy, Bipasha; Luderman, Kathryn D.; Stokes, Stephanie L.; Beg, Asim; DeFelice, Louis J.

    2014-01-01

    The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [3H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants’ altered function with respect to membrane conductance and Na+ sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP+ [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na+e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na+e stimulation of [3H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na+]e stimulated [3H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [3H]DA efflux in T62D-hDAT cells was independent of Na+e and commensurate with [3H]DA efflux attained in WT-hDAT cells, either by removal of Na+e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na+-primed orientation—possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT. PMID:24753048

  2. LXR/RXR ligand activation enhances basolateral efflux of beta-sitosterol in CaCo-2 cells.

    PubMed

    Field, F Jeffrey; Born, Ella; Mathur, Satya N

    2004-05-01

    To examine whether intestinal ABCA1 was responsible for the differences observed between cholesterol and beta-sitosterol absorption, ABCA1-facilitated beta-sitosterol efflux was investigated in CaCo-2 cells following liver X receptor/retinoid X receptor (LXR/RXR) activation. Both the LXR agonist T0901317 and the natural RXR/LXR agonists 22-hydroxycholesterol and 9-cis retinoic acid enhanced the basolateral efflux of beta-sitosterol without altering apical efflux. LXR-mediated enhanced beta-sitosterol efflux occurred between 6 h and 12 h after activation, suggesting that transcription, protein synthesis, and trafficking was likely necessary prior to facilitating efflux. The transcription inhibitor actinomycin D prevented the increase in beta-sitosterol efflux by T0901317. Glybenclamide, an inhibitor of ABCA1 activity, and arachidonic acid, a fatty acid that interferes with LXR activation, also prevented beta-sitosterol efflux in response to the LXR ligand activation. Influx of beta-sitosterol mass did not alter the basolateral or apical efflux of the plant sterol, nor did it alter ABCA1, ABCG1, ABCG5, or ABCG8 gene expression or ABCA1 mass. Similar to results observed with intestinal ABCA1-facilitated cholesterol efflux, LXR/RXR ligand activation enhanced the basolateral efflux of beta-sitosterol without affecting apical efflux. The results suggest that ABCA1 does not differentiate between cholesterol and beta-sitosterol and thus is not responsible for the selectivity of sterol absorption by the intestine. ABCA1, however, may play a role in beta-sitosterol absorption.

  3. 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

  4. Hydrophobic amino acids in the hinge region of the 5A apolipoprotein mimetic peptide are essential for promoting cholesterol efflux by the ABCA1 transporter.

    PubMed

    Sviridov, Denis O; Andrianov, Alexander M; Anishchenko, Ivan V; Stonik, John A; Amar, Marcelo J A; Turner, Scott; Remaley, Alan T

    2013-01-01

    The bihelical apolipoprotein mimetic peptide 5A effluxes cholesterol from cells and reduces inflammation and atherosclerosis in animal models. We investigated how hydrophobic residues in the hinge region between the two helices are important in the structure and function of this peptide. By simulated annealing analysis and molecular dynamics modeling, two hydrophobic amino acids, F-18 and W-21, in the hinge region were predicted to be relatively surface-exposed and to interact with the aqueous solvent. Using a series of 5A peptide analogs in which F-18 or W-21 was changed to either F, W, A, or E, only peptides with hydrophobic amino acids in these two positions were able to readily bind and solubilize phospholipid vesicles. Compared with active peptides containing F or W, peptides containing E in either of these two positions were more than 10-fold less effective in effluxing cholesterol by the ABCA1 transporter. Intravenous injection of 5A in C57BL/6 mice increased plasma-free cholesterol (5A: 89.9 ± 13.6 mg/dl; control: 38.7 ± 4.3 mg/dl (mean ± S.D.); P < 0.05) and triglycerides (5A: 887.0 ± 172.0 mg/dl; control: 108.9 ± 9.9 mg/dl; P < 0.05), whereas the EE peptide containing E in both positions had no effect. Finally, 5A increased cholesterol efflux approximately 2.5-fold in vivo from radiolabeled macrophages, whereas the EE peptide was inactive. These results provide a rationale for future design of therapeutic apolipoprotein mimetic peptides and provide new insights into the interaction of hydrophobic residues on apolipoproteins with phospholipids in the lipid microdomain created by the ABCA1 transporter during the cholesterol efflux process.

  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. 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.

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

    PubMed

    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

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

    PubMed Central

    Chen, Rujin; Hilson, Pierre; Sedbrook, John; Rosen, Elizabeth; Caspar, Timothy; Masson, Patrick 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. PMID:9844024

  9. Celecoxib Up Regulates the Expression of Drug Efflux Transporter ABCG2 in Breast Cancer Cell Lines

    PubMed Central

    Kalalinia, Fatemeh; Elahian, Fatemeh; Mosaffa, Fatemeh; Behravan, Javad

    2014-01-01

    Elevated expression of the drug efflux transporter ABCG2 seems to correlate with multidrug resistance of cancer cells. Specific COX-2 inhibitor celecoxib has been shown to enhance the sensitivity of cancer cells to anticancer drugs. To clarify whether ABCG2 inhibition is involved in the sensitizing effect of celecoxib, we investigated whether the expression of ABCG2 in breast cancer cell lines, could be modulated by celecoxib. The expression of the multidrug resistant gene (ABCG2) at mRNA and protein level was detected by real-time quantitative reverse transcription-polymerase chain reaction and flow cytometry analysis, respectively. Among three human breast cancer cell lines ABCG2 and COX-2 were highly expressed in MCF7-MX and MDA-MB-231 cells, respectively. The COX-2 inhibitor celecoxib up-regulated the expression of ABCG2 mRNA in MCF-7 and MCF7-MX cells, which was accompanied by increased ABCG2 protein expression. While celecoxib was able to block the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated increase in COX-2 expression in MDA-MB-231 cells, it increased the expression of ABCG2 up to 4.27 times to the control level at mRNA level and with less intensity at protein level. Our findings provide evidence that celecoxib up-regulates ABCG2 expression in human breast cancer cells and proposed that ABCG2 is not involved in chemosensitizing effects of celecoxib. PMID:25587329

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

    PubMed Central

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

    2016-01-01

    Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases1. To date, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-182,3. The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases4,5 as well as Cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations6,7. Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by multiple stimuli8,9. Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. We report here the identification of Nek7, a member of the family of mammalian NIMA-related kinases (Neks)10, as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of Nek7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasome. NLRP3-activating stimuli promoted the NLRP3-Nek7 interaction in a process dependent on potassium efflux. NLRP3 associated with the catalytic domain of Nek7, but the catalytic activity of Nek7 was dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3-Nek7 complex, which along with ASC oligomerization and ASC speck formation were abrogated in the absence of Nek7. Nek7 was required for macrophages harboring the CAPS-associated NLRP3R258W activating mutation to activate caspase-1. Mouse chimeras reconstituted with wild-type, Nek7−/− or Nlrp3−/− hematopoietic cells revealed that Nek7 was required for NLRP3 inflammasome activation in vivo. These studies demonstrate that Nek7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome

  11. 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

  12. Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters

    PubMed Central

    Uto-Kondo, Harumi; Ayaori, Makoto; Nakaya, Kazuhiro; Takiguchi, Shunichi; Yakushiji, Emi; Ogura, Masatsune; Terao, Yoshio; Ozasa, Hideki; Sasaki, Makoto; Komatsu, Tomohiro; Sotherden, Grace Megumi; Hosoai, Tamaki; Sakurada, Masami; Ikewaki, Katsunori

    2014-01-01

    Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30–49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL. PMID:25120277

  13. 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.

  14. Quantitative investigation of the brain-to-cerebrospinal fluid unbound drug concentration ratio under steady-state conditions in rats using a pharmacokinetic model and scaling factors for active efflux transporters.

    PubMed

    Kodaira, Hiroshi; Kusuhara, Hiroyuki; Fuse, Eiichi; Ushiki, Junko; Sugiyama, Yuichi

    2014-06-01

    A pharmacokinetic model was constructed to explain the difference in brain- and cerebrospinal fluid (CSF)-to-plasma and brain-to-CSF unbound drug concentration ratios (Kp,uu,brain, Kp,uu,CSF, and Kp,uu,CSF/brain, respectively) of drugs under steady-state conditions in rats. The passive permeability across the blood-brain barrier (BBB), PS1, was predicted by two methods using log(D/molecular weight(0.5)) for PS1(1) or the partition coefficient in octanol/water at pH 7.4 (LogD), topologic van der Waals polar surface area, and van der Waals surface area of the basic atoms for PS1(2). The coefficients of each parameter were determined using previously reported in situ rat BBB permeability. Active transport of drugs by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) measured in P-gp- and Bcrp-overexpressing cells was extrapolated to in vivo by introducing scaling factors. Brain- and CSF-to-plasma unbound concentration ratios (Kp,uu,brain and Kp,uu,CSF, respectively) of 19 compounds, including P-gp and Bcrp substrates (daidzein, dantrolene, flavopiridol, genistein, loperamide, quinidine, and verapamil), were simultaneously fitted to the equations in a three-compartment model comprising blood, brain, and CSF compartments. The calculated Kp,uu,brain and Kp,uu,CSF of 17 compounds were within a factor of three of experimental values. Kp,uu,CSF values of genistein and loperamide were outliers of the prediction, and Kp,uu,brain of dantrolene also became an outlier when PS1(2) was used. Kp,uu,CSF/brain of the 19 compounds was within a factor of three of experimental values. In conclusion, the Kp,uu,CSF/brain of drugs, including P-gp and Bcrp substrates, could be successfully explained by a kinetic model using scaling factors combined with in vitro evaluation of P-gp and Bcrp activities. PMID:24644297

  15. Facilitated transporters mediate net efflux of amino acids to the fetus across the basal membrane of the placental syncytiotrophoblast.

    PubMed

    Cleal, J K; Glazier, J D; Ntani, G; Crozier, S R; Day, P E; Harvey, N C; Robinson, S M; Cooper, C; Godfrey, K M; Hanson, M A; Lewis, R M

    2011-02-15

    Fetal growth depends on placental transfer of amino acids from maternal to fetal blood. The mechanisms of net amino acid efflux across the basal membrane (BM) of the placental syncytiotrophoblast to the fetus, although vital for amino acid transport, are poorly understood. We examined the hypothesis that facilitated diffusion by the amino acid transporters TAT1, LAT3 and LAT4 plays an important role in this process, with possible effects on fetal growth. Amino acid transfer was measured in isolated perfused human placental cotyledons (n = 5 per experiment) using techniques which distinguish between different transport processes. Placental TAT1, LAT3 and LAT4 proteins were measured, and mRNA expression levels (measured using real-time quantitative-PCR) were related to fetal and neonatal anthropometry and dual-energy X-ray absorptiometry measurements of neonatal lean mass in 102 Southampton Women's Survey (SWS) infants. Under conditions preventing transport by amino acid exchangers, all amino acids appearing in the fetal circulation were substrates of TAT1, LAT3 or LAT4. Western blots demonstrated the presence of TAT1, LAT3 and LAT4 in placental BM preparations. Placental TAT1 and LAT3 mRNA expression were positively associated with measures of fetal growth in SWS infants (P < 0.05). We provide evidence that the efflux transporters TAT1, LAT3 and LAT4 are present in the human placental BM, and may play an important role in the net efflux of amino acids to the fetus. Unlike other transporters they can increase fetal amino acid concentrations. Consistent with a role in placental amino acid transfer capacity and fetal growth TAT1 and LAT3 mRNA expression showed positive associations with infant size at birth.

  16. Pharmacokinetic simulations to explore dissolution criteria of BCS I and III biowaivers with and without MDR-1 efflux transporter.

    PubMed

    Kortejärvi, H; Malkki, J; Shawahna, R; Scherrmann, J-M; Urtti, A; Yliperttula, M

    2014-09-30

    In this study, a pharmacokinetic simulation model was used to explore the dissolution acceptance criteria for BCS I and III biowaivers and to examine the risk of MDR-1 efflux transporter on bioequivalence of substrates. The compartmental absorption and transit (CAT) model with one- or two systemic compartments was used. The parameter values used in the simulations were based on the pharmacokinetics of existing 70 BCS I and III drugs. Based on the simulations BCS I drug products with Tmax of >0.9 h, both dissolution criteria "very rapid" and "rapid and similar" were acceptable. For rapidly absorbed and distributed BCS I drug products with Tmax of 0.6-0.9 h, the dissolution criterion "very rapid" is preferred. If Tmax is less than 0.6 h there is a risk of bioinequivalence for the BCS I drug products regardless of the dissolution criteria. Based on the simulations, all BCS III drug products were good biowaiver candidates with both dissolution criteria. Almost all the BCS III drug products (>89%) and many BCS I products (9-57%) showed risks of bioinequivalence, if an excipient in either product inhibits MDR1-efflux transport of the drug. To eliminate these risks excipients with prior use in bioequivalent products should be used for MDR-1 efflux substrates.

  17. The ABCG2 Efflux Transporter in the Mammary Gland Mediates Veterinary Drug Secretion across the Blood-Milk Barrier into Milk of Dairy Cows.

    PubMed

    Mahnke, Hanna; Ballent, Mariana; Baumann, Sven; Imperiale, Fernanda; von Bergen, Martin; Lanusse, Carlos; Lifschitz, Adrian L; Honscha, Walther; Halwachs, Sandra

    2016-05-01

    In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2 excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2 transport across polarized Madin-Darby canine kidney II cells-bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2(B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk. PMID:26956640

  18. Ciprofloxacin Is Actively Transported across Bronchial Lung Epithelial Cells Using a Calu-3 Air Interface Cell Model

    PubMed Central

    Ong, Hui Xin; Traini, Daniela; Bebawy, Mary

    2013-01-01

    Ciprofloxacin is a well-established broad-spectrum fluoroquinolone antibiotic that penetrates well into the lung tissues; still, the mechanisms of its transepithelial transport are unknown. The contributions of specific transporters, including multidrug efflux transporters, organic cation transporters, and organic anion-transporting polypeptide transporters, to the uptake of ciprofloxacin were investigated in vitro using an air interface bronchial epithelial model. Our results demonstrate that ciprofloxacin is subject to predominantly active influx and a slight efflux component. PMID:23507281

  19. Bioluminescent imaging of drug efflux at the blood-brain barrier mediated by the transporter ABCG2.

    PubMed

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

    2013-12-17

    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.

  20. PK11195, a peripheral benzodiazepine receptor (pBR) ligand, broadly blocks drug efflux to chemosensitize leukemia and myeloma cells by a pBR-independent, direct transporter-modulating mechanism.

    PubMed

    Walter, Roland B; Pirga, Jason L; Cronk, Michelle R; Mayer, Sasha; Appelbaum, Frederick R; Banker, Deborah E

    2005-11-15

    The peripheral benzodiazepine receptor (pBR) ligand, PK11195, promotes mitochondrial apoptosis and blocks P-glycoprotein (Pgp)-mediated drug efflux to chemosensitize cancer cells at least as well or better than the Pgp modulator, cyclosporine A (CSA). We now show that PK11195 broadly inhibits adenosine triphosphate (ATP)-binding cassette (ABC) transporters in hematologic cancer cell lines and primary leukemia-cell samples, including multidrug resistance protein (MRP), breast cancer resistance protein (BCRP), and/or Pgp. Ectopic expression models confirmed that pBR can directly mediate chemosensitizing by PK11195, presumably via mitochondrial activities, but showed that pBR expression is unnecessary to PK11195-mediated efflux inhibition. PK11195 binds plasma-membrane sites in Pgp-expressing cells, stimulates Pgp-associated adenosine triphosphatase (ATPase) activity, and causes conformational changes in Pgp, suggesting that PK11195 modulates Pgp-mediated efflux by direct transporter interaction(s). PK11195 and CSA bind noncompetitively in Pgp-expressing cells, indicating that PK11195 interacts with Pgp at sites that are distinct from CSA-binding sites. Importantly, PK11195 concentrations that were effective in these in vitro assays can be safely achieved in patients. Because PK11195 promotes chemotherapy-induced apoptosis by a pBR-dependent mitochondrial mechanism and broadly blocks drug efflux by an apparently pBR-independent, ABC transporter-dependent mechanism, PK11195 may be a useful clinical chemosensitizer in cancer patients.

  1. Rice SPX-Major Facility Superfamily3, a Vacuolar Phosphate Efflux Transporter, Is Involved in Maintaining Phosphate Homeostasis in Rice.

    PubMed

    Wang, Chuang; Yue, Wenhao; Ying, Yinghui; Wang, Shoudong; Secco, David; Liu, Yu; Whelan, James; Tyerman, Stephen D; Shou, Huixia

    2015-12-01

    To maintain a stable cytosol phosphate (Pi) concentration, plant cells store Pi in their vacuoles. When the Pi concentration in the cytosol decreases, Pi is exported from the vacuole into the cytosol. This export is mediated by Pi transporters on the tonoplast. In this study, we demonstrate that SYG1, PHO81, and XPR1 (SPX)-Major Facility Superfamily (MFS) proteins have a similar structure with yeast (Saccharomyces cerevisiae) low-affinity Pi transporters Phosphatase87 (PHO87), PHO90, and PHO91. OsSPX-MFS1, OsSPX-MFS2, and OsSPX-MFS3 all localized on the tonoplast of rice (Oryza sativa) protoplasts, even in the absence of the SPX domain. At high external Pi concentration, OsSPX-MFS3 could partially complement the yeast mutant strain EY917 under pH 5.5, which lacks all five Pi transporters present in yeast. In oocytes, OsSPX-MFS3 was shown to facilitate Pi influx or efflux depending on the external pH and Pi concentrations. In contrast to tonoplast localization in plants cells, OsSPX-MFS3 was localized to the plasma membrane when expressed in both yeast and oocytes. Overexpression of OsSPX-MFS3 results in decreased Pi concentration in the vacuole of rice tissues. We conclude that OsSPX-MFS3 is a low-affinity Pi transporter that mediates Pi efflux from the vacuole into cytosol and is coupled to proton movement. PMID:26424157

  2. Human intestinal transporter database: QSAR modeling and virtual profiling of drug uptake, efflux and interactions

    PubMed Central

    Sedykh, Alexander; Fourches, Denis; Duan, Jianmin; Hucke, Oliver; Garneau, Michel; Zhu, Hao; Bonneau, Pierre; Tropsha, Alexander

    2013-01-01

    Purpose Membrane transporters mediate many biological effects of chemicals and play a major role in pharmacokinetics and drug resistance. The selection of viable drug candidates among biologically active compounds requires the assessment of their transporter interaction profiles. Methods Using public sources, we have assembled and curated the largest, to our knowledge, human intestinal transporter database (>5,000 interaction entries for >3,700 molecules). This data was used to develop thoroughly validated classification Quantitative Structure-Activity Relationship (QSAR) models of transport and/or inhibition of several major transporters including MDR1, BCRP, MRP1-4, PEPT1, ASBT, OATP2B1, OCT1, and MCT1. Results & Conclusions QSAR models have been developed with advanced machine learning techniques such as Support Vector Machines, Random Forest, and k Nearest Neighbors using Dragon and MOE chemical descriptors. These models afforded high external prediction accuracies of 71–100% estimated by 5-fold external validation, and showed hit retrieval rates with up to 20-fold enrichment in the virtual screening of DrugBank compounds. The compendium of predictive QSAR models developed in this study can be used for virtual profiling of drug candidates and/or environmental agents with the optimal transporter profiles. PMID:23269503

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  5. Enhancement of antibiotic activity against multidrug-resistant bacteria by the efflux pump inhibitor 3,4-dibromopyrrole-2,5-dione isolated from a Pseudoalteromonas sp.

    PubMed

    Whalen, Kristen E; Poulson-Ellestad, Kelsey L; Deering, Robert W; Rowley, David C; Mincer, Tracy J

    2015-03-27

    Members of the resistance nodulation cell division (RND) of efflux pumps play essential roles in multidrug resistance (MDR) in Gram-negative bacteria. Here, we describe the search for new small molecules from marine microbial extracts to block efflux and thus restore antibiotic susceptibility in MDR bacterial strains. We report the isolation of 3,4-dibromopyrrole-2,5-dione (1), an inhibitor of RND transporters, from Enterobacteriaceae and Pseudomonas aeruginosa, from the marine bacterium Pseudoalteromonas piscicida. 3,4-Dibromopyrrole-2,5-dione decreased the minimum inhibitory concentrations (MICs) of two fluoroquinolones, an aminoglycoside, a macrolide, a beta-lactam, tetracycline, and chloramphenicol between 2- and 16-fold in strains overexpressing three archetype RND transporters (AcrAB-TolC, MexAB-OprM, and MexXY-OprM). 3,4-Dibromopyrrole-2,5-dione also increased the intracellular accumulation of Hoechst 33342 in wild-type but not in transporter-deficient strains and prevented H33342 efflux (IC50 = 0.79 μg/mL or 3 μM), a hallmark of efflux pump inhibitor (EPI) functionality. A metabolomic survey of 36 Pseudoalteromonas isolates mapped the presence of primarily brominated metabolites only within the P. piscicida phylogenetic clade, where a majority of antibiotic activity was also observed, suggesting a link between halogenation and enhanced secondary metabolite biosynthetic potential. In sum, 3,4-dibromopyrrole-2,5-dione is a potent EPI and deserves further attention as an adjuvant to enhance the effectiveness of existing antibiotics. PMID:25646964

  6. 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.

  7. Fungicide efflux and the MgMFS1 transporter contribute to the multidrug resistance phenotype in Zymoseptoria tritici field isolates.

    PubMed

    Omrane, Selim; Sghyer, Hind; Audéon, Colette; Lanen, Catherine; Duplaix, Clémentine; Walker, Anne-Sophie; Fillinger, Sabine

    2015-08-01

    Septoria leaf blotch is mainly controlled by fungicides. Zymoseptoria tritici, which is responsible for this disease, displays strong adaptive capacity to fungicide challenge. It developed resistance to most fungicides due to target site modifications. Recently, isolated strains showed cross-resistance to fungicides with unrelated modes of action, suggesting a resistance mechanism known as multidrug resistance (MDR). We show enhanced prochloraz efflux, sensitive to the modulators amitryptiline and chlorpromazine, for two Z. tritici strains, displaying an MDR phenotype in addition to the genotypes CYP51(I381V Y461H) or CYP51(I381V ΔY459/) (G460) , respectively, hereafter named MDR6 and MDR7. Efflux was also inhibited by verapamil in the MDR7 strain. RNA sequencing lead to the identification of several transporter genes overexpressed in both MDR strains. The expression of the MgMFS1 gene was the strongest and constitutively high in MDR field strains. Its inactivation in the MDR6 strain abolished resistance to fungicides with different modes of action supporting its involvement in MDR in Z. tritici. A 519 bp insert in the MgMFS1 promoter was detected in half of the tested MDR field strains, but absent from sensitive field strains, suggesting that the insert is correlated with the observed MDR phenotype. Besides MgMfs1, other transporters and mutations may be involved in MDR in Z. tritici.

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

    PubMed

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

    2015-02-01

    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.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    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

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

    SciTech Connect

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

    1990-05-01

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

  13. Ofloxacin resistance in Mycobacterium tuberculosis is associated with efflux pump activity independent of resistance pattern and genotype.

    PubMed

    Sun, Zhaogang; Xu, Yuhui; Sun, Yong; Liu, Yi; Zhang, Xuxia; Huang, Hairong; Li, Chuanyou

    2014-12-01

    Drug-resistance to ofloxacin (OFX) in Mycobacterium tuberculosis is due to missense mutations in gyrA and other factors, such as alterations in the activity of drug efflux pumps. In this study, we identified 8 extensively drug resistant tuberculosis (XDR-TB), 40 multidrug resistant TB (MDR-TB), 38 polydrug resistant TB (PDR-TB), and 16 single OFX-resistant TB from 102 clinical isolates. We tested the effect of three efflux inhibitors, reserpine, verapamil, and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), on changes in the OFX minimum inhibitory concentration (MIC) using Resazurin microtitre assay. These three inhibitors changed the MICs from 2- to 32-fold, with CCCP having the strongest effect. A total of 55%, 74%, and 83% of the tested isolates had changes in MIC of more than two-fold by reserpine, verapamil, and CCCP, respectively. The inhibitors led to similar fold-changes of OFX MICs in the XDR, MDR, PDR, and single OFX-resistant isolates. For each inhibitor, a higher resistance to OFX was associated with the greater efflux pump activity. There were no significant differences in the effect of efflux pump inhibitors upon Beijing and non-Beijing M. tuberculosis genotypes. Taken together, these results indicate that the efflux pump activity was greater in the isolates higher resistant to OFX and had similar effects on isolates with different drug resistant pattern, and had similar effects on Beijing and non-Beijing genotypes.

  14. Inhibition mechanism of P-glycoprotein mediated efflux by mPEG-PLA and influence of PLA chain length on P-glycoprotein inhibition activity.

    PubMed

    Li, Wenjing; Li, Xinru; Gao, Yajie; Zhou, Yanxia; Ma, Shujin; Zhao, Yong; Li, Jinwen; Liu, Yan; Wang, Xinglin; Yin, Dongdong

    2014-01-01

    The present study aimed to investigate the effect of monomethoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) (mPEG-PLA) on the activity of P-glycoprotein (P-gp) in Caco-2 cells and further unravel the relationship between PLA chain length in mPEG-PLA and influence on P-gp efflux and the action mechanism. The transport results of rhodamine 123 (R123) across Caco-2 cell monolayers suggested that mPEG-PLA unimers were responsible for its P-gp inhibitory effect. Furthermore, transport studies of R123 revealed that the inhibitory potential of P-gp efflux by mPEG-PLA analogues was strongly correlated with their structural features and showed that the hydrophilic mPEG-PLA copolymers with an intermediate PLA chain length and 10.20 of hydrophilic-lipophilic balance were more effective at inhibiting P-gp efflux in Caco-2 cells. The fluorescence polarization measurement results ruled out the plasma membrane fluidization as a contributor for inhibition of P-gp by mPEG-PLA. Concurrently, mPEG-PLA inhibited neither basal P-gp ATPase (ATP is adenosine triphosphate) activity nor substrate stimulated P-gp ATPase activity, suggesting that mPEG-PLA seemed not to be a substrate of P-gp and a competitive inhibitor. No evident alteration in P-gp surface level was detected by flow cytometry upon exposure of the cells to mPEG-PLA. The depletion of intracellular ATP, which was likely to be a result of partial inhibition of cellular metabolism, was directly correlated with inhibitory potential for P-gp mediated efflux by mPEG-PLA analogues. Hence, intracellular ATP-depletion appeared to be possible explanation to the inhibition mechanism of P-gp by mPEG-PLA. Taken together, the establishment of a relationship between PLA chain length and impact on P-gp efflux activity and interpretation of action mechanism of mPEG-PLA on P-gp are of fundamental importance and will facilitate future development of mPEG-PLA in the drug delivery area.

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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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 Fe2+ and manganese, not Fe3+ or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe2+ 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. PMID:26208637

  16. Multidrug efflux pump MdtBC of Escherichia coli is active only as a B2C heterotrimer.

    PubMed

    Kim, Hong-Suk; Nagore, Daniel; Nikaido, Hiroshi

    2010-03-01

    RND (resistance-nodulation-division) family transporters in Gram-negative bacteria frequently pump out a wide range of inhibitors and often contribute to multidrug resistance to antibiotics and biocides. An archetypal RND pump of Escherichia coli, AcrB, is known to exist as a homotrimer, and this construction is essential for drug pumping through the functionally rotating mechanism. MdtBC, however, appears different because two pump genes coexist within a single operon, and genetic deletion data suggest that both pumps must be expressed in order for the drug efflux to occur. We have expressed the corresponding genes, with one of them in a His-tagged form. Copurification of MdtB and MdtC under these conditions showed that they form a complex, with an average stoichiometry of 2:1. Unequivocal evidence that only the trimer containing two B protomers and one C protomer is active was obtained by expressing all possible combinations of B and C in covalently linked forms. Finally, conversion into alanine of the residues, known to form a proton translocation pathway in AcrB, inactivated transport only when made in MdtB, not when made in MdtC, a result suggesting that MdtC plays a different role not directly involved in drug binding and extrusion.

  17. 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 Central

    Zimmermann, Estevan Sonego; Laureano, João Victor; dos Santos, Camila Neris; Schmidt, Stephan; Lagishetty, Chakradhar V.; de Castro, Whocely Victor

    2015-01-01

    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

  18. Establishment of a set of double transfectants coexpressing organic anion transporting polypeptide 1B3 and hepatic efflux transporters for the characterization of the hepatobiliary transport of telmisartan acylglucuronide.

    PubMed

    Ishiguro, Naoki; Maeda, Kazuya; Saito, Asami; Kishimoto, Wataru; Matsushima, Soichiro; Ebner, Thomas; Roth, Willy; Igarashi, Takashi; Sugiyama, Yuichi

    2008-04-01

    In the hepatic uptake of organic anions, organic anion transporting polypeptide (OATP) 1B1 is believed to be mainly involved. We have constructed a set of double-transfected cells coexpressing OATP1B1 and hepatic efflux transporters and characterized the transcellular transport of several anions. Recent reports have also suggested the importance of OATP1B3 in the hepatic uptake of some compounds. However, there is little information about OATP1B3-selective substrate and no good tool for the evaluation of efflux transporters of OATP1B3 substrates. In the present study, we found an OATP1B3-selective substrate and established a novel set of double transfectants expressing OATP1B3. Telmisartan acylglucuronide (tel-glu) is a main metabolite of telmisartan, an angiotensin II receptor antagonist. Tel-glu is recognized by hepatobiliary transport systems and efficiently distributed to liver. Several studies using rat and human hepatocytes and transporter-expressing cells revealed that OATP1B3 was responsible for the hepatic uptake of tel-glu in humans. By using double transfectants expressing OATP1B3, we investigated the transcellular transport of tel-glu as well as estradiol 17beta-d-glucuronide (E(2)17betaG) and cholecystokinin octapeptide (CCK-8) to identify the responsible efflux transporters in their biliary excretion. Vectorial basal-to-apical transport of tel-glu was observed in all kinds of double transfectants expressing OATP1B3. In contrast, basal-to-apical transport of E(2)17betaG and CCK-8 was seen only in the OATP1B3/MRP2 double transfectant compared with OATP1B3-expressing cells. Therefore, the newly established set of double transfectants expressing OATP1B3 combined with OATP1B1-expressing double transfectants can be used as a powerful tool for the rapid identification of hepatic uptake and efflux transporters of organic anions.

  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. Active Efflux Influences the Potency of Quorum Sensing Inhibitors in Pseudomonas aeruginosa

    PubMed Central

    Moore, Joseph D.; Gerdt, Joseph P.; Eibergen, Nora R.; Blackwell, Helen E.

    2014-01-01

    Many bacteria regulate gene expression through a cell-cell signaling process called quorum sensing (QS). In proteobacteria, QS is largely mediated by signaling molecules known as N-acylated L-homoserine lactones (AHLs) and their associated intracellular LuxR-type receptors. The design of non-native small molecules capable of inhibiting LuxR-type receptors, and thereby QS, in proteobacteria is an active area of research, and numerous lead compounds are AHL derivatives that mimic native AHL signals. Much of this past work has focused on the pathogen Pseudomonas aeruginosa, which controls an arsenal of virulence factors and biofilm formation through QS. The MexAB-OprM drug efflux pump has been shown to play a role in the secretion of the major AHL signal in P. aeruginosa, N-(3-oxododecanoyl) L-homoserine lactone. In the current study, we show that a variety of non-native AHLs and related derivatives capable of inhibiting LuxR-type receptors in P. aeruginosa display significantly higher potency in a P. aeruginosa Δ(mexAB-oprM) mutant, suggesting that MexAB-OprM also recognizes these compounds as substrates. We also demonstrate that the potency of 5,6-dimethyl-2-aminobenzimidazole, recently shown to be a QS and biofilm inhibitor in P. aeruginosa, is not affected by the presence or absence of the MexAB-OprM pump. These results have implications for the use of non-native AHLs and related derivatives as QS modulators in P. aeruginosa and other bacteria, and provide a potential design strategy for the development of new QS modulators that are resistant to active efflux. PMID:24478193

  1. A novel Na(+) -Independent alanine-serine-cysteine transporter 1 inhibitor inhibits both influx and efflux of D-Serine.

    PubMed

    Sakimura, Katsuya; Nakao, Kenji; Yoshikawa, Masato; Suzuki, Motohisa; Kimura, Haruhide

    2016-10-01

    NMDA receptor dysfunctions are hypothesized to underlie the pathophysiology of schizophrenia, and treatment with D-serine (D-Ser), an NMDA receptor coagonist, may improve the clinical symptoms of schizophrenia. Thus, upregulating the synaptic D-Ser level is a novel strategy for schizophrenia treatment. Na(+) -independent alanine-serine-cysteine transporter 1 (asc-1) is a transporter responsible for regulating the extracellular D-Ser levels in the brain. In this study, we discovered a novel asc-1 inhibitor, (+)-amino(1-(3,5-dichlorophenyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (ACPP), and assessed its pharmacological profile. ACPP inhibited the D-[(3) H]Ser uptake in human asc-1-expressing CHO cells and rat primary neurons with IC50 values of 0.72 ± 0.13 and 0.89 ± 0.30 μM, respectively. In accordance with the lower asc-1 expression levels in astrocytes, ACPP did not inhibit D-Ser uptake in rat primary astrocytes. In a microdialysis study, ACPP dose dependently decreased the extracellular D-Ser levels in the rat hippocampus under the same conditions in which the asc-1 inhibitor S-methyl-L-cysteine (SMLC) increased it. To obtain insights into this difference, we conducted a D-[(3) H]Ser efflux assay using asc-1-expressing CHO cells. ACPP inhibited D-[(3) H]Ser efflux, whereas SMLC increased it. These results suggest that ACPP is a novel inhibitor of asc-1. © 2016 Wiley Periodicals, Inc. PMID:27302861

  2. The role of PIN auxin efflux carriers in polar auxin transport and accumulation and their effect on shaping maize development.

    PubMed

    Forestan, Cristian; Varotto, Serena

    2012-07-01

    In plants, proper seed development and the continuing post-embryonic organogenesis both require that different cell types are correctly differentiated in response to internal and external stimuli. Among internal stimuli, plant hormones and particularly auxin and its polar transport (PAT) have been shown to regulate a multitude of plant physiological processes during vegetative and reproductive development. Although our current auxin knowledge is almost based on the results from researches on the eudicot Arabidopsis thaliana, during the last few years, many studies tried to transfer this knowledge from model to crop species, maize in particular. Applications of auxin transport inhibitors, mutant characterization, and molecular and cell biology approaches, facilitated by the sequencing of the maize genome, allowed the identification of genes involved in auxin metabolism, signaling, and particularly in polar auxin transport. PIN auxin efflux carriers have been shown to play an essential role in regulating PAT during both seed and post-embryonic development in maize. In this review, we provide a summary of the recent findings on PIN-mediated polar auxin transport during maize development. Similarities and differences between maize and Arabidopsis are analyzed and discussed, also considering that their different plant architecture depends on the differentiation of structures whose development is controlled by auxins. PMID:22186966

  3. 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...

  4. 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

  5. Tunicamycin depresses P-glycoprotein glycosylation without an effect on its membrane localization and drug efflux activity in L1210 cells.

    PubMed

    Sereš, 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.

  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 Tomato 14-3-3 protein TFT4 modulates H+ efflux, basipetal auxin transport, and the PKS5-J3 pathway in the root growth response to alkaline stress.

    PubMed

    Xu, Weifeng; Jia, Liguo; Shi, Weiming; Baluska, Frantisek; Kronzucker, Herbert J; Liang, Jiansheng; Zhang, Jianhua

    2013-12-01

    Alkaline stress is a common environmental stress, in particular in salinized soils. Plant roots respond to a variety of soil stresses by regulating their growth, but the nature of the regulatory pathways engaged in the alkaline stress response (ASR) is not yet understood. Previous studies show that PIN-FORMED2, an auxin (indole-3-acetic acid [IAA]) efflux transporter, PKS5, a protein kinase, and DNAJ HOMOLOG3 (J3), a chaperone, play key roles in root H(+) secretion by regulating plasma membrane (PM) H(+)-ATPases directly or by targeting 14-3-3 proteins. Here, we investigated the expression of all 14-3-3 gene family members (TOMATO 14-3-3 PROTEIN1 [TFT1]-TFT12) in tomato (Solanum lycopersicum) under ASR, showing the involvement of four of them, TFT1, TFT4, TFT6, and TFT7. When these genes were separately introduced into Arabidopsis (Arabidopsis thaliana) and overexpressed, only the growth of TFT4 overexpressors was significantly enhanced when compared with the wild type under stress. H(+) efflux and the activity of PM H(+)-ATPase were significantly enhanced in the root tips of TFT4 overexpressors. Microarray analysis and pharmacological examination of the overexpressor and mutant plants revealed that overexpression of TFT4 maintains primary root elongation by modulating PM H(+)-ATPase-mediated H(+) efflux and basipetal IAA transport in root tips under alkaline stress. TFT4 further plays important roles in the PKS5-J3 signaling pathway. Our study demonstrates that TFT4 acts as a regulator in the integration of H(+) efflux, basipetal IAA transport, and the PKS5-J3 pathway in the ASR of roots and coordinates root apex responses to alkaline stress for the maintenance of primary root elongation. PMID:24134886

  8. 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.

  9. Understanding the structural requirements for activators of the Kef bacterial potassium efflux system.

    PubMed

    Healy, Jessica; Ekkerman, Silvia; Pliotas, Christos; Richard, Morgiane; Bartlett, Wendy; Grayer, Samuel C; Morris, Garrett M; Miller, Samantha; Booth, Ian R; Conway, Stuart J; Rasmussen, Tim

    2014-04-01

    The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of ∼10-20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10-20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system. PMID:24601535

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Machado, Diana; Pires, David; Perdigão, João; Couto, Isabel; Portugal, Isabel; Martins, Marta; Amaral, Leonard; Anes, Elsa; Viveiros, Miguel

    2016-01-01

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

  12. 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

    2013-05-01

    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.

  13. 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

    2013-05-01

    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. PMID:23586460

  14. PI3K signaling supports amphetamine-induced dopamine efflux.

    PubMed

    Lute, Brandon J; Khoshbouei, Habibeh; Saunders, Christine; Sen, Namita; Lin, Richard Z; Javitch, Jonathan A; Galli, Aurelio

    2008-08-01

    The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efflux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efflux evoked by AMPH, since only active DAT at the plasma membrane can efflux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efflux.

  15. Functional characterization of common protein variants in the efflux transporter ABCC11 and identification of T546M as functionally damaging variant.

    PubMed

    Arlanov, R; Lang, T; Jedlitschky, G; Schaeffeler, E; Ishikawa, T; Schwab, M; Nies, A T

    2016-04-01

    Multidrug resistance protein 8 (ABCC11) is an efflux transporter for anionic lipophilic compounds, conferring resistance to antiviral and anticancer agents like 5-fluorouracil (5-FU). ABCC11 missense variants may contribute to variability in drug response but functional consequences, except for the 'earwax variant' c.538G>A, are unknown. Using the 'Screen and Insert' technology, we generated human embryonic kidney 293 cells stably expressing ABCC11 missense variants frequently occurring in different ethnic populations: c.57G>A, c.538G>A, c.950C>A, c.1637C>T, c.1942G>A, c.4032A>G. A series of in silico prediction analyses and in vitro plasma membrane vesicle uptake, immunoblotting and immunolocalization experiments were undertaken to investigate functional consequences. We identified c.1637C>T (T546M), previously associated with 5-FU-related toxicity, as a novel functionally damaging ABCC11 variant exhibiting markedly reduced transport function of 5-FdUMP, the active cytotoxic metabolite of 5-FU. Detailed analysis of 14 subpopulations revealed highest allele frequencies of c.1637C>T in Europeans and Americans (up to 11%) compared with Africans and Asians (up to 3%).

  16. 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%).

  17. 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}.

  18. Structure-activity relationships of a novel pyranopyridine series of Gram-negative bacterial efflux pump inhibitors.

    PubMed

    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

    2015-05-01

    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.

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

    PubMed

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

    2005-09-01

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

  1. 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

  2. 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...

  3. Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells: An insight into the efflux-metabolism alliance.

    PubMed

    Hua, Fang; Shi, Mei-jun; Zhu, Xiao-lu; Li, Meng; Wang, Hong-xu; Yu, Xiao-ming; Li, Yan; Zhu, Chuan-jiang

    2015-11-01

    The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide (CLA) enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients (Papp) of (-) and (+)CLA were higher in the absorptive direction than those in the secretory direction with efflux ratios (ER) of 0.709±0.411 and 0.867±0.250 (×10(-6)cm/s), respectively. Their bidirectional transports were significantly reduced by 75.6-87.5% after treatment with verapamil (a P-glycoprotein inhibitor) that increased the rate of metabolism by CYP3A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of (-) and (+)CLA with ERs being 2.934±1.432 and 1.877±0.148(×10(-6)cm/s) respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enantiomers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following (-) or (+)-isomer treatment alone. Furthermore, the uptake of (-)CLA was more than that of (+)CLA in the transfected cells. Incubation with ketoconazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexamethasone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharmacology of (-)CLA as a candidate drug for treatment of Alzheimer's disease. PMID:26301745

  4. Transport and uptake of clausenamide enantiomers in CYP3A4-transfected Caco-2 cells: An insight into the efflux-metabolism alliance.

    PubMed

    Hua, Fang; Shi, Mei-jun; Zhu, Xiao-lu; Li, Meng; Wang, Hong-xu; Yu, Xiao-ming; Li, Yan; Zhu, Chuan-jiang

    2015-11-01

    The present study developed a CYP3A4-expressed Caco-2 monolayer model at which effects of the efflux-metabolism alliance on the transport and uptake of clausenamide (CLA) enantiomers as CYP3A4 substrates were investigated. The apparent permeability coefficients (Papp) of (-) and (+)CLA were higher in the absorptive direction than those in the secretory direction with efflux ratios (ER) of 0.709±0.411 and 0.867±0.250 (×10(-6)cm/s), respectively. Their bidirectional transports were significantly reduced by 75.6-87.5% after treatment with verapamil (a P-glycoprotein inhibitor) that increased the rate of metabolism by CYP3A4, whereas the CYP3A4 inhibitor ketoconazole treatment markedly enhanced the basolateral to apical flux of (-) and (+)CLA with ERs being 2.934±1.432 and 1.877±0.148(×10(-6)cm/s) respectively. These changes could be blocked by the duel CYP3A4/P-glycoprotein inhibitor cyclosporine A, consequently, Papp values for CLA enantiomers in both directions were significantly greater than those obtained by using verapamil or ketoconazole, and their ERs were similar to those following (-) or (+)-isomer treatment alone. Furthermore, the uptake of (-)CLA was more than that of (+)CLA in the transfected cells. Incubation with ketoconazole decreased the intracellular concentrations of the two enantiomers. This effect disappeared in the presence of a CYP3A4 inducer dexamethasone. These results indicated that CYP3A4 could influence P-gp efflux, transport and uptake of CLA enantiomers as CYP3A4 substrates and that a duel inhibition to CYP3A4/ P-glycoprotein could enhance their absorption and bioavailability, which provides new insight into the efflux-metabolism alliance and will benefit the clinical pharmacology of (-)CLA as a candidate drug for treatment of Alzheimer's disease.

  5. 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.

  6. Microbial Efflux Pump Inhibition: Tactics and Strategies

    PubMed Central

    Tegos, George P.; Haynes, Mark; Strouse, J. Jacob; Khan, Mohiuddin Md. T.; Bologa, Cristian G.; Oprea, Tudor I.; Sklar, Larry A.

    2013-01-01

    Traditional antimicrobials are increasingly suffering from the emergence of multidrug resistance among pathogenic microorganisms. To overcome these deficiencies, a range of novel approaches to control microbial infections are under investigation as potential alternative treatments. Multidrug efflux is a key target of these efforts. Efflux mechanisms are broadly recognized as major components of resistance to many classes of chemotherapeutic agents as well as antimicrobials. Efflux occurs due to the activity of membrane transporter proteins widely known as Multidrug Efflux Systems (MES). They are implicated in a variety of physiological roles other than efflux and identifying natural substrates and inhibitors is an active and expanding research discipline. One plausible alternative is the combination of conventional antimicrobial agents/antibiotics with small molecules that block MES known as multidrug efflux pump inhibitors (EPIs). An array of approaches in academic and industrial research settings, varying from high-throughput screening (HTS) ventures to bioassay guided purification and determination, have yielded a number of promising EPIs in a series of pathogenic systems. This synergistic discovery platform has been exploited in translational directions beyond the potentiation of conventional antimicrobial treatments. This venture attempts to highlight different tactical elements of this platform, identifying the need for highly informative and comprehensive EPI-discovery strategies. Advances in assay development genomics, proteomics as well as the accumulation of bioactivity and structural information regarding MES facilitates the basis for a new discovery era. This platform is expanding drastically. A combination of chemogenomics and chemoinformatics approaches will integrate data mining with virtual and physical HTS ventures and populate the chemical-biological interface with a plethora of novel chemotypes. This comprehensive step will expedite the

  7. Multidrug efflux pumps: the structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.

    PubMed

    Kerr, Ian D; Jones, Peter M; George, Anthony M

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.

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

    PubMed

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

    2016-09-10

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

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

    PubMed

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

    2015-01-01

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

  10. Reduction of cellular stress by TolC-dependent efflux pumps in Escherichia coli indicated by BaeSR and CpxARP activation of spy in efflux mutants.

    PubMed

    Rosner, Judah L; Martin, Robert G

    2013-03-01

    Escherichia coli has nine inner membrane efflux pumps which complex with the outer membrane protein TolC and cognate membrane fusion proteins to form tripartite transperiplasmic pumps with diverse functions, including the expulsion of antibiotics. We recently observed that tolC mutants have elevated activities for three stress response regulators, MarA, SoxS, and Rob, and we suggested that TolC-dependent efflux is required to prevent the accumulation of stressful cellular metabolites. Here, we used spy::lacZ fusions to show that two systems for sensing/repairing extracytoplasmic stress, BaeRS and CpxARP, are activated in the absence of TolC-dependent efflux. In either tolC mutants or bacteria with mutations in the genes for four TolC-dependent efflux pumps, spy expression was increased 6- to 8-fold. spy encodes a periplasmic chaperone regulated by the BaeRS and CpxARP stress response systems. The overexpression of spy in tolC or multiple efflux pump mutants also depended on these systems. spy overexpression was not due to acetate, ethanol, or indole accumulation, since external acetate had only a minor effect on wild-type cells, ethanol had a large effect that was not CpxA dependent, and a tolC tnaA mutant which cannot accumulate internal indole overexpressed spy. We propose that, unless TolC-dependent pumps excrete certain metabolites, the metabolites accumulate and activate at least five different stress response systems.

  11. 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

  12. EPAct Transportation Regulatory Activities

    SciTech Connect

    2011-11-21

    The U.S. Department of Energy's (DOE) Vehicle Technologies Program manages several transportation regulatory activities established by the Energy Policy Act of 1992 (EPAct), as amended by the Energy Conservation Reauthorization Act of 1998, EPAct 2005, and the Energy Independence and Security Act of 2007 (EISA).

  13. 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

  14. Rice SPX-Major Facility Superfamily3, a Vacuolar Phosphate Efflux Transporter, Is Involved in Maintaining Phosphate Homeostasis in Rice1[OPEN

    PubMed Central

    Ying, Yinghui; Wang, Shoudong; Secco, David; Liu, Yu; Whelan, James; Tyerman, Stephen D.; Shou, Huixia

    2015-01-01

    To maintain a stable cytosol phosphate (Pi) concentration, plant cells store Pi in their vacuoles. When the Pi concentration in the cytosol decreases, Pi is exported from the vacuole into the cytosol. This export is mediated by Pi transporters on the tonoplast. In this study, we demonstrate that SYG1, PHO81, and XPR1 (SPX)-Major Facility Superfamily (MFS) proteins have a similar structure with yeast (Saccharomyces cerevisiae) low-affinity Pi transporters Phosphatase87 (PHO87), PHO90, and PHO91. OsSPX-MFS1, OsSPX-MFS2, and OsSPX-MFS3 all localized on the tonoplast of rice (Oryza sativa) protoplasts, even in the absence of the SPX domain. At high external Pi concentration, OsSPX-MFS3 could partially complement the yeast mutant strain EY917 under pH 5.5, which lacks all five Pi transporters present in yeast. In oocytes, OsSPX-MFS3 was shown to facilitate Pi influx or efflux depending on the external pH and Pi concentrations. In contrast to tonoplast localization in plants cells, OsSPX-MFS3 was localized to the plasma membrane when expressed in both yeast and oocytes. Overexpression of OsSPX-MFS3 results in decreased Pi concentration in the vacuole of rice tissues. We conclude that OsSPX-MFS3 is a low-affinity Pi transporter that mediates Pi efflux from the vacuole into cytosol and is coupled to proton movement. PMID:26424157

  15. Increase in multidrug transport activity is associated with oocyte maturation in sea stars.

    PubMed

    Roepke, Troy A; Hamdoun, Amro M; Cherr, Gary N

    2006-12-01

    In this study, we report on the presence of efflux transporter activity before oocyte maturation in sea stars and its upregulation after maturation. This activity is similar to the multidrug resistance (MDR) activity mediated by ATP binding cassette (ABC) efflux transporters. In sea star oocytes the efflux activity, as measured by exclusion of calcein-am, increased two-fold 3 h post-maturation. Experiments using specific and non-specific dyes and inhibitors demonstrated that the increase in transporter activity involves an ABCB protein, P-glycoprotein (P-gp), and an ABCC protein similar to the MDR-associated protein (MRP)-like transporters. Western blots using an antibody directed against mammalian P-gp recognized a 45 kDa protein in sea star oocytes that increased in abundance during maturation. An antibody directed against sea urchin ABCC proteins (MRP) recognized three proteins in immature oocytes and two in mature oocytes. Experiments using inhibitors suggest that translation and microtubule function are both required for post-maturation increases in transporter activity. Immunolabeling revealed translocation of stored ABCB proteins to the plasma cell membrane during maturation, and this translocation coincided with increased transport activity. These MDR transporters serve protective roles in oocytes and eggs, as demonstrated by sensitization of the oocytes to the maturation inhibitor, vinblastine, by MRP and PGP-specific transporter inhibitors.

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

    PubMed Central

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

    2015-01-01

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

  17. 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

  18. Random Mutagenesis of the Multidrug Transporter AcrB from Escherichia coli for Identification of Putative Target Residues of Efflux Pump Inhibitors

    PubMed Central

    Kohler, Samay; Buck, Annika; Dambacher, Christine; König, Armin; Bohnert, Jürgen A.; Kern, Winfried V.

    2014-01-01

    Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-β-naphthylamide (PAβN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAβN, which is consistent with the modes of action of PAβN differing from those of NMP. PMID:25182653

  19. Efflux transport of chrysin and apigenin sulfates in HEK293 cells overexpressing SULT1A3: The role of multidrug resistance-associated protein 4 (MRP4/ABCC4).

    PubMed

    Li, Wan; Sun, Hua; Zhang, Xingwang; Wang, Huan; Wu, Baojian

    2015-11-01

    Efflux transport is a critical determinant to the pharmacokinetics of sulfate conjugates. Here we aimed to establish SULT1A3 stably transfected HEK293 cells, and to determine the contributions of BCRP and MRP transporters to excretion of chrysin and apigenin sulfates. The cDNA of SULT1A3 was stably introduced into HEK293 cells using a lentiviral vector, generating a sulfonation active cell line (i.e., SULT293 cells). Identification of sulfate transporters was achieved through chemical inhibition (using chemical inhibitors) and biological inhibition (using short-hairpin RNAs (shRNAs)) methods. Sulfated metabolites were rapidly generated and excreted upon incubation of SULT293 cells with chrysin and apigenin. Ko143 (a selective BCRP inhibitor) did not show inhibitory effects on sulfate disposition, whereas the pan-MRP inhibitor MK-571 caused significant reductions (38.5-64.3%, p<0.001) in sulfate excretion and marked elevations (160-243%, p<0.05) in sulfate accumulation. Further, two efflux transporters (BCRP and MRP4) expressed in the cells were knocked-down by shRNA-mediated silencing. Neither sulfate excretion nor sulfate accumulation was altered in BCRP knocked-down cells as compared to scramble cells. By contrast, MRP4 knock-down led to moderate decreases (17.1-20.6%, p<0.05) in sulfate excretion and increases (125-135%, p<0.05) in sulfate accumulation. In conclusion, MRP4 was identified as an exporter for chrysin and apigenin sulfates. The SULT1A3 modified HEK293 cells were an appropriate tool to study SULT1A3-mediated sulfonation and to characterize BCRP/MRP4-mediated sulfate transport.

  20. 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

  1. Impact of passive permeability and gut efflux transport on the oral bioavailability of novel series of piperidine-based renin inhibitors in rodents.

    PubMed

    Lévesque, Jean-François; Bleasby, Kelly; Chefson, Amandine; Chen, Austin; Dubé, Daniel; Ducharme, Yves; Fournier, Pierre-André; Gagné, Sébastien; Gallant, Michel; Grimm, Erich; Hafey, Michael; Han, Yongxin; Houle, Robert; Lacombe, Patrick; Laliberté, Sébastien; MacDonald, Dwight; Mackay, Bruce; Papp, Robert; Tschirret-Guth, Richard

    2011-09-15

    An oral bioavailability issue encountered during the course of lead optimization in the renin program is described herein. The low F(po) of pyridone analogs was shown to be caused by a combination of poor passive permeability and gut efflux transport. Substitution of pyridone ring for a more lipophilic moiety (logD>1.7) had minimal effect on rMdr1a transport but led to increased passive permeability (P(app)>10 × 10(-6) cm/s), which contributed to overwhelm gut transporters and increase rat F(po). LogD and in vitro passive permeability determination were found to be key in guiding SAR and improve oral exposure of renin inhibitors.

  2. Intestinal absorption mechanism of mirabegron, a potent and selective β₃-adrenoceptor agonist: involvement of human efflux and/or influx transport systems.

    PubMed

    Takusagawa, Shin; Ushigome, Fumihiko; Nemoto, Hiroyuki; Takahashi, Yutaka; Li, Qun; Kerbusch, Virginie; Miyashita, Aiji; Iwatsubo, Takafumi; Usui, Takashi

    2013-05-01

    Mirabegron, a weak-basic compound, is a potent and selective β3-adrenoceptor agonist for the treatment of overactive bladder. Mirabegron extended release formulation shows dose-dependent oral bioavailability in humans, which is likely attributable to saturation of intestinal efflux abilities leading to higher absorption with higher doses. This study evaluated the membrane permeability of mirabegron and investigated the involvement of human intestinal transport proteins in the membrane permeation of mirabegron. Transcellular transport and cellular/vesicular uptake assays were performed using Caco-2 cells and/or human intestinal efflux (P-glycoprotein [P-gp], breast cancer resistance protein [BCRP], and multidrug resistance associated protein 2 [MRP2]) and influx (peptide transporter 1 [PEPT1], OATP1A2, and OATP2B1) transporter-expressing cells, vesicles, or Xenopus laevis oocytes. The absorptive permeability coefficients of mirabegron in Caco-2 cells (1.68-1.83 × 10(-6) cm/s) at the apical and basal pH of 6.5 and 7.4, respectively, were slightly higher than those of nadolol (0.97-1.41 × 10(-6) cm/s), a low permeability reference standard, but lower than those of metoprolol and propranolol (both ranged from 8.49 to 11.6 × 10(-6) cm/s), low/high permeability boundary reference standards. Increasing buffer pH at the apical side from 5.5 to 8.0 gradually increased the absorptive permeation of mirabegron from 0.226 to 1.66 × 10(-6) cm/s, but was still less than the value in the opposite direction (11.0-14.2 × 10(-6) cm/s). The time- and concentration-dependent transport of mirabegron was observed in P-gp-expressing cells and OATP1A2-expressing oocytes with apparent Km values of 294 and 8.59 μM, respectively. In contrast, no clear BCRP-, MRP2-, PEPT1-, or OATP2B1-mediated uptake of mirabegron was observed in their expressing vesicles or cells. These findings suggest that mirabegron has low-to-moderate membrane permeability and P-gp is likely to be involved in its

  3. The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification

    PubMed Central

    Neufeld, Edward B.; O’Brien, Katherine; Walts, Avram D.; Stonik, John A.; Malide, Daniela; Combs, Christian A.; Remaley, Alan T.

    2014-01-01

    We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM) and in late endosomes (LE) mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM)-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated) and lysenin-induced (SM-mediated) cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo) and disordered (Ld) membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification. PMID:25485894

  4. PDE5 inhibitors, sildenafil and vardenafil, reverse multidrug resistance by inhibiting the efflux function of multidrug resistance protein 7 (ATP-binding Cassette C10) transporter.

    PubMed

    Chen, Jun-Jiang; Sun, Yue-Li; Tiwari, Amit K; Xiao, Zhi-Jie; Sodani, Kamlesh; Yang, Dong-Hua; Vispute, Saraubh G; Jiang, Wen-Qi; Chen, Si-Dong; Chen, Zhe-Sheng

    2012-08-01

    Phosphodiesterase type 5 (PDE5) inhibitors are widely used in the treatment of male erectile dysfunction and pulmonary hypertension. Recently, several groups have evaluated the ability of PDE5 inhibitors for their anticancer activities. Previously, we had shown that sildenafil, vardenafil and tadalafil could reverse P-glycoprotein (ATP-binding cassette B1)-mediated MDR. In the present study, we determined whether these PDE5 inhibitors have the potential to reverse multidrug resistance protein 7 (MRP7; ATP-binding cassette C10)-mediated MDR. We found that sildenafil and vardenafil dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to paclitaxel, docetaxel and vinblastine, while tadalafil had only a minimal effect. Accumulation and efflux experiments demonstrated that sildenafil and vardenafil increased the intracellular accumulation of [(3)H]-paclitaxel by inhibiting the efflux of [(3 H]-paclitaxel in HEK/MRP7 cells. In addition, immunoblot and immunofluorescence analyses indicated that no significant alterations of MRP7 protein expression and localization in plasma membranes were found after treatment with sildenafil, vardenafil or tadalafil. These results demonstrate that sildenafil and vardenafil reverse MRP7-mediated a MDR through inhibition of the drug efflux function of MRP7. Our findings indicate a potentially novel use of PDE5 inhibitors as an adjuvant chemotherapeutic agent in clinical practice. PMID:22578167

  5. In Vitro Drug Response and Efflux Transporters Associated with Drug Resistance in Pediatric High Grade Glioma and Diffuse Intrinsic Pontine Glioma

    PubMed Central

    Veringa, Susanna J. E.; Biesmans, Dennis; van Vuurden, Dannis G.; Jansen, Marc H. A.; Wedekind, Laurine E.; Horsman, Ilona; Wesseling, Pieter; Vandertop, William Peter; Noske, David P.; Kaspers, GertJan J. L.; Hulleman, Esther

    2013-01-01

    Pediatric high-grade gliomas (pHGG), including diffuse intrinsic pontine gliomas (DIPG), are the leading cause of cancer-related death in children. While it is clear that surgery (if possible), and radiotherapy are beneficial for treatment, the role of chemotherapy for these tumors is still unclear. Therefore, we performed an in vitro drug screen on primary glioma cells, including three DIPG cultures, to determine drug sensitivity of these tumours, without the possible confounding effect of insufficient drug delivery. This screen revealed a high in vitro cytotoxicity for melphalan, doxorubicine, mitoxantrone, and BCNU, and for the novel, targeted agents vandetanib and bortezomib in pHGG and DIPG cells. We subsequently determined the expression of the drug efflux transporters P-gp, BCRP1, and MRP1 in glioma cultures and their corresponding tumor tissues. Results indicate the presence of P-gp, MRP1 and BCRP1 in the tumor vasculature, and expression of MRP1 in the glioma cells themselves. Our results show that pediatric glioma and DIPG tumors per se are not resistant to chemotherapy. Treatment failure observed in clinical trials, may rather be contributed to the presence of drug efflux transporters that constitute a first line of drug resistance located at the blood-brain barrier or other resistance mechanism. As such, we suggest that alternative ways of drug delivery may offer new possibilities for the treatment of pediatric high-grade glioma patients, and DIPG in particular. PMID:23637844

  6. Dopamine transporters are involved in the onset of hypoxia-induced dopamine efflux in striatum as revealed by in vivo microdialysis.

    PubMed

    Orset, Cyrille; Parrot, Sandrine; Sauvinet, Valérie; Cottet-Emard, Jean-Marie; Bérod, Anne; Pequignot, Jean-Marc; Denoroy, Luc

    2005-06-01

    Although many studies have revealed alterations in neurotransmission during ischaemia, few works have been devoted to the neurochemical effects of mild hypoxia, a situation encountered during life in altitude or in several pathologies. In that context, the present work was undertaken to determine the in vivo mechanisms underlying the striatal dopamine efflux induced by mild hypoxaemic hypoxia. For that purpose, the extracellular concentrations of dopamine and its metabolite 3,4-dihydroxyphenyl acetic acid were simultaneously measured using brain microdialysis during acute hypoxic exposure (10% O(2), 1h) in awake rats. Hypoxia induced a +80% increase in dopamine. Application of the dopamine transporters inhibitor, nomifensine (10 microM), just before the hypoxia prevented the rise in dopamine during the early part of hypoxia; in contrast the application of nomifensine after the beginning of hypoxia, failed to alter the increase in dopamine. Application of the voltage-dependent Na(+) channel blocker tetrodotoxin abolished the increase in dopamine, whether administered just before or after the beginning of hypoxia. These data show that the neurochemical mechanisms of the dopamine efflux may change over the course of the hypoxic exposure, dopamine transporters being involved only at the beginning of hypoxia.

  7. 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.

  8. 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

  9. 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.

  10. 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. PMID:27274079

  11. Multidrug efflux pumps of Gram-positive bacteria.

    PubMed

    Schindler, Bryan D; Kaatz, Glenn W

    2016-07-01

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

  12. [Efflux systems in Serratia marcescens].

    PubMed

    Mardanova, A M; Bogomol'naia, L M; Romanova, Iu D; Sharipova, M R

    2014-01-01

    A widespread bacterium Serratia marcescens (family Enterobacteriaceae) is an opportunistic and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from pathogen and exhibits multiple drug resistance. Active removal of antibiotics and other antimicrobials from the cells by efflux systems is one of the mechanisms responsible for microbial resistance to these compounds. Among enterobacteria, efflux systems of Escherichia coli and Salmonella enterica var. Typhimurium have been studied most extensively. Few efflux systems that belong to different families have been reported for S. marcescens. In this review, we analyzed available literature about S. marcescens efflux systems and carried out the comparative analysis of the genes encoding the RND type systems in different Serratia species and in other enterobacteria. Bioinformatical analysis of the S. marcescens genome allowed us to identify the previously unknown efflux systems based on their homology with the relevant E. coli genes. Identification of additional efflux systems in S. marcescens genome will promote our understanding of physiology of these bacteria, will detect new molecular mechanisms of resistance and will reveal their resistance potential.

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

    PubMed Central

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

    2010-01-01

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

  14. 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.

  15. Effect of venlafaxine and desvenlafaxine on drug efflux protein expression and biodistribution in vivo.

    PubMed

    Bachmeier, Corbin; Levin, Gary M; Beaulieu-Abdelahad, David; Reed, Jon; Mullan, Michael

    2013-10-01

    Venlafaxine, and to a lesser extent desvenlafaxine, has previously been shown to induce the expression of the drug efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in whole cells and alter the cellular permeability of a known drug efflux probe (rhodamine 123). To validate these in vitro findings, wild-type mice were treated for 4 days with 10 mg/kg venlafaxine or desvenlafaxine, and drug efflux transporter expression was examined in the brain, liver, and intestine. P-gp and BCRP expression was significantly upregulated in the intestine, following a treatment with venlafaxine (2.6- and 6.7-fold, respectively) or desvenlafaxine (2.3- and 4.8-fold, respectively). In addition, venlafaxine increased the BCRP expression in the brain (40%) and liver (60%), whereas desvenlafaxine had no effect on drug efflux transporter levels in these tissues. Using the same treatment paradigm, we observed a minimal impact of either drug on the brain disposition of the known drug efflux probe, topotecan. However, in the periphery, venlafaxine treatment significantly reduced the topotecan oral bioavailability by nearly 40%, whereas the impact of desvenlafaxine on topotecan plasma levels was more modest (23%). These studies demonstrate an effect of venlafaxine on the drug efflux transport activity and the potential for clinical drug-drug interactions.

  16. Dual action antifungal small molecule modulates multidrug efflux and TOR signaling.

    PubMed

    Shekhar-Guturja, Tanvi; Gunaherath, G M Kamal B; Wijeratne, E M Kithsiri; 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; Gunatilaka, A A Leslie; Cowen, Leah E

    2016-10-01

    There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, 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 antifungal-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 the 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 treating fungal infectious disease that evades resistance.

  17. Dual action antifungal small molecule modulates multidrug efflux and TOR signaling.

    PubMed

    Shekhar-Guturja, Tanvi; Gunaherath, G M Kamal B; Wijeratne, E M Kithsiri; 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; Gunatilaka, A A Leslie; Cowen, Leah E

    2016-10-01

    There is an urgent need for new strategies to treat invasive fungal infections, which are a leading cause of human mortality. Here, 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 antifungal-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 the 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 treating fungal infectious disease that evades resistance. PMID:27571477

  18. 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

  19. The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity

    PubMed Central

    Hürlimann, Hans Caspar; Pinson, Benoît; Stadler-Waibel, Martha; Zeeman, Samuel C; Freimoser, Florian M

    2009-01-01

    Yeast has two phosphate-uptake systems that complement each other: the high-affinity transporters (Pho84 and Pho89) are active under phosphate starvation, whereas Pho87 and Pho90 are low-affinity transporters that function when phosphate is abundant. Here, we report new regulatory functions of the amino-terminal SPX domain of Pho87 and Pho90. By studying truncated versions of Pho87 and Pho90, we show that the SPX domain limits the phosphate-uptake velocity, suppresses phosphate efflux and affects the regulation of the phosphate signal transduction pathway. Furthermore, split-ubiquitin assays and co-immunoprecipitation suggest that the SPX domain of both Pho90 and Pho87 interacts physically with the regulatory protein Spl2. This work suggests that the SPX domain inhibits low-affinity phosphate transport through a physical interaction with Spl2. PMID:19590579

  20. 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

    2016-09-01

    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 [(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.

  1. 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

  2. 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. PMID:24201377

  3. 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...

  4. 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

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

    PubMed

    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

  6. 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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  9. 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

  10. 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…

  11. 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...

  12. Efflux-Mediated Antifungal Drug Resistance†

    PubMed Central

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

    2009-01-01

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

  13. 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

  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. 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

  16. 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

  17. Activated transport in AMTEC electrodes

    NASA Astrophysics Data System (ADS)

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

    1992-08-01

    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 the 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.

  18. Assembly and operation of bacterial tripartite multidrug efflux pumps.

    PubMed

    Du, Dijun; van Veen, Hendrik W; Luisi, Ben F

    2015-05-01

    Microorganisms encode several classes of transmembrane pumps that can expel an enormous range of toxic substances, thereby improving their fitness in harsh environments and contributing to resistance against antimicrobial agents. In Gram-negative bacteria these pumps can take the form of tripartite assemblies that actively efflux drugs and other harmful compounds across the cell envelope. We describe recent structural and functional data that have provided insights into the transport mechanisms of these intricate molecular machines.

  19. 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 (...

  20. In Vivo Bioluminescent Imaging of ATP-Binding Cassette Transporter-Mediated Efflux at the Blood-Brain Barrier.

    PubMed

    Bakhsheshian, Joshua; Wei, Bih-Rong; Hall, Matthew D; Simpson, R Mark; Gottesman, Michael M

    2016-01-01

    We provide a detailed protocol for imaging ATP-binding cassette subfamily G member 2 (ABCG2) function at the blood-brain barrier (BBB) of transgenic mice. D-Luciferin is specifically transported by ABCG2 found on the apical side of endothelial cells at the BBB. The luciferase-luciferin enzymatic reaction produces bioluminescence, which allows a direct measurement of ABCG2 function at the BBB. Therefore bioluminescence imaging (BLI) correlates with ABCG2 function at the BBB and this can be measured by administering luciferin in a mouse model that expresses luciferase in the brain parenchyma. BLI allows for a relatively low-cost alternative for studying transporter function in vivo compared to other strategies such as positron emission tomography. This method for imaging ABCG2 function at the BBB can be used to investigate pharmacokinetic inhibition of the transporter. PMID:27424909

  1. 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

  2. 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. PMID:25036090

  3. 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...

  4. Killing Them with Kindness? In-Hive Medications May Inhibit Xenobiotic Efflux Transporters and Endanger Honey Bees

    PubMed Central

    Hawthorne, David J.; Dively, Galen P.

    2011-01-01

    Background Honey bees (Apis mellifera) have recently experienced higher than normal overwintering colony losses. Many factors have been evoked to explain the losses, among which are the presence of residues of pesticides and veterinary products in hives. Multiple residues are present at the same time, though most often in low concentrations so that no single product has yet been associated with losses. Involvement of a combination of residues to losses may however not be excluded. To understand the impact of an exposure to combined residues on honey bees, we propose a mechanism-based strategy, focusing here on Multi-Drug Resistance (MDR) transporters as mediators of those interactions. Methodology/Principal Findings Using whole-animal bioassays, we demonstrate through inhibition by verapamil that the widely used organophosphate and pyrethroid acaricides coumaphos and τ-fluvalinate, and three neonicotinoid insecticides: imidacloprid, acetamiprid and thiacloprid are substrates of one or more MDR transporters. Among the candidate inhibitors of honey bee MDR transporters is the in-hive antibiotic oxytetracycline. Bees prefed oxytetracycline were significantly sensitized to the acaricides coumaphos and τ-fluvalinate, suggesting that the antibiotic may interfere with the normal excretion or metabolism of these pesticides. Conclusions/Significance Many bee hives receive regular treatments of oxytetracycline and acaricides for prevention and treatment of disease and parasites. Our results suggest that seasonal co-application of these medicines to bee hives could increase the adverse effects of these and perhaps other pesticides. Our results also demonstrate the utility of a mechanism-based strategy. By identifying pesticides and apicultural medicines that are substrates and inhibitors of xenobiotic transporters we prioritize the testing of those chemical combinations most likely to result in adverse interactions. PMID:22073195

  5. Biochemistry of Bacterial Multidrug Efflux Pumps

    PubMed Central

    Kumar, Sanath; Varela, Manuel F.

    2012-01-01

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

  6. P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity.

    PubMed

    Nervi, Pierluigi; Li-Blatter, Xiaochun; Aänismaa, Päivi; Seelig, Anna

    2010-03-01

    We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux. PMID:20004641

  7. 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

    Parrish, Karen E; Pokorny, Jenny; Mittapalli, Rajendar K; Bakken, Katrina; Sarkaria, Jann N; Elmquist, William F

    2015-11-01

    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

  8. Segmental dependent transport of low permeability compounds along the small intestine due to P-glycoprotein: the role of efflux transport in the oral absorption of BCS class III drugs.

    PubMed

    Dahan, Arik; Amidon, Gordon L

    2009-01-01

    The purpose of this study was to investigate the role of P-gp efflux in the in vivo intestinal absorption process of BCS class III P-gp substrates, i.e. high-solubility low-permeability drugs. The in vivo permeability of two H (2)-antagonists, cimetidine and famotidine, was determined by the single-pass intestinal perfusion model in different regions of the rat small intestine, in the presence or absence of the P-gp inhibitor verapamil. The apical to basolateral (AP-BL) and the BL-AP transport of the compounds in the presence or absence of various efflux transporters inhibitors (verapamil, erythromycin, quinidine, MK-571 and fumitremorgin C) was investigated across Caco-2 cell monolayers. P-gp expression levels in the different intestinal segments were confirmed by immunoblotting. Cimetidine and famotidine exhibited segmental dependent permeability through the gut wall, with decreased P(eff) in the distal ileum in comparison to the proximal regions of the intestine. Coperfusion of verapamil with the drugs significantly increased the permeability in the ileum, while no significant change in the jejunal permeability was observed. Both drugs exhibited significantly greater BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. Concentration dependent decrease of this secretion was obtained by the P-gp inhibitors verapamil, erythromycin and quinidine, while no effect was evident by the MRP2 inhibitor MK-571 and the BCRP inhibitor FTC, indicating that P-gp is the transporter mediates the intestinal efflux of cimetidine and famotidine. P-gp levels throughout the intestine were inversely related to the in vivo permeability of the drugs from the different segments. The data demonstrate that for these high-solubility low-permeability P-gp substrates, P-gp limits in vivo intestinal absorption in the distal segments of the small intestine; however P-gp plays a minimal role in the proximal intestinal segments due to significant lower P-gp expression levels

  9. 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

  10. 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.

  11. 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.

  12. [11C]Flumazenil brain uptake is influenced by the blood-brain barrier efflux transporter P-glycoprotein

    PubMed Central

    2012-01-01

    Background [11C]Flumazenil and positron emission tomography (PET) are used clinically to assess gamma-aminobutyric acid (GABA)-ergic function and to localize epileptic foci prior to resective surgery. Enhanced P-glycoprotein (P-gp) activity has been reported in epilepsy and this may confound interpretation of clinical scans if [11C]flumazenil is a P-gp substrate. The purpose of this study was to investigate whether [11C]flumazenil is a P-gp substrate. Methods [11C]Flumazenil PET scans were performed in wild type (WT) (n = 9) and Mdr1a/1b, (the genes that encode for P-gp) double knockout (dKO) (n = 10) mice, and in naive rats (n = 10). In parallel to PET scanning, [11C]flumazenil plasma concentrations were measured in rats. For 6 of the WT and 6 of the dKO mice a second, [11C]flumazenil scan was acquired after administration of the P-gp inhibitor tariquidar. Cerebral [11C]flumazenil concentrations in WT and Mdr1a/1b dKO mice were compared (genetic disruption model). Furthermore, pre and post P-gp-blocking cerebral [11C]flumazenil concentrations were compared in all animals (pharmacological inhibition model). Results Mdr1a/1b dKO mice had approximately 70% higher [11C]flumazenil uptake in the brain than WT mice. After administration of tariquidar, cerebral [11C]flumazenil uptake in WT mice increased by about 80% in WT mice, while it remained the same in Mdr1a/1b dKO mice. In rats, cerebral [11C]flumazenil uptake increased by about 60% after tariquidar administration. Tariquidar had only a small effect on plasma clearance of flumazenil. Conclusions The present study showed that [11C]flumazenil is a P-gp substrate in rodents. Consequently, altered cerebral [11C]flumazenil uptake, as observed in epilepsy, may not reflect solely GABAA receptor density changes but also changes in P-gp activity. PMID:22455873

  13. 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. PMID:26824720

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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. PMID:27463566

  19. Signal focusing through active transport.

    PubMed

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing-faster and more precise signaling-are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  20. Signal focusing through active transport

    NASA Astrophysics Data System (ADS)

    Godec, Aljaž; Metzler, Ralf

    2015-07-01

    The accuracy of molecular signaling in biological cells and novel diagnostic devices is ultimately limited by the counting noise floor imposed by the thermal diffusion. Motivated by the fact that messenger RNA and vesicle-engulfed signaling molecules transiently bind to molecular motors and are actively transported in biological cells, we show here that the random active delivery of signaling particles to within a typical diffusion distance to the receptor generically reduces the correlation time of the counting noise. Considering a variety of signaling particle sizes from mRNA to vesicles and cell sizes from prokaryotic to eukaryotic cells, we show that the conditions for active focusing—faster and more precise signaling—are indeed compatible with observations in living cells. Our results improve the understanding of molecular cellular signaling and novel diagnostic devices.

  1. 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

  2. Pathways of arsenic uptake and efflux.

    PubMed

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

    2012-01-01

    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.

  3. Effects of efflux-pump inducers and genetic variation of the multidrug transporter cmeB in biocide resistance of Campylobacter jejuni and Campylobacter coli.

    PubMed

    Mavri, Ana; Smole Možina, Sonja

    2013-03-01

    Multidrug efflux pumps, such as CmeABC and CmeDEF, are involved in the resistance of Campylobacter to a broad spectrum of antimicrobials. The aim of this study was to analyse the effects of two putative efflux-pump inducers, bile salts and sodium deoxycholate, on the resistance of Campylobacter to biocides (triclosan, benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and trisodium phosphate), SDS and erythromycin. The involvement of the CmeABC and CmeDEF efflux pumps in this resistance was studied on the basis of the effects of bile salts and sodium deoxycholate in Campylobacter cmeB, cmeF and cmeR mutants. The genetic variation in the cmeB gene was also examined, to see whether this polymorphism is related to the function of the efflux pump. In 15 Campylobacter jejuni and 23 Campylobacter coli strains, bile salts and sodium deoxycholate increased the MICs of benzalkonium chloride, chlorhexidine diacetate, cetylpyridinium chloride and SDS, and decreased the MICs of triclosan, trisodium phosphate and erythromycin. Bile salts and sodium deoxycholate further decreased or increased the MICs of biocides and erythromycin in the cmeF and cmeR mutants. For cmeB polymorphisms, 17 different cmeB-specific PCR-RFLP patterns were identified: six within C. jejuni only, nine within C. coli only and two in both species. In conclusion, bile salts and sodium deoxycholate can increase or decrease bacterial resistance to structurally unrelated antimicrobials. The MIC increases in the cmeF and cmeR mutants indicated that at least one non-CmeABC efflux system is involved in resistance to biocides. These results indicate that the cmeB gene polymorphism identified is not associated with biocide and erythromycin resistance in Campylobacter.

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

    SciTech Connect

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

    2013-11-15

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

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

    PubMed Central

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

    2012-01-01

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

  6. Discovery of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides active against efflux-mediated resistant Streptococcus pneumoniae.

    PubMed

    Bukvić Krajačić, Mirjana; Dumić, Miljenko; Novak, Predrag; Cindrić, Mario; Koštrun, Sanja; Fajdetić, Andrea; Alihodžić, Sulejman; Brajša, Karmen; Kujundžić, Nedjeljko

    2011-01-15

    A series of novel ureas and thioureas of 3-decladinosyl-3-hydroxy 15-membered azalides, were discovered, structurally characterized and biologically evaluated. They have shown good antibacterial activity against selected Gram-positive and Gram-negative bacterial strains. These include N″ substituted 9a-(N'-carbamoyl-γ-aminopropyl)- (6a,c), 9a-(N'-thiocarbamoyl-γ-aminopropyl)- (7a,e), 9a-[N'-(β-cyanoethyl)-N'-(carbamoyl-γ-aminopropyl)]- (9a-c, 9g) 9a-[N'-(β-cyanoethyl)-N'-(thiocarbamoyl-γ-aminopropyl)]-derivatives (10d-f) of 5-O-desosaminyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythronolide A (3). Among the synthesized compounds thiourea 7a and urea 9b have shown substantially improved activity comparable to azithromycin (1) and significantly better activity than the 3-decladinosyl-azithromycin (2) and the parent 3-cladinosyl analogues against efflux-mediated resistant S. pneumoniae.

  7. Causes and Consequences of Variability in Drug Transporter Activity in Pediatric Drug Therapy.

    PubMed

    Rodieux, Frédérique; Gotta, Verena; Pfister, Marc; van den Anker, Johannes N

    2016-07-01

    Drug transporters play a key role in mediating the uptake of endo- and exogenous substances into cells as well as their efflux. Therefore, variability in drug transporter activity can influence pharmaco- and toxicokinetics and be a determinant of drug safety and efficacy. In children, particularly in neonates and young infants, the contribution of tissue-specific drug transporters to drug absorption, distribution, and excretion may differ from that in adults. In this review 5 major factors and their interdependence that may influence drug transporter activity in children are discussed: developmental differences, genetic polymorphisms, pediatric comorbidities, interacting comedication, and environmental factors. Even if data are sparse, altered drug transporter activity due to those factors have been associated with clinically relevant differences in drug disposition, efficacy, and safety in pediatric patients. Single nucleotide polymorphisms in drug transporter-encoding genes were the most studied source of drug transporter variability in children. However, in the age group where drug transporter activity has been reported to differ from that in adults, namely neonates and young infants, hardly any studies have been performed. Longitudinal studies in this young population are required to investigate the age- and disease-dependent genotype-phenotype relationships and relevance of drug transporter drug-drug interactions. Physiologically based pharmacokinetic modeling approaches can integrate drug- and patient-specific parameters, including drug transporter ontogeny, and may further improve in silico predictions of pediatric-specific pharmacokinetics. PMID:27385174

  8. Import and efflux of flubendazole in Haemonchus contortus strains susceptible and resistant to anthelmintics.

    PubMed

    Bártíková, Hana; Vokřál, Ivan; Kubíček, Vladimír; Szotáková, Barbora; Prchal, Lukáš; Lamka, Jiří; Várady, Marián; Skálová, Lenka

    2012-07-01

    Drug entry into the body of a helminth is a key factor in the efficacy of anthelmintics. The present project was designed to study the ex vivo uptake and efflux of the benzimidazole anthelmintic flubendazole (FLU) in four strains of H. contortus: the ISE strain (fully susceptible to anthelmintics), the ISE-S strain (resistant to ivermectin), the BR strain (resistant to benzimidazoles) and the WR strain (multi-resistant). The transport of FLU between dead and living nematodes was also compared as well as the effect of verapamil, an inhibitor of the main efflux ABCB1 transporter (P-glycoprotein), on FLU accumulation in nematodes. The obtained results showed that FLU is able to effectively enter H. contortus adults due to high FLU lipophilicity. Passive diffusion is probably the only mechanism in both FLU import and efflux from nematodes. No differences in FLU transport were found among four H. contortus strains with different sensitivity to anthelmintics. No active FLU efflux from H. contortus and no effect of verapamil were observed, indicating that H. contortus cannot protect itself against FLU by the active removal of this anthelmintic from its body.

  9. 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.

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

    PubMed

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

    2016-07-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Sodium dependency of active chloride transport across isolated fish skin (Gillichthys mirabilis).

    PubMed Central

    Marshall, W S

    1981-01-01

    1. The effects of thiocyanate, ouabain, ion-substituted Ringer solution and electrochemical gradients on Na+ and Cl- transport were examined using the isolated skin of the marine teleost, Gillichthys mirabilis. 2. Bilateral replacement of Na+ with choline in the bathing solutions reduces net Cl- flux by 93%, indicating that active Cl- transport by the skin is Na-dependent. 3. Thiocyanate inhibits short-circuit current with an ED50 of 6.4 x 10(-4)M, and, at 10(-2)M, decreases Cl-efflux, influx, net flux and short-circuit current by 68, 33, 74 and 81%, respectively. 4. Ouabain (10(-5)M) reduces Cl- efflux and net flux by 56 and 86%, respectively, indicating that the Cl- transport requires Na,K-ATPase. 5. Subsequent addition of thiocyanate to ouabain-treated skin reduces Cl- efflux, net flux and short-circuit current, suggesting that the two agents operate at different sites involved in Cl- transport. 6. Unilateral substitution of gluconate for Cl- on the serosal side does not affect Cl- influx, indicating that Cl- passive transport is via Fickean diffusion, not Cl-Cl exchange diffusion. 7. The addition of NaCl to the mucosal side, which mimics the in vivo sea-water condition, increases Cl- influx and transepithelial potential and decreases tissue resistance. The net flux (secretion) of Cl- with hypertonic saline on the mucosal side (0.51 +/- 0.06 muequiv/cm2 . hr) demonstrates that the skin could secrete Cl- in vivo. 8. Na+ fluxes across the skin are passive, as the observed flux ration (efflux/influx) is similar to that predicted by the Ussing-Teorell equation under both closed- and open-circuit conditions. 9. The permeability ratio (PNa:PCl) in approximately 5.4:1.0, indicating that the skin is more permeable to Na+, and that at least part of the serosa-positive transepithelial potential may be a Na+ diffusion potential. 10. The results suggest that Cl- secretion by Gillichthys skin is secondary active transport involving Na,K-ATPase and serosal Na+. PMID:7320911

  14. 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.

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

    PubMed

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

    2013-11-15

    Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. PMID:24140409

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

    PubMed Central

    Shao, Fei; Ford, David A.

    2013-01-01

    Trans fatty acid consumption is associated with an increased risk of coronary heart disease. This increased risk has been attributed to decreased levels of HDL cholesterol and increased levels of LDL cholesterol. However, the mechanism by which trans fatty acid modulates cholesterol transit remains poorly defined. ATP-binding cassette transporter A1 (ABCA1)-mediated macrophage cholesterol efflux is the rate-limiting step initiating apolipoprotein A-I lipidation. In this study, elaidic acid, the most abundant trans fatty acid in partially hydrogenated vegetable oil, was shown to stabilize macrophage ABCA1 protein levels in comparison to that of its cis fatty acid isomer, oleic acid. The mechanism responsible for the disparate effects of oleic and elaidic acid on ABCA1 levels was through accelerated ABCA1 protein degradation in cells treated with oleic acid. In contrast, no apparent differences were observed in ABCA1 mRNA levels, and only minor changes were observed in Liver X receptor/Retinoic X receptor promoter activity in cells treated with elaidic and oleic acid. Efflux of both tracers and cholesterol mass revealed that elaidic acid slightly increased ABCA1-mediated cholesterol efflux, while oleic acid led to decreased ABCA1-mediated efflux. In conclusion, these studies sho that cis and trans structural differences in eighteen carbon n-9 monoenoic fatty acids variably impact cholesterol efflux through disparate effects on ABCA1 protein degradation. PMID:23800855

  17. The E3 Ubiquitin Ligase SCFTIR1/AFB and Membrane Sterols Play Key Roles in Auxin Regulation of Endocytosis, Recycling, and Plasma Membrane Accumulation of the Auxin Efflux Transporter PIN2 in Arabidopsis thaliana[C][W][OA

    PubMed Central

    Pan, Jianwei; Fujioka, Shozo; Peng, Jianling; Chen, Jianghua; Li, Guangming; Chen, Rujin

    2009-01-01

    The PIN family of auxin efflux transporters exhibit polar plasma membrane (PM) localization and play a key role in auxin gradient-mediated developmental processes. Auxin inhibits PIN2 endocytosis and promotes its PM localization. However, the underlying mechanisms remain elusive. Here, we show that the inhibitory effect of auxin on PIN2 endocytosis was impaired in SCFTIR1/AFB auxin signaling mutants. Similarly, reducing membrane sterols impaired auxin inhibition of PIN2 endocytosis. Gas chromatography–mass spectrometry analyses indicate that membrane sterols were significantly reduced in SCFTIR1/AFB mutants, supporting a link between membrane sterols and auxin signaling in regulating PIN2 endocytosis. We show that auxin promoted PIN2 recycling from endosomes to the PM and increased PIN2 steady state levels in the PM fraction. Furthermore, we show that the positive effect of auxin on PIN2 levels in the PM was impaired by inhibiting membrane sterols or auxin signaling. Consistent with this, the sterol biosynthetic mutant fk-J79 exhibited pronounced defects in primary root elongation and gravitropic response. Our data collectively indicate that, although there are distinct processes involved in endocytic regulation of specific PM-resident proteins, the SCFTIR1/AFB-dependent processes are required for auxin regulation of endocytosis, recycling, and PM accumulation of the auxin efflux transporter PIN2 in Arabidopsis thaliana. PMID:19218398

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

    PubMed

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

    2016-09-01

    The multi-drug resistance offered by Pseudomonas aeruginosa to antibiotics can be attributed towards its propensity to develop biofilm, modification in cell membrane and to efflux antibacterial drugs. The present study explored the activity of Glycyrrhiza glabra and one of its pure compounds, glycyrrhizic acid against P. aeruginosa and their mechanism of action in terms of the effect on membrane permeability, efflux activity, and biofilm formation were determined. Minimum inhibitory concentrations were determined by using broth dilution technique. The minimum bactericidal concentrations were assessed on agar plate. The MIC of the extract and glycyrrhizic acid was found to be 200 and 100 μg ml(-1), respectively. The MBC was found to be 800 and 400 μg ml(-1) in the case of extract and glycyrrhizic acid, respectively. Time -dependent killing efficacy was also estimated. Flowcytometric analysis with staining methods was used to determine the effect of extract and glycyrrhizic acid at 2 × MIC on different physiological parameters and compared it with the standard (antibiotic). The growth of P. aeruginosa was significantly inhibited by extract and the pure compound. The herbal extract and the glycyrrhic acid were also found to effective in targeting the physiological parameters of the bacteria that involve cell membrane permeabilization, efflux activity, and biofilm formation. This study reports the antipseudomonal action of Glycyrrhiza glabra and one of its compound and provides insight into their mode of action. PMID:27392698

  19. On the mechanism of A23187-induced potassium efflux in rat liver mitochondria.

    PubMed

    Dordick, R S; Brierley, G P; Garlid, K D

    1980-11-10

    1. Rat liver mitochondria undergo a spontaneous, respiration-dependent K+ extrusion which is accelerated by citrate. This K+ efflux is electroneutral and is considered to occur on an endogenous K/H exchanger. The spontaneous efflux, but not nigericin-induced K/H exchange, is always preceded by a lag phase, suggesting that the lag phase is a characteristic property of the endogenous exchange reaction. 2. K+ extrusion induced by ionophore A23187 also has the characteristics of K/H exchange. The rate of K+ efflux is faster and the lag time is shorter when compared to endogenous K+ efflux. The effects of A23187 on the lag phase suggest that the ionophore acts by unmasking the endogenous exchanger. This conclusion is supported by the finding that K+ efflux rates reach a maximum which cannot be exceeded by increasing the dose of A23187 but is exceeded by adding nigericin. 3. Steady state perturbation studies were carried out on respiring mitochondria in which electrophoretic K+ influx was balanced by electroneutral K+ efflux. These steady states were appropriately shifted in opposite directions by additions of nigericin or valinomycin. In contrast, addition of A23187 had no effect. It is concluded that A23187 is incapable of transporting K+ in rat liver mitochondria. 4. These results are consistent with a model in which free matrix Mg2+ acts as a K/H carrier "brake." The proposed role of this carrier-brake mechanism is to provide volume homeostasis with minimal energy expenditure. According to this model, both citrate and A23187 stimulate K/H exchange by reducing Mg2+ activity within the matrix. Citrate acts by complexation of Mg2+, while A23187 acts by transporting Mg2+ out of the matrix.

  20. 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. PMID:21343453

  1. Mitochondria influence CDR1 efflux pump activity, Hog1-mediated oxidative stress pathway, iron homeostasis, and ergosterol levels in Candida albicans.

    PubMed

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

    2013-11-01

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

  2. 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

  3. 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

  4. Efflux Systems in Bacteria and their Metabolic Engineering Applications

    PubMed Central

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

    2015-01-01

    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. PMID:26363557

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. Transport aircraft flying qualities activities

    NASA Technical Reports Server (NTRS)

    Moul, M. T.

    1981-01-01

    The optimal control model for pilot vehicle systems was used to develop a methodology for predicting pilot ratings for commercial transports. The method was tested by applying it to a family of transport configurations for which subjective pilot ratings were obtained. Specific attention is given to the development of the simulator program and procedures so as to yield objective and subjective performance data useful for a critical evaluation of the analytical method.

  10. 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

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

    PubMed

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

    2014-03-01

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

  12. 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. PMID:22819804

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

    PubMed Central

    Piddock, Laura J. V.

    2006-01-01

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

  14. Dendritic cells phenotype fitting under hypoxia or lipopolysaccharide; adenosine 5′-triphosphate-binding cassette transporters far beyond an efflux pump

    PubMed Central

    Lloberas, N; Rama, I; Llaudó, I; Torras, J; Cerezo, G; Cassis, L; Franquesa, M; Merino, A; Benitez-Ribas, D; Cruzado, J M; Herrero-Fresneda, I; Bestard, O; Grinyó, J M

    2013-01-01

    This study examines adenosine 5′-triphosphate-binding cassette (ABC) transporters as a potential therapeutic target in dendritic cell (DC) modulation under hypoxia and lipopolysaccharide (LPS). Functional capacity of dendritic cells (DCs) (mixed lymphocyte reaction: MLR) and maturation of iDCs were evaluated in the presence or absence of specific ABC-transporter inhibitors. Monocyte-derived DCs were cultured in the presence of interleukin (IL)-4/granulocyte–macrophage colony-stimulating factor (GM-CSF). Their maturation under hypoxia or LPS conditions was evaluated by assessing the expression of maturation phenotypes using flow cytometry. The effect of ABC transporters on DC maturation was determined using specific inhibitors for multi-drug resistance (MDR1) and multi-drug resistance proteins (MRPs). Depending on their maturation status to elicit T cell alloresponses, the functional capacity of DCs was studied by MLR. Mature DCs showed higher P-glycoprotein (Pgp) expression with confocal microscopy. Up-regulation of maturation markers was observed in hypoxia and LPS-DC, defining two different DC subpopulation profiles, plasmacytoid versus conventional-like, respectively, and different cytokine release T helper type 2 (Th2) versus Th1, depending on the stimuli. Furthermore, hypoxia-DCs induced more B lymphocyte proliferation than control-iDC (56% versus 9%), while LPS-DCs induced more CD8-lymphocyte proliferation (67% versus 16%). ABC transporter-inhibitors strongly abrogated DC maturation [half maximal inhibitory concentration (IC50): P-glycoprotein inhibition using valspodar (PSC833) 5 μM, CAS 115104-28-4 (MK571) 50 μM and probenecid 2·5 μM], induced significantly less lymphocyte proliferation and reduced cytokine release compared with stimulated-DCs without inhibitors. We conclude that diverse stimuli, hypoxia or LPS induce different profiles in the maturation and functionality of DC. Pgp appears to play a role in these DC events. Thus, ABC-transporters

  15. 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

  16. 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.

  17. 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

    Chen, Wei-Ming; Sheu, Wayne H-H; 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

  18. Stimulation of lateral hypothalamic glutamate and acetylcholine efflux by nicotine: implications for mechanisms of nicotine-induced activation of orexin neurons.

    PubMed

    Pasumarthi, Ravi K; Fadel, Jim

    2010-05-01

    The hypothalamus is a prominent target of nicotine action. We have previously shown that acute systemic nicotine treatment induces Fos expression in the lateral hypothalamus and perifornical area (LH/PFA), with orexin/hypocretin neurons being particularly responsive. However, the neurochemical correlates of acute nicotine treatment in the LH/PFA have not been described. Anatomical studies have revealed that this area receives afferents from cholinergic, glutamatergic, and GABAergic telencephalic brain regions, suggesting a potential role for these neurotransmitters in mediating the hypothalamic component of nicotine effects on homeostatic phenomena, such as arousal and appetite. Here, we used in vivo microdialysis to determine the effect of acute systemic or local nicotine on glutamate, acetylcholine, and GABA efflux in the LH/PFA of rats. Local administration of nicotine significantly increased acetylcholine and glutamate, but not GABA, in the LH/PFA. Thus, we further tested the role of afferent sources of glutamate and acetylcholine in mediating acute nicotine-induced activation of orexin neurons by unilaterally lesioning the prefrontal cortex or basal forebrain cholinergic regions. Lesioned animals showed reduced Fos-positive orexin neurons following nicotine treatment. These data suggest that both acetylcholine and glutamate may mediate the effects of acute nicotine on the activity of hypothalamic neurons, including orexin/hypocretin cells. Changes in cholinergic or glutamatergic transmission in this region with chronic nicotine may contribute to long-term alterations in functions mediated by LH/PFA neurons, including feeding and arousal.

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

    PubMed Central

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

    2013-01-01

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

  20. Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions.

    PubMed

    Zahrádka, Jaromír; Sychrová, Hana

    2012-06-01

    Saccharomyces cerevisiae extrudes K(+) cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K(+) exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.

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

    PubMed

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

    2013-01-01

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

  2. 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. 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...

  5. Curcumin promotes cholesterol efflux from adipocytes related to PPARgamma-LXRalpha-ABCA1 passway.

    PubMed

    Dong, Shao-zhuang; Zhao, Shui-ping; Wu, Zhi-hong; Yang, Jun; Xie, Xiang-zhu; Yu, Bi-lian; Nie, Sai

    2011-12-01

    Curcumin affects the functions of adipocytes. But it is not known whether curcumin has some effect on the cholesterol efflux process of adipocytes. Rabbit subcutaneous adipocytes were incubated with 5, 10 and 20 μg/ml curcumin for 24 h. The cholesterol efflux onto apoAI was assessed, and the peroxisome proliferators-activated receptor (PPAR) γ, liver X receptor (LXR) α and ATP-binding cassette transporter A1 (ABCA1) mRNA expression in adipocytes were quantified by reverse-transcription polymerase chain reaction (RT-PCR). Curcumin increased the cholesterol efflux from adipocytes in dose-dependent manner. The increased expression of PPARγ, LXRα and ABCA1 caused by curcumin were parallel. When the adipocytes were pre-treated by GW9662, the increased expression of PPARγ induced by curcumin was partially prevented, subsequent to the down-regulation of LXRα and ABCA1. Curcumin can affect the cholesterol efflux from adipocytes by regulating the PPARγ-LXR-ABCA1 passway.

  6. 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.

  7. Fragment-Based Strategy for Investigating and Suppressing the Efflux of Bioactive Small Molecules.

    PubMed

    Compton, Corey L; Carney, Daniel W; Groomes, Patrice V; Sello, Jason K

    2015-01-01

    Membrane protein-mediated drug efflux is a phenomenon that compromises our ability to treat both infectious diseases and cancer. Accordingly, there is much interest in the development of strategies for suppression of the mechanisms by which therapeutic agents are effluxed. Here, using resistance to the cyclic acyldepsipeptide (ADEP) antibacterial agents as a model, we demonstrate a new counter-efflux strategy wherein a fragment of an actively exported bioactive compound competitively interferes with its efflux and potentiates its activity. A fragment comprising the N-heptenoyldifluorophenylalanine side chain of the pharmacologically optimized ADEPs potentiates the antibacterial activity of the ADEPs against actinobacteria to a greater extent than reserpine, a well-known efflux inhibitor. Beyond their validation of a new approach to studying molecular recognition by drug efflux pumps, our findings have important implications for killing Mycobacterium tuberculosis with ADEPs and reclaiming the efficacies of therapeutic agents whose activity has been compromised by efflux pumps. PMID:27620145

  8. Structural perspectives on secondary active transporters

    PubMed Central

    Boudker, Olga; Verdon, Grégory

    2010-01-01

    Secondary active transporters catalyze concentrative transport of substrates across lipid membranes by harnessing the energy of electrochemical ion gradients. These transporters bind their ligands on one side of the membrane, and undergo a global conformational change to release them on the other side of the membrane. Over the last few years, crystal structures have captured several bacterial secondary transporters in different states along their transport cycle, providing insight into possible molecular mechanisms. In this review, we will summarize recent findings focusing on the emerging structural and mechanistic similarities between evolutionary diverse transporters. We will also discuss the structural basis of substrate binding, ion coupling and inhibition viewed from the perspective of these similarities. PMID:20655602

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

    SciTech Connect

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

    2014-09-05

    Highlights: • Lipoprotein hydrolysis products were produced by lipoprotein lipase. • Hydrolysis products lowers expression of macrophage cholesterol transporters. • Hydrolysis products reduces expression of select nuclear receptors. • Fatty acid products lowers cholesterol transporters and select nuclear receptors. • Fatty acid products reduces cholesterol efflux from macrophages. - Abstract: Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

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

    PubMed

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

    2014-09-01

    Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL.

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

    PubMed

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

    2014-09-01

    Lipoprotein lipase (LPL) is an extracellular lipase that primarily hydrolyzes triglycerides within circulating lipoproteins. Macrophage LPL contributes to atherogenesis, but the mechanisms behind it are poorly understood. We hypothesized that the products of lipoprotein hydrolysis generated by LPL promote atherogenesis by inhibiting the cholesterol efflux ability by macrophages. To test this hypothesis, we treated human THP-1 macrophages with total lipoproteins that were hydrolyzed by LPL and we found significantly reduced transcript levels for the cholesterol transporters ATP binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor BI. These decreases were likely due to significant reductions for the nuclear receptors liver-X-receptor-α, peroxisome proliferator activated receptor (PPAR)-α, and PPAR-γ. We prepared a mixture of free fatty acids (FFA) that represented the ratios of FFA species within lipoprotein hydrolysis products, and we found that the FFA mixture also significantly reduced cholesterol transporters and nuclear receptors. Finally, we tested the efflux of cholesterol from THP-1 macrophages to apolipoprotein A-I, and we found that the treatment of THP-1 macrophages with the FFA mixture significantly attenuated cholesterol efflux. Overall, these data show that the FFA component of lipoprotein hydrolysis products generated by LPL may promote atherogenesis by inhibiting cholesterol efflux, which partially explains the pro-atherogenic role of macrophage LPL. PMID:25130461

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

    PubMed

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

    2016-04-15

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

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

    PubMed

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

    2016-04-15

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

  14. Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

    NASA Astrophysics Data System (ADS)

    Borisova, T. A.; Himmelreich, N. H.

    The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

  15. 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.

  16. 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.

  17. 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

  18. Ratchet transport powered by chiral active particles.

    PubMed

    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.

  19. 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

  20. 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

  1. Cost, effectiveness and environmental relevance of multidrug transporters in sea urchin embryos.

    PubMed

    Cole, Bryan J; Hamdoun, Amro; Epel, David

    2013-10-15

    ATP-binding cassette transporters protect cells via efflux of xenobiotics and endogenous byproducts of detoxification. While the cost of this ATP-dependent extrusion is known at the molecular level, i.e. the ATP used for each efflux event, the overall cost to a cell or organism of operating this defense is unclear, especially as the cost of efflux changes depending on environmental conditions. During prolonged exposure to xenobiotics, multidrug transporter activity could be costly and ineffective because effluxed substrate molecules are not modified in the process and could thus undergo repeated cycles of efflux and re-entry. Here we use embryos of the purple sea urchin, Strongylocentrotus purpuratus, as a model to determine transport costs and benefits under environmentally relevant xenobiotic concentrations. Strikingly, our results show that efflux transporter activity costs less than 0.2% of total ATP usage, as a proportion of oxygen consumption. The benefits of transport, defined as the reduction in substrate accumulation due to transporter activity, depended largely, but not entirely, on the rate of passive flux of each substrate across the plasma membrane. One of the substrates tested exhibited rapid membrane permeation coupled with high rates of efflux, thus inducing rapid and futile cycles of efflux followed by re-entry of the substrate. This combination significantly reduced transporter effectiveness as a defense and increased costs even at relatively low substrate concentrations. Despite these effects with certain substrates, our results show that efflux transporters are a remarkably effective and low-cost first line of defense against exposure to environmentally relevant concentrations of xenobiotics.

  2. Cost, effectiveness and environmental relevance of multidrug transporters in sea urchin embryos

    PubMed Central

    Cole, Bryan J.; Hamdoun, Amro; Epel, David

    2013-01-01

    SUMMARY ATP-binding cassette transporters protect cells via efflux of xenobiotics and endogenous byproducts of detoxification. While the cost of this ATP-dependent extrusion is known at the molecular level, i.e. the ATP used for each efflux event, the overall cost to a cell or organism of operating this defense is unclear, especially as the cost of efflux changes depending on environmental conditions. During prolonged exposure to xenobiotics, multidrug transporter activity could be costly and ineffective because effluxed substrate molecules are not modified in the process and could thus undergo repeated cycles of efflux and re-entry. Here we use embryos of the purple sea urchin, Strongylocentrotus purpuratus, as a model to determine transport costs and benefits under environmentally relevant xenobiotic concentrations. Strikingly, our results show that efflux transporter activity costs less than 0.2% of total ATP usage, as a proportion of oxygen consumption. The benefits of transport, defined as the reduction in substrate accumulation due to transporter activity, depended largely, but not entirely, on the rate of passive flux of each substrate across the plasma membrane. One of the substrates tested exhibited rapid membrane permeation coupled with high rates of efflux, thus inducing rapid and futile cycles of efflux followed by re-entry of the substrate. This combination significantly reduced transporter effectiveness as a defense and increased costs even at relatively low substrate concentrations. Despite these effects with certain substrates, our results show that efflux transporters are a remarkably effective and low-cost first line of defense against exposure to environmentally relevant concentrations of xenobiotics. PMID:23913944

  3. Proton and cation transport activity of the M2 proton channel from influenza A virus.

    PubMed

    Leiding, Thom; Wang, Jun; Martinsson, Jonas; DeGrado, William F; Arsköld, Sindra Peterson

    2010-08-31

    The M2 protein is a small, single-span transmembrane (TM) protein from the influenza A virus. This virus enters cells via endosomes; as the endosomes mature and become more acidic M2 facilitates proton transport into the viral interior, thereby disrupting matrix protein/RNA interactions required for infectivity. A mystery has been how protons can accumulate in the viral interior without developing a large electrical potential that impedes further inward proton translocation. Progress in addressing this question has been limited by the availability of robust methods of unidirectional insertion of the protein into virus-like vesicles. Using an optimized procedure for reconstitution, we show that M2 has antiporter-like activity, facilitating K(+) or Na(+) efflux when protons flow down a concentration gradient into the vesicles. Cation efflux is very small except under conditions mimicking those encountered by the endosomally entrapped virus, in which protons are flowing through the channel. This proton/cation exchange function is consistent with the known high proton selectivity of the channel. Thus, M2 acts as a proton uniporter that occasionally allows K(+) to flow to maintain electrical neutrality. Remarkably, as the pH inside M2-containing vesicles (pH(in)) decreases, the proton channel activity of M2 is inhibited, but its cation transport activity is activated. This reciprocal inhibition of proton flux and activation of cation flux with decreasing pH(in) first allows accumulation of protons in the early stages of acidification, then trapping of protons within the virus when low pH(in) is achieved.

  4. 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

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

    PubMed

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

    2016-09-01

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

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

    PubMed

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

    2016-09-01

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

  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. Evidence for an increased rate of choline efflux across erythrocyte membranes in Alzheimer's disease.

    PubMed

    Butterfield, D A; Nicholas, M M; Markesbery, W R

    1985-07-01

    Alzheimer's disease (AD), the major dementing disorder of the elderly, is associated with cholinergic neuronal loss and decreased activity of choline acetyltransferase (CAT). Previous biophysical studies had suggested an altered conformation of membrane proteins in AD erythrocyte ghosts. Since erythrocytes have a choline transport system and cholinergic neurons are implicated in AD, the present experiments were undertaken to determine if the efflux rate of [14C]choline was altered in AD erythrocytes. The mean efflux rate constant was highly significantly increased (P less than 0.01) by greater than 25% in 9 drug-free AD patients compared to 9 sex-matched, drug-free controls of similar age. These results are discussed in terms of potential molecular mechanisms to account for cholinergic neuronal loss in AD.

  9. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier

    PubMed Central

    Li, Yangfang; Wu, Qian; Li, Chen; Liu, Ling; Du, Kun; Shen, Jin; Wu, Yuqin; Zhao, Xiaofen; Zhao, Mei; Bao, Lingyun; Gao, Jin; Keep, Richard F.; Xiang, Jianming

    2016-01-01

    While the blood-brain barrier (BBB) protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC) transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP) but not those overexpressing human P-gp (MDCKII-MDR cells) had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated. PMID:27300692

  10. 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

  11. Health Impacts of Active Transportation in Europe.

    PubMed

    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.

  12. Health Impacts of Active Transportation in Europe.

    PubMed

    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

  13. Genomic Analysis of ATP Efflux in Saccharomyces cerevisiae.

    PubMed

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

    2015-11-19

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

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

    PubMed

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

    2016-01-01

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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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. PMID:26318642

  20. Biomarker versus environmental factors: seasonal variations and modelling multixenobiotic defence (MXD) transport activity in transplanted zebra mussels.

    PubMed

    Pain, Sandrine; Devin, Simon; Parant, Marc

    2007-02-01

    The occurrence of biomarker temporal variations linked to environmental factors makes it difficult to distinguish the specific effect of pollution. The present work aims to investigate the seasonal variations of the transport activity of the multixenobiotic defence (MXD), which is used as a biological tool for the monitoring of pollution in aquatic ecosystems. The MXD transport activity was monitored monthly from August 2001 to October 2002 in zebra mussels (Dreissena polymorpha) transplanted to three sites in the Moselle River. The 'efflux method' was used to evaluate functional activity of MXD by assessing rhodamine B efflux with or without an inhibitor (verapamil). Environmental parameters were provided by a French regulatory agency (Water Agency) that monitors river water quality. The results of a principal components analysis describe the seasonal cycle of water characteristics and demonstrate that MXD activity is subjected to significant temporal variations. These data were described with a generalised linear model that enables it to link MXD variability to the seasonal variations of environmental parameters such as temperature or levels of organic contamination. This work proposes a modelling approach and highlights that the occurrence of seasonal variations in MXD response has to be taken into account in the interpretation of in situ monitoring studies. PMID:17210171

  1. Biomarker versus environmental factors: seasonal variations and modelling multixenobiotic defence (MXD) transport activity in transplanted zebra mussels.

    PubMed

    Pain, Sandrine; Devin, Simon; Parant, Marc

    2007-02-01

    The occurrence of biomarker temporal variations linked to environmental factors makes it difficult to distinguish the specific effect of pollution. The present work aims to investigate the seasonal variations of the transport activity of the multixenobiotic defence (MXD), which is used as a biological tool for the monitoring of pollution in aquatic ecosystems. The MXD transport activity was monitored monthly from August 2001 to October 2002 in zebra mussels (Dreissena polymorpha) transplanted to three sites in the Moselle River. The 'efflux method' was used to evaluate functional activity of MXD by assessing rhodamine B efflux with or without an inhibitor (verapamil). Environmental parameters were provided by a French regulatory agency (Water Agency) that monitors river water quality. The results of a principal components analysis describe the seasonal cycle of water characteristics and demonstrate that MXD activity is subjected to significant temporal variations. These data were described with a generalised linear model that enables it to link MXD variability to the seasonal variations of environmental parameters such as temperature or levels of organic contamination. This work proposes a modelling approach and highlights that the occurrence of seasonal variations in MXD response has to be taken into account in the interpretation of in situ monitoring studies.

  2. 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.

  3. 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. PMID:26812157

  4. Cyclic AMP efflux inhibitors as potential therapeutic agents for leukemia

    PubMed Central

    Perez, Dominique R.; Smagley, Yelena; Garcia, Matthew; Carter, Mark B.; Evangelisti, Annette; Matlawska-Wasowska, Ksenia; Winter, Stuart S.; Sklar, Larry A.; Chigaev, Alexandre

    2016-01-01

    Apoptotic evasion is a hallmark of cancer. We propose that some cancers may evade cell death by regulating 3′-5′-cyclic adenosine monophosphate (cAMP), which is associated with pro-apoptotic signaling. We hypothesize that leukemic cells possess mechanisms that efflux cAMP from the cytoplasm, thus protecting them from apoptosis. Accordingly, cAMP efflux inhibition should result in: cAMP accumulation, activation of cAMP-dependent downstream signaling, viability loss, and apoptosis. We developed a novel assay to assess cAMP efflux and performed screens to identify inhibitors. In an acute myeloid leukemia (AML) model, several identified compounds reduced cAMP efflux, appropriately modulated pathways that are responsive to cAMP elevation (cAMP-responsive element-binding protein phosphorylation, and deactivation of Very Late Antigen-4 integrin), and induced mitochondrial depolarization and caspase activation. Blocking adenylyl cyclase activity was sufficient to reduce effects of the most potent compounds. These compounds also decreased cAMP efflux and viability of B-lineage acute lymphoblastic leukemia (B-ALL) cell lines and primary patient samples, but not of normal primary peripheral blood mononuclear cells. Our data suggest that cAMP efflux is a functional feature that could be therapeutically targeted in leukemia. Furthermore, because some of the identified drugs are currently used for treating other illnesses, this work creates an opportunity for repurposing. PMID:27129155

  5. 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.

  6. 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.

  7. Coupled Na/K/Cl efflux. "Reverse" unidirectional fluxes in squid giant axons

    PubMed Central

    1987-01-01

    Studies of unidirectional Cl-, Na+, and K+ effluxes were performed on isolated, internally dialyzed squid giant axons. The studies were designed to determine whether the coupled Na/K/Cl co-transporter previously identified as mediating influxes (Russell. 1983. Journal of General Physiology. 81:909-925) could also mediate the reverse fluxes (effluxes). We found that 10 microM bumetanide blocked 7-8 pmol/cm2 X s of Cl- efflux from axons containing ATP, Na+, and K+. However, if any one of these solutes was removed from the internal dialysis fluid, Cl- efflux was reduced by 7-8 pmol/cm2 X s and the remainder was insensitive to bumetanide. About 5 pmol/cm2 X s of Na+ efflux was inhibited by 10 microM bumetanide in the continuous presence of 10(-5) M ouabain and 10(-7) M tetrodotoxin if Cl-, K+, and ATP were all present in the internal dialysis fluid. However, the omission of Cl- or K+ or ATP reduced the Na+ efflux, leaving it bumetanide insensitive. K+ efflux had to be studied under voltage-clamp conditions with the membrane potential held at -90 mV because the dominant pathway for K+ efflux (the delayed rectifier) has a high degree of voltage sensitivity. Under this voltage-clamped condition, 1.8 pmol/cm2 X s of K+ efflux could be inhibited by 10 microM bumetanide. All of these results are consistent with a tightly coupled Na/K/Cl co-transporting efflux mechanism. Furthermore, the requirements for cis-side co-ions and intracellular ATP are exactly like those previously described for the coupled Na/K/Cl influx process. We propose that the same transporter mediates both influx and efflux, hence demonstrating "reversibility," a necessary property for an ion-gradient-driven transport process. PMID:3598557

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

    PubMed

    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

  9. 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

  10. Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

    SciTech Connect

    Puri, Nidhi; Manoharlal, Raman; Sharma, Monika; Sanglard, Dominique; Prasad, Rajendra

    2011-01-07

    Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte-factual concerns

  11. 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.

  12. A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

    PubMed

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

    2016-07-01

    TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. PMID:27039280

  13. Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells

    PubMed Central

    Kalayda, Ganna V; Wagner, Christina H; Buß, Irina; Reedijk, Jan; Jaehde, Ulrich

    2008-01-01

    Background Copper homeostasis proteins ATP7A and ATP7B are assumed to be involved in the intracellular transport of cisplatin. The aim of the present study was to assess the relevance of sub cellular localisation of these transporters for acquired cisplatin resistance in vitro. For this purpose, localisation of ATP7A and ATP7B in A2780 human ovarian carcinoma cells and their cisplatin-resistant variant, A2780cis, was investigated. Methods Sub cellular localisation of ATP7A and ATP7B in sensitive and resistant cells was investigated using confocal fluorescence microscopy after immunohistochemical staining. Co-localisation experiments with a cisplatin analogue modified with a carboxyfluorescein-diacetate residue were performed. Cytotoxicity of the fluorescent cisplatin analogue in A2780 and A2780cis cells was determined using an MTT-based assay. The significance of differences was analysed using Student's t test or Mann-Whitney test as appropriate, p values of < 0.05 were considered significant. Results In the sensitive cells, both transporters are mainly localised in the trans-Golgi network, whereas they are sequestrated in more peripherally located vesicles in the resistant cells. Altered localisation of ATP7A and ATP7B in A2780cis cells is likely to be a consequence of major abnormalities in intracellular protein trafficking related to a reduced lysosomal compartment in this cell line. Changes in sub cellular localisation of ATP7A and ATP7B may facilitate sequestration of cisplatin in the vesicular structures of A2780cis cells, which may prevent drug binding to genomic DNA and thereby contribute to cisplatin resistance. Conclusion Our results indicate that alterations in sub cellular localisation of transport proteins may contribute to cisplatin resistance in vitro. Investigation of intracellular protein localisation in primary tumour cell cultures and tumour tissues may help to develop markers of clinically relevant cisplatin resistance. Detection of resistant

  14. The BnALMT1 and BnALMT2 genes from rape encode aluminum-activated malate transporters that enhance the aluminum resistance of plant cells.

    PubMed

    Ligaba, Ayalew; Katsuhara, Maki; Ryan, Peter R; Shibasaka, Mineo; Matsumoto, Hideaki

    2006-11-01

    The release of organic anions from roots can protect plants from aluminum (Al) toxicity and help them overcome phosphorus (P) deficiency. Our previous findings showed that Al treatment induced malate and citrate efflux from rape (Brassica napus) roots, and that P deficiency did not induce the efflux. Since this response is similar to the malate efflux from wheat (Triticum aestivum) that is controlled by the TaALMT1 gene, we investigated whether homologs of TaALMT1 are present in rape and whether they are involved in the release of organic anions. We isolated two TaALMT1 homologs from rape designated BnALMT1 and BnALMT2 (B. napus Al-activated malate transporter). The expression of these genes was induced in roots, but not shoots, by Al treatment but P deficiency had no effect. Several other cations (lanthanum, ytterbium, and erbium) also increased BnALMT1 and BnALMT2 expression in the roots. The function of the BnALMT1 and BnALMT2 proteins was investigated by heterologous expression in cultured tobacco (Nicotiana tabacum) cells and in Xenopus laevis oocytes. Both transfection systems showed an enhanced capacity for malate efflux but not citrate efflux, when exposed to Al. Smaller malate fluxes were also activated by ytterbium and erbium treatment. Transgenic tobacco cells grew significantly better than control cells following an 18 h treatment with Al, indicating that the expression of BnALMT1 and BnALMT2 increased the resistance of these plant cells to Al stress. This report demonstrates that homologs of the TaALMT1 gene from wheat perform similar functions in other species.

  15. 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.

  16. Identification of efflux proteins using efficient radial basis function networks with position-specific scoring matrices and biochemical properties.

    PubMed

    Ou, Yu-Yen; Chen, Shu-An; Chang, Yun-Min; Velmurugan, Devadasan; Fukui, Kazuhiko; Michael Gromiha, M

    2013-09-01

    Efflux proteins are membrane proteins, which are involved in the transportation of multidrugs. The annotation of efflux proteins in genomic sequences would aid to understand the function. Although the percentage of membrane proteins in genomes is estimated to be 25-30%, there is no information about the content of efflux proteins. For annotating such class of proteins it is necessary to develop a reliable method to identify efflux proteins from amino acid sequence information. In this work, we have developed a method based on radial basis function networks using position specific scoring matrices (PSSM) and amino acid properties. We noticed that the C-terminal domain of efflux proteins contain vital information for discrimination. Our method showed an accuracy of 78 and 92% in discriminating efflux proteins from transporters and membrane proteins, respectively using fivefold cross-validation. We utilized our method for annotating the genomes E. coli and P. aeruginosa and it predicted 8.7 and 9.2% of proteins as efflux proteins in these genomes, respectively. The predicted efflux proteins have been compared with available experimental data and we observed a very good agreement between them. Further, we developed a web server for classifying efflux proteins and it is freely available at http://rbf.bioinfo.tw/∼sachen/EFFLUXpredict/Efflux-RBF.php. We suggest that our method could be an effective tool for annotating efflux proteins in genomic sequences.

  17. 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

  18. 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.

  19. Walnut oil increases cholesterol efflux through inhibition of stearoyl CoA desaturase 1 in THP-1 macrophage-derived foam cells

    PubMed Central

    2011-01-01

    Background Walnuts significantly decrease total and low-density lipoprotein cholesterol in normo- and hypercholesterolemic individuals. No study to date has evaluated the effects of walnuts on cholesterol efflux, the initial step in reverse cholesterol transport, in macrophage-derived foam cells (MDFC). The present study was conducted to investigate the mechanisms by which walnut oil affects cholesterol efflux. Methods The extract of English walnuts (walnut oil) was dissolved in DMSO and applied to cultured THP-1 MDFC cells (0.5 mg/mL). THP-1 MDFC also were treated with human sera (10%, v:v) taken from subjects in a walnut feeding study. Cholesterol efflux was examined by liquid scintillation counting. Changes in gene expression were quantified by real time PCR. Results Walnut oil treatment significantly increased cholesterol efflux through decreasing the expression of the lipogenic enzyme stearoyl CoA desaturase 1 (SCD1) in MDFC. Alpha-linolenic acid (ALA), the major n-3 polyunsaturated fatty acids found in walnuts, recaptured SCD1 reduction in MDFC, a mechanism mediated through activation of nuclear receptor farnesoid-X-receptor (FXR). Postprandial serum treatment also increased cholesterol efflux in MDFC. When categorized by baseline C-reactive protein (CRP; cut point of 2 mg/L), subjects in the lower CRP sub-group benefited more from dietary intervention, including a more increase in cholesterol efflux, a greater reduction in SCD1, and a blunted postprandial lipemia. Conclusion In conclusion, walnut oil contains bioactive molecules that significantly improve cholesterol efflux in MDFC. However, the beneficial effects of walnut intake may be reduced by the presence of a pro-inflammatory state. Trial Registration ClinicalTrials.gov: NCT00938340 PMID:21871057

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

    PubMed

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

    2016-06-24

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

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

    PubMed Central

    2016-01-01

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

  2. 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

  3. Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae

    PubMed Central

    Srinivasan, Vijaya Bharathi; Singh, Bharat Bhushan; Priyadarshi, Nitesh; Chauhan, Neeraj Kumar; Rajamohan, Govindan

    2014-01-01

    Background Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS) efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance. Principal Findings Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology. Conclusions and Significance The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae. PMID:24823362

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

    PubMed Central

    Manna, Pulak R.

    2016-01-01

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

  5. Functional cloning and characterization of the multidrug efflux pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli.

    PubMed

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

    2008-09-01

    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.

  6. Functional cloning and characterization of the multidrug efflux pumps NorM from Neisseria gonorrhoeae and YdhE from Escherichia coli.

    PubMed

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

    2008-09-01

    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

  7. Putative mycobacterial efflux inhibitors from the seeds of Aframomum melegueta.

    PubMed

    Gröblacher, Barbara; Maier, Veronika; Kunert, Olaf; Bucar, Franz

    2012-07-27

    In order to identify new putative efflux pump inhibitors that represent an appropriate target in antimycobacterial chemotherapy, nine paradol- and gingerol-related compounds (1-9) isolated from the seeds of Aframomum melegueta were assessed for their potential to inhibit ethidium bromide (EtBr) efflux in a Mycobacterium smegmatis model. Five of the compounds from A. melegueta and NMR spectroscopic data of the diketone 6-gingerdione (2) and its enolic tautomers, methyl-6-gingerol (5) and rac-6-dihydroparadol (7), are presented herein for the first time. After determination of their antimycobacterial activities and modulatory effects on the MIC of antibiotics as well as their synergistic effects in combination with antibiotics against M. smegmatis mc(2) 155, their impact on EtBr accumulation and efflux was evaluated using a microtiter plate-based fluorometric assay. The compounds exhibited moderate to weak antimycobacterial activities, and the best modulators induced a 4- to 16-fold decrease of the MICs of EtBr and rifampicin as well as a reduction of the MIC of isoniazid with fractional inhibitory concentration index values indicating synergistic activities in some cases. 6-Paradol (3), 8-gingerol (6), and rac-6-dihydroparadol (7) were the most potent EtBr efflux inhibitors in M. smegmatis mc(2) 155, displaying EtBr efflux inhibiting activities comparable to reference inhibitors.

  8. 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

  9. Boosting Effect of 2-Phenylquinoline Efflux Inhibitors in Combination with Macrolides against Mycobacterium smegmatis and Mycobacterium avium.

    PubMed

    Machado, Diana; Cannalire, Rolando; Santos Costa, Sofia; Manfroni, Giuseppe; Tabarrini, Oriana; Cecchetti, Violetta; Couto, Isabel; Viveiros, Miguel; Sabatini, Stefano

    2015-12-11

    The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4'-propoxyphenyl)quinoline Staphylococcus aureus NorA efflux inhibitors (1-4) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, O-ethylpiperazinyl derivative 2 showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different M. avium strains. The contribution of the M. avium efflux pumps MAV_1406 and MAV_1695 to clarithromycin resistance was proved because they were found to be overexpressed in two M. avium 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound 2. Additionally, compound 2 showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against M. avium 104-infected macrophages. In summary, the 2-(4'-propoxyphenyl)quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both S. aureus and nontuberculous mycobacteria. PMID:27623057

  10. A second mechanism for aluminum resistance in wheat relies on the constitutive efflux of citrate from roots.

    PubMed

    Ryan, Peter R; Raman, Harsh; Gupta, Sanjay; Horst, Walter J; Delhaize, Emmanuel

    2009-01-01

    The first confirmed mechanism for aluminum (Al) resistance in plants is encoded by the wheat (Triticum aestivum) gene, TaALMT1, on chromosome 4DL. TaALMT1 controls the Al-activated efflux of malate from roots, and this mechanism is widespread among Al-resistant genotypes of diverse genetic origins. This study describes a second mechanism for Al resistance in wheat that relies on citrate efflux. Citrate efflux occurred constitutively from the roots of Brazilian cultivars Carazinho, Maringa, Toropi, and Trintecinco. Examination of two populations segregating for this trait showed that citrate efflux was controlled by a single locus. Whole-genome linkage mapping using an F(2) population derived from a cross between Carazinho (citrate efflux) and the cultivar EGA-Burke (no citrate efflux) identified a major locus on chromosome 4BL, Xce(c), which accounts for more than 50% of the phenotypic variation in citrate efflux. Mendelizing the quantitative variation in citrate efflux into qualitative data, the Xce(c) locus was mapped within 6.3 cM of the microsatellite marker Xgwm495 locus. This linkage was validated in a second population of F(2:3) families derived from a cross between Carazinho and the cultivar Egret (no citrate efflux). We show that expression of an expressed sequence tag, belonging to the multidrug and toxin efflux (MATE) gene family, correlates with the citrate efflux phenotype. This study provides genetic and physiological evidence that citrate efflux is a second mechanism for Al resistance in wheat.

  11. Glial cell ceruloplasmin and hepcidin differentially regulate iron efflux from brain microvascular endothelial cells.

    PubMed

    McCarthy, Ryan C; Kosman, Daniel J

    2014-01-01

    We have used an in vitro model system to probe the iron transport pathway across the brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). This model consists of human BMVEC (hBMVEC) and C6 glioma cells (as an astrocytic cell line) grown in a transwell, a cell culture system commonly used to quantify metabolite flux across a cell-derived barrier. We found that iron efflux from hBMVEC through the ferrous iron permease ferroportin (Fpn) was stimulated by secretion of the soluble form of the multi-copper ferroxidase, ceruloplasmin (sCp) from the co-cultured C6 cells. Reciprocally, expression of sCp mRNA in the C6 cells was increased by neighboring hBMVEC. In addition, data indicate that C6 cell-secreted hepcidin stimulates internalization of hBMVEC Fpn but only when the end-feet projections characteristic of this glia-derived cell line are proximal to the endothelial cells. This hepcidin-dependent loss of Fpn correlated with knock-down of iron efflux from the hBMVEC; this result was consistent with the mechanism by which hepcidin regulates iron efflux in mammalian cells. In summary, the data support a model of iron trafficking across the BBB in which the capillary endothelium induce the underlying astrocytes to produce the ferroxidase activity needed to support Fpn-mediated iron efflux. Reciprocally, astrocyte proximity modulates the effective concentration of hepcidin at the endothelial cell membrane and thus the surface expression of hBMVEC Fpn. These results are independent of the source of hBMVEC iron (transferrin or non-transferrin bound) indicating that the model developed here is broadly applicable to brain iron homeostasis.

  12. Tripartite assembly of RND multidrug efflux pumps

    PubMed Central

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

    2016-01-01

    Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB–OprM and Escherichia coli AcrAB–TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA–MexB–TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components. PMID:26867482

  13. 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.

  14. 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

  15. Adenosine monophosphate-activated protein kinase activation, substrate transporter translocation, and metabolism in the contracting hyperthyroid rat heart.

    PubMed

    Heather, Lisa C; Cole, Mark A; Atherton, Helen J; Coumans, Will A; Evans, Rhys D; Tyler, Damian J; Glatz, Jan F C; Luiken, Joost J F P; Clarke, Kieran

    2010-01-01

    Thyroid hormones can modify cardiac metabolism via multiple molecular mechanisms, yet their integrated effect on overall substrate metabolism is poorly understood. Here we determined the effect of hyperthyroidism on substrate metabolism in the isolated, perfused, contracting rat heart. Male Wistar rats were injected for 7 d with T(3) (0.2 mg/kg x d ip). Plasma free fatty acids increased by 97%, heart weights increased by 33%, and cardiac rate pressure product, an indicator of contractile function, increased by 33% in hyperthyroid rats. Insulin-stimulated glycolytic rates and lactate efflux rates were increased by 33% in hyperthyroid rat hearts, mediated by an increased insulin-stimulated translocation of the glucose transporter GLUT4 to the sarcolemma. This was accompanied by a 70% increase in phosphorylated AMP-activated protein kinase (AMPK) and a 100% increase in phosphorylated acetyl CoA carboxylase, confirming downstream signaling from AMPK. Fatty acid oxidation rates increased in direct proportion to the increased heart weight and rate pressure product in the hyperthyroid heart, mediated by synchronized changes in mitochondrial enzymes and respiration. Protein levels of the fatty acid transporter, fatty acid translocase (FAT/CD36), were reduced by 24% but were accompanied by a 19% increase in the sarcolemmal content of fatty acid transport protein 1 (FATP1). Thus, the relationship between fatty acid metabolism, cardiac mass, and contractile function was maintained in the hyperthyroid heart, associated with a sarcolemmal reorganization of fatty acid transporters. The combined effects of T(3)-induced AMPK activation and insulin stimulation were associated with increased sarcolemmal GLUT4 localization and glycolytic flux in the hyperthyroid heart. PMID:19940039

  16. Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3'-deoxyadenosine (cordycepin).

    PubMed

    Agutter, P S; McCaldin, B

    1979-05-15

    The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed.

  17. Inhibition of ribonucleic acid efflux from isolated SV40-3T3 cell nuclei by 3'-deoxyadenosine (cordycepin).

    PubMed Central

    Agutter, P S; McCaldin, B

    1979-01-01

    The effect of 3'-deoxyadenosine (cordycepin) on mRNA efflux from isolated SV40-3T3 cell nuclei has been studied and compared with its effect on the nucleoside triphosphatase activity in the isolated nuclear envelope. Inhibition of mRNA efflux occurs rapidly, but is dependent on the presence of ATP. Half-maximal inhibition occurs with 40 microM-cordycepin. The effect is not simulated by 2'-deoxyadenosine or by actinomycin D, and adenosine provides a substantial degree of protection against it. Cordycepin does not directly inhibit the nucleoside triphosphatase. The stimulation of this enzyme by poly(A) is not affected unless the poly(A) and cordycepin are incubated together with nuclear lysate in the presence of ATP; in this case the stimulation is significantly reduced. Possible interpretations of these results and their relevance for understanding the system in vivo for nucleo-cytoplasmic messenger transport are discussed. PMID:226073

  18. 3-(Benzo[d][1,3]dioxol-5-ylamino)-N-(4-fluorophenyl)thiophene-2-carboxamide overcomes cancer chemoresistance via inhibition of angiogenesis and P-glycoprotein efflux pump activity.

    PubMed

    Mudududdla, Ramesh; Guru, Santosh K; Wani, Abubakar; Sharma, Sadhana; Joshi, Prashant; Vishwakarma, Ram A; Kumar, Ajay; Bhushan, Shashi; Bharate, Sandip B

    2015-04-14

    3-((Quinolin-4-yl)methylamino)-N-(4-(trifluoromethoxy)phenyl)thiophene-2-carboxamide (OSI-930, 1) is a potent inhibitor of c-kit and VEGFR2, currently under phase I clinical trials in patients with advanced solid tumors. In order to understand the structure-activity relationship, a series of 3-arylamino N-aryl thiophene 2-carboxamides were synthesized by modifications at both quinoline and amide domains of the OSI-930 scaffold. All the synthesized compounds were screened for in vitro cytotoxicity in a panel of cancer cell lines and for VEGFR1 and VEGFR2 inhibition. Thiophene 2-carboxamides substituted with benzo[d][1,3]dioxol-5-yl and 2,3-dihydrobenzo[b][1,4]dioxin-6-yl groups 1l and 1m displayed inhibition of VEGFR1 with IC50 values of 2.5 and 1.9 μM, respectively. Compounds 1l and 1m also inhibited the VEGF-induced HUVEC cell migration, indicating its anti-angiogenic activity. OSI-930 along with compounds 1l and 1m showed inhibition of P-gp efflux pumps (MDR1, ABCB1) with EC50 values in the range of 35-74 μM. The combination of these compounds with doxorubicin led to significant enhancement of the anticancer activity of doxorubicin in human colorectal carcinoma LS180 cells, which was evident from the improved IC50 of doxorubicin, the increased activity of caspase-3 and the significant reduction in colony formation ability of LS180 cells after treatment with doxorubicin. Compound 1l showed a 13.8-fold improvement in the IC50 of doxorubicin in LS180 cells. The ability of these compounds to display dual inhibition of VEGFR and P-gp efflux pumps demonstrates the promise of this scaffold for its development as multi-drug resistance-reversal agents.

  19. 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.

  20. 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.

  1. In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

    PubMed Central

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

    2014-01-01

    There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient. PMID:24638061

  2. Blonanserin, a novel atypical antipsychotic agent not actively transported as substrate by P-glycoprotein.

    PubMed

    Inoue, Tomoko; Osada, Kenichi; Tagawa, Masaaki; Ogawa, Yuriko; Haga, Toshiaki; Sogame, Yoshihisa; Hashizume, Takanori; Watanabe, Takashi; Taguchi, Atsushi; Katsumata, Takashi; Yabuki, Masashi; Yamaguchi, Noboru

    2012-10-01

    Although blonanserin, a novel atypical antipsychotic agent with dopamine D(2)/serotonin 5-HT(2A) antagonistic properties, displays good brain distribution, the mechanism of this distribution has not been clarified. P-glycoprotein [(P-gp) or multidrug resistance protein 1 (MDR1)] is an efflux transporter expressed in the brain and plays an important role in limiting drug entry into the central nervous system (CNS). In particular, P-gp can affect the pharmacokinetics and efficacy of antipsychotics, and exacerbate or soothe their adverse effects. In this study, we conducted in vitro and in vivo experiments to determine whether blonanserin is a P-gp substrate. Risperidone and its active metabolite 9-hydroxyrisperidone, both of which are P-gp substrates, were used as reference drugs. Affinity of blonanserin, risperidone, and 9-hydroxyrisperidone for P-gp was evaluated by in vitro transcellular transport across LLC-PK1, human MDR1 cDNA-transfected LLC-PK1 (LLC-MDR1), and mouse Mdr1a cDNA-transfected LLC-PK1 (LLC-Mdr1a). In addition, pharmacokinetic parameters in the brain and plasma (B/P ratio) of test compounds were measured in mdr1a/1b knockout (KO) and wild-type (WT) mice. The results of in vitro experiments revealed that P-gp does not actively transport blonanserin as a substrate in humans or mice. In addition, blonanserin displayed comparable B/P ratios in KO and WT mice, whereas B/P ratios of risperidone and 9-hydroxyrisperidone differed markedly in these animals. Our results indicate that blonanserin is not a P-gp substrate and therefore its brain distribution is unlikely to be affected by this transporter.

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

    PubMed Central

    Bernhardt, Julius; Neumann, Eberhard

    1978-01-01

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

  4. Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Staphylococcus aureus

    PubMed Central

    2011-01-01

    Background Antimicrobial resistance mediated by efflux systems is still poorly characterized in Staphylococcus aureus, despite the description of several efflux pumps (EPs) for this bacterium. In this work we used several methodologies to characterize the efflux activity of 52 S. aureus isolates resistant to ciprofloxacin collected in a hospital in Lisbon, Portugal, in order to understand the role played by these systems in the resistance to fluoroquinolones. Results Augmented efflux activity was detected in 12 out of 52 isolates and correlated with increased resistance to fluoroquinolones. Addition of efflux inhibitors did not result in the full reversion of the fluoroquinolone resistance phenotype, yet it implied a significant decrease in the resistance levels, regardless of the type(s) of mutation(s) found in the quinolone-resistance determining region of grlA and gyrA genes, which accounted for the remaining resistance that was not efflux-mediated. Expression analysis of the genes coding for the main efflux pumps revealed increased expression only in the presence of inducing agents. Moreover, it showed that not only different substrates can trigger expression of different EP genes, but also that the same substrate can promote a variable response, according to its concentration. We also found isolates belonging to the same clonal type that showed different responses towards drug exposure, thus evidencing that highly related clinical isolates may diverge in the efflux-mediated response to noxious agents. The data gathered by real-time fluorometric and RT-qPCR assays suggest that S. aureus clinical isolates may be primed to efflux antimicrobial compounds. Conclusions The results obtained in this work do not exclude the importance of mutations in resistance to fluoroquinolones in S. aureus, yet they underline the contribution of efflux systems for the emergence of high-level resistance. All together, the results presented in this study show the potential role

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

    SciTech Connect

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

    1990-02-26

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

  6. Transport of biologically active material in laser cutting.

    PubMed

    Frenz, M; Mathezloic, F; Stoffel, M H; Zweig, A D; Romano, V; Weber, H P

    1988-01-01

    The transport of biologically active material during laser cutting with CO2 and Er lasers is demonstrated. This transport mechanism removes particles from the surface of gelatin, agar, and liver samples into the depth of the laser-formed craters. The transport phenomenon is explained by a contraction and condensation of enclosed hot water vapor. We show by cultivating transported bacteria in agar that biological particles can survive the shock of the transport. Determination of the numbers of active cells evidences a more pronounced activity of the cultivated bacteria after impact with an Er laser than with a CO2 laser.

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

    PubMed

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

    2014-05-22

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

  8. Structure of the AcrAB-TolC multidrug efflux pump

    PubMed Central

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

    2015-01-01

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

  9. 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

  10. 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)

  11. 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.

  12. 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

  13. The accumulation and efflux of lead partly depend on ATP-dependent efflux pump-multidrug resistance protein 1 and glutathione in testis Sertoli cells.

    PubMed

    Huang, Shaoxin; Ye, Jingping; Yu, Jun; Chen, Li; Zhou, Langhuan; Wang, Hong; Li, Zhen; Wang, Chunhong

    2014-05-01

    Since lead accumulation is toxic to cells, its excretion is crucial for organisms to survive the toxicity. In this study, mouse testis sertoli (TM4) and Mrp1 lower-expression TM4-sh cells were used to explore the lead accumulation characteristics, and the role of ATP-dependent efflux pump-multidrug resistance protein 1 (Mrp1) in lead excretion. TM4 cells possess Mrp-like transport activity. The expression levels of mrp1 mRNA and Mrp1 increased after lead treatments at first and then decreased. The maximum difference of relative mRNA expression reached 10 times. In the presence of lead acetate, the amount of cumulative lead in TM4-sh was much higher than that in TM4. After the treatment with lead acetate at 10-40 μM for 12h or 24h, the differences were about 2-8 times. After with the switch to lead-free medium, the cellular lead content in TM4-sh remains higher than that in TM4 cells at 1,3, 6, and 9h time points (P<0.01). Energy inhibitor sodium azide, Mrp inhibitors MK571 and glutathione (GSH) biosynthesis inhibitor BSO could block lead efflux from TM4 cells significantly. These results indicate that lead excretion may be mediated by Mrp1 and GSH in TM4 cells. Mrp1 could be one of the important intervention points for lead detoxification.

  14. The role of the rice aquaporin Lsi1 in arsenite efflux from roots.

    PubMed

    Zhao, Fang-Jie; Ago, Yukiko; Mitani, Namiki; Li, Ren-Ying; Su, Yu-Hong; Yamaji, Naoki; McGrath, Steve P; Ma, Jian Feng

    2010-04-01

    *When supplied with arsenate (As(V)), plant roots extrude a substantial amount of arsenite (As(III)) to the external medium through as yet unidentified pathways. The rice (Oryza sativa) silicon transporter Lsi1 (OsNIP2;1, an aquaporin channel) is the major entry route of arsenite into rice roots. Whether Lsi1 also mediates arsenite efflux was investigated. *Expression of Lsi1 in Xenopus laevis oocytes enhanced arsenite efflux, indicating that Lsi1 facilitates arsenite transport bidirectionally. *Arsenite was the predominant arsenic species in arsenate-exposed rice plants. During 24-h exposure to 5 mum arsenate, rice roots extruded arsenite to the external medium rapidly, accounting for 60-90% of the arsenate uptake. A rice mutant defective in Lsi1 (lsi1) extruded significantly less arsenite than the wild-type rice and, as a result, accumulated more arsenite in the roots. By contrast, Lsi2 mutation had little effect on arsenite efflux to the external medium. *We conclude that Lsi1 plays a role in arsenite efflux in rice roots exposed to arsenate. However, this pathway accounts for only 15-20% of the total efflux, suggesting the existence of other efflux transporters.

  15. Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Escherichia coli.

    PubMed

    Paltansing, Sunita; Tengeler, Anouk C; Kraakman, Margriet E M; Claas, Eric C J; Bernards, Alexandra T

    2013-12-01

    Resistance to ciprofloxacin in Escherichia coli is increasing parallel to increased use of fluoroquinolones both in The Netherlands and in other European countries. The objective was to investigate the contribution of active efflux and expression of outer membrane proteins (OMPs) in a collection of clinical E. coli isolates collected at a clinical microbiology department in a Dutch hospital. Forty-seven E. coli isolates a wide range of ciprofloxacin minimum inhibitory concentrations and known mutations in the quinolone resistance determining region were included. A fluorometric determination of bisbenzimide efflux was used two different efflux pump inhibitors and compared to quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the expression levels of acrA, acrB, tolC, yhiV, and mdfA efflux pump genes and the OMPs ompF and ompX. Six isolates (12.7%) showed increased efflux. Although in 35 isolates (76%), overexpression of ≥1 efflux pump genes using qRT-PCR was present. Only the combined overexpression of acrAB-TolC and mdfA correlated with the phenotypic efflux assay using glucose/carbonyl cyanide m-chlorophenylhydrazone with glucose. Thus, efflux was involved in ciprofloxacin resistance in a limited number of E. coli isolates collected at a clinical microbiology department in a Dutch hospital complementing other resistance mechanisms.

  16. P-glycoprotein Mediated Efflux Modulators of Plant Origin: A Short Review.

    PubMed

    Silva, Nuno; Salgueiro, Lígia; Fortuna, Ana; Cavaleiro, Carlos

    2016-05-01

    Drug efflux transporters such as P-glycoprotein (P-gp) help maintain cellular homeostasis but are also major contributors to the development of multidrug resistance (MDR) phenomena. Since P-gp was associated with MDR, several compounds showing potential to inhibit this transporter have been identified. Particular attention has been given to natural products, namely those of plant origin, looking for highly effective and safe P-gp inhibitors with little to no interaction with other cellular or metabolic processes. Here we abridge several examples of plant compounds from distinct classes, polyketides, lignans, anthraquinones, coumarins, alkaloids, mono- and sesqui-terpenes, steroids and limonoids, which have shown the ability to modulate in vitro or in vivo the P-gp activity.

  17. 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

  18. 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

  19. HDL-apolipoprotein A-I exchange is independently associated with cholesterol efflux capacity.

    PubMed

    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

    2015-10-01

    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.

  20. Copper transport in the yeast Saccharomyces cerevisiae

    SciTech Connect

    Martinez, L.D.; Connelly, J.L.

    1987-05-01

    Biochemical processes involved in the movement of copper (Cu) into and out of the yeast Saccharomyces Cerevisiae have been investigated. Overall uptake of Cu was measured by disappearance of Cu from the reaction mixture by atomic absorption sensitive to 10/sup -10/M. The process of Cu influx is composed of a prerequisite binding and subsequent transport. The binding is non-energetic but is competitively inhibited by zinc(Zn). Transport is energetic as shown by an increased influx in the presence of added glucose. This process is prevented by 2,4-dinitrophenol(DNP). Cu influx is accompanied by an exchange for potassium(K) in a ratio of K:Cu=2:1. The process of Cu efflux involves a second type of binding site, probably of low affinity but large capacity. The presence of glucose causes the binding of extracellular Cu to these sites in a non-energy-dependent mechanism which prevents Cu efflux. Zn does not compete. DNP has no effect. The K:Cu ratio of 4:1 observed in the absence of glucose suggests a lowered net Cu uptake as a result of concomitant efflux activity. Finally, in the absence but not the presence of glucose, the pH of the extracellular solution increases. These observations are consistent with the idea that (a) yeast membrane has two Cu-binding sites, one of which participates in influx and one in efflux; (b) Cu exchanges with K during influx and with protons during efflux.

  1. 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

  2. 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…

  3. 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

  4. Mechanism of coupling drug transport reactions located in two different membranes

    PubMed Central

    Zgurskaya, Helen I.; Weeks, Jon W.; Ntreh, Abigail T.; Nickels, Logan M.; Wolloscheck, David

    2015-01-01

    Gram- negative bacteria utilize a diverse array of multidrug transporters to pump toxic compounds out of the cell. Some transporters, together with periplasmic membrane fusion proteins (MFPs) and outer membrane channels, assemble trans-envelope complexes that expel multiple antibiotics across outer membranes of Gram-negative bacteria and into the external medium. Others further potentiate this efflux by pumping drugs across the inner membrane into the periplasm. Together these transporters create a powerful network of efflux that protects bacteria against a broad range of antimicrobial agents. This review is focused on the mechanism of coupling transport reactions located in two different membranes of Gram-negative bacteria. Using a combination of biochemical, genetic and biophysical approaches we have reconstructed the sequence of events leading to the assembly of trans-envelope drug efflux complexes and characterized the roles of periplasmic and outer membrane proteins in this process. Our recent data suggest a critical step in the activation of intermembrane efflux pumps, which is controlled by MFPs. We propose that the reaction cycles of transporters are tightly coupled to the assembly of the trans-envelope complexes. Transporters and MFPs exist in the inner membrane as dormant complexes. The activation of complexes is triggered by MFP binding to the outer membrane channel, which leads to a conformational change in the membrane proximal domain of MFP needed for stimulation of transporters. The activated MFP-transporter complex engages the outer membrane channel to expel substrates across the outer membrane. The recruitment of the channel is likely triggered by binding of effectors (substrates) to MFP or MFP-transporter complexes. This model together with recent structural and functional advances in the field of drug efflux provides a fairly detailed understanding of the mechanism of drug efflux across the two membranes. PMID:25759685

  5. 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

  6. Proteasome Regulator Marizomib (NPI-0052) Exhibits Prolonged Inhibition, Attenuated Efflux, and Greater Cytotoxicity than Its Reversible Analogs

    PubMed Central

    Obaidat, Amanda; Weiss, Jeffrey; Wahlgren, Brett; Manam, Rama R.; Macherla, Venkat R.; McArthur, Katherine; Chao, Ta-Hsiang; Palladino, Michael A.; Lloyd, G. Kenneth; Potts, Barbara C.; Enna, Salvatore J.; Neuteboom, Saskia T. C.

    2011-01-01

    The present study was undertaken to compare the cellular transport characteristics of [3H]NPI-0052 (1R,4R,5S)-4-(2-chloroethyl)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione (marizomib; salinosporamide A) and [3H]NPI-0047 (1R,4R, 5S)-1-((S)-((S)-cyclohex-2-enyl)(hydroxy)methyl)-4-ethyl-5-methyl-6-oxa-2-azabicyclo[3.2.0]heptane-3,7-dione in RPMI 8226 multiple myeloma and PC-3 prostate adenocarcinoma cells to determine whether these properties explain differences in the cytotoxic potencies of these chemical analogs. The results indicate that marizomib, which possesses a chemical-leaving group, is more cytotoxic to both cell lines and inhibits proteasome activity more completely at lower concentrations than NPI-0047, a nonleaving-group analog. Moreover, it was found that both compounds accumulate in these cells by simple diffusion and the same carrier-mediated transport system. Although the rate of uptake is similar, the cellular efflux, which does not seem to be mediated by a major ATP-binding cassette (ABC)-efflux transporter, is more rapid for NPI-0047 than for marizomib. Experiments revealed that the irreversible binding of marizomib to the proteasome is responsible for its slower efflux, longer duration of action, and greater cytotoxicity compared with NPI-0047. The discovery that major ABC transporters of the multidrug resistance-associated protein family do not seem to be involved in the accumulation or removal of these agents suggests they may not be affected by multidrug resistance mechanisms during prolonged administration. PMID:21303921

  7. 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.

  8. The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of "off Target" Gene (glnE) Expression.

    PubMed

    Johnson, Paul J T; Shafer, William M

    2015-06-01

    MtrR is a well-characterized repressor of the Neisseria gonorrhoeae mtrCDE efflux pump operon. However, results from a previous transcriptional profiling study suggested that MtrR also represses or activates expression of at least sixty genes outside of the mtr locus. Evidence that MtrR can directly repress so-called "off target" genes has previously been reported; in particular, MtrR was shown to directly repress glnA, which encodes glutamine synthetase. In contrast, evidence for the ability of MtrR to directly activate expression of gonococcal genes has been lacking; herein, we provide such evidence. We now report that MtrR has the ability to directly activate expression of glnE, which encodes the dual functional adenyltransferase/deadenylase enzyme GlnE that modifies GlnA resulting in regulation of its role in glutamine biosynthesis. With its capacity to repress expression of glnA, the results presented herein emphasize the diverse and often opposing regulatory properties of MtrR that likely contributes to the overall physiology and metabolism of N. gonorrhoeae. PMID:26078871

  9. 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

  10. The effect of N-acetylcysteine on chloride efflux from airway epithelial cells.

    PubMed

    Varelogianni, Georgia; Oliynyk, Igor; Roomans, Godfried M; Johannesson, Marie

    2010-03-01

    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. PMID:19947928

  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. Sodium efflux from perfused giant algal cells.

    PubMed

    Clint, G M; Macrobbie, E A

    1987-06-01

    Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including (22)Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 μM N,N'-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.

  13. Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton

    PubMed Central

    Venosa, R A

    2003-01-01

    Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (π) from its normal value (π= 1) to one-half (π= 0.5), but also in muscles that are returned to π= 1 after equilibration in π= 2 medium. The equilibration in π= 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+–K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 μm). Under those conditions, the π= 1 →π= 0.5 transfer produced a 43 % increase in pump sites, while the π= 2 →π= 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 μm) and intermediate filaments by acrylamide (4 mm) did not alter the hypotonic effect. Likewise, genistein (100 μm), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 μm) and latrunculin B (10 μm) reduced the increase in Na+ efflux induced by π= 1 →π= 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by π= 1 →π= 0.5 and π= 2 →π= 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport. PMID:12598593

  14. 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.

  15. High-Density Lipoprotein Function Measurement in Human Studies: Focus on Cholesterol Efflux Capacity.

    PubMed

    Rohatgi, Anand

    2015-01-01

    A low plasma level of high-density lipoprotein (HDL) cholesterol (HDL-C) is a major risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). However, several observations have highlighted the shortcomings of using cholesterol content as the sole reflection of HDL metabolism. In particular, several large randomized controlled trials of extended release niacin and cholesteryl-ester transfer protein (CETP) inhibitors on background statin therapy have failed to show improvement in ASCVD outcomes despite significant increases in HDL-C. Reverse cholesterol transport (RCT) is the principal HDL function that impacts macrophage foam cell formation and other functions such as endothelial activation of endothelial nitric oxide synthase, monocyte adhesion, and platelet aggregation. Cholesterol efflux from macrophages to plasma/serum reflects the first critical step of RCT and is considered a key anti-atherosclerotic function of HDL. Whether this function is operative in humans remains to be seen, but recent studies assessing cholesterol efflux in humans suggest that the cholesterol efflux capacity (CEC) of human plasma or serum is a potent marker of ASCVD risk. This review describes the methodology of measuring CEC ex vivo from human samples and the findings to date linking CEC to human disease. Studies to date confirm that CEC can be reliably measured using stored human blood samples as cholesterol acceptors and suggest that CEC may be a promising new biomarker for atherosclerotic and metabolic diseases. Further studies are needed to standardize measurements and clarify the role CEC may play in predicting risk of developing disease and response to therapies. PMID:25968932

  16. High-Density Lipoprotein Function Measurement in Human Studies: Focus on Cholesterol Efflux Capacity

    PubMed Central

    Rohatgi, Anand

    2015-01-01

    A low plasma level of high-density lipoprotein (HDL) cholesterol (HDL-C) is a major risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). However, several observations have highlighted the shortcomings of using cholesterol content as the sole reflection of HDL metabolism. In particular, several large randomized controlled trials of extended release niacin and cholesteryl-ester transfer protein (CETP) inhibitors on background statin therapy have failed to show improvement in ASCVD outcomes despite significant increases in HDL-C. Reverse cholesterol transport (RCT) is the principal HDL function that impacts macrophage foam cell formation and other functions such as endothelial activation of endothelial nitric oxide synthase, monocyte adhesion, and platelet aggregation. Cholesterol efflux from macrophages to plasma/serum reflects the first critical step of RCT and is considered a key anti-atherosclerotic function of HDL. Whether this function is operative in humans remains to be seen, but recent studies assessing cholesterol efflux in humans suggest that the cholesterol efflux capacity (CEC) of human plasma or serum is a potent marker of ASCVD risk. This review describes the methodology of measuring CEC ex vivo from human samples and the findings to date linking CEC to human disease. Studies to date confirm that CEC can be reliably measured using stored human blood samples as cholesterol acceptors and suggest that CEC may be a promising new biomarker for atherosclerotic and metabolic diseases. Further studies are needed to standardize measurements and clarify the role CEC may play in predicting risk of developing disease and response to therapies. PMID:25968932

  17. Clonal relatedness is a predictor of spontaneous multidrug efflux pump gene overexpression in Staphylococcus aureus.

    PubMed

    Schindler, Bryan D; Jacinto, Pauline L; Buensalido, Joseph Adrian L; Seo, Susan M; Kaatz, Glenn W

    2015-05-01

    Increased expression of genes encoding multidrug resistance efflux pumps (MDR-EPs) contributes to antimicrobial agent and biocide resistance in Staphylococcus aureus. Previously identified associations between norA overexpression and spa type t002 meticillin-resistant S. aureus (MRSA), and a similar yet weaker association between mepA overexpression and type t008 meticillin-susceptible S. aureus (MSSA), in clinical isolates are suggestive of clonal dissemination. It is also possible that related strains are prone to mutations resulting in overexpression of specific MDR-EP genes. Exposure of non-MDR-EP-overexpressing clinical isolates to biocides and dyes can select for MDR-EP-overexpressing mutants. spa types t002 and t008 isolates are predominated by multilocus sequencing typing sequence types (STs) 5 and 8, respectively. In this study, non-MDR-EP gene-overexpressing clinical isolates (MRSA and MSSA) representing ST5 and ST8 were subjected to single exposures of ethidium bromide (EtBr) to select for EtBr-resistant mutants. Measurements of active EtBr transport among mutants were used to demonstrate an efflux-proficient phenotype. Using quantitative reverse-transcription PCR, it was found that EtBr-resistant mutants of ST5 and ST8 parental strains predominantly overexpressed mepA (100%) and mdeA (83%), respectively, regardless of meticillin sensitivity. Associations between clonal lineage and MDR-EP gene overexpression differed from those previously observed and suggest the latter is due to clonal spread of efflux-proficient strains. The predilection of in vitro-selected mutants of related strains to overexpress the same MDR-EP gene indicates the presence of a consistent mutational process.

  18. High-Density Lipoprotein Function Measurement in Human Studies: Focus on Cholesterol Efflux Capacity.

    PubMed

    Rohatgi, Anand

    2015-01-01

    A low plasma level of high-density lipoprotein (HDL) cholesterol (HDL-C) is a major risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). However, several observations have highlighted the shortcomings of using cholesterol content as the sole reflection of HDL metabolism. In particular, several large randomized controlled trials of extended release niacin and cholesteryl-ester transfer protein (CETP) inhibitors on background statin therapy have failed to show improvement in ASCVD outcomes despite significant increases in HDL-C. Reverse cholesterol transport (RCT) is the principal HDL function that impacts macrophage foam cell formation and other functions such as endothelial activation of endothelial nitric oxide synthase, monocyte adhesion, and platelet aggregation. Cholesterol efflux from macrophages to plasma/serum reflects the first critical step of RCT and is considered a key anti-atherosclerotic function of HDL. Whether this function is operative in humans remains to be seen, but recent studies assessing cholesterol efflux in humans suggest that the cholesterol efflux capacity (CEC) of human plasma or serum is a potent marker of ASCVD risk. This review describes the methodology of measuring CEC ex vivo from human samples and the findings to date linking CEC to human disease. Studies to date confirm that CEC can be reliably measured using stored human blood samples as cholesterol acceptors and suggest that CEC may be a promising new biomarker for atherosclerotic and metabolic diseases. Further studies are needed to standardize measurements and clarify the role CEC may play in predicting risk of developing disease and response to therapies.

  19. Monocytes from HIV+ individuals show impaired cholesterol efflux and increased foam cell formation after transendothelial migration

    PubMed Central

    MAISA, Anna; HEARPS, Anna C.; ANGELOVICH, Thomas A.; PEREIRA, Candida F.; ZHOU, Jingling; SHI, Margaret D.Y.; PALMER, Clovis S.; MULLER, William A.; CROWE, Suzanne M.; JAWOROWSKI, Anthony

    2016-01-01

    Design HIV+ individuals have an increased risk of atherosclerosis and cardiovascular disease which is independent of antiretroviral therapy and traditional risk factors. Monocytes play a central role in the development of atherosclerosis, and HIV-related chronic inflammation and monocyte activation may contribute to increased atherosclerosis, but the mechanisms are unknown. Methods Using an in vitro model of atherosclerotic plaque formation, we measured the transendothelial migration of purified monocytes from age-matched HIV+ and uninfected donors and examined their differentiation into foam cells. Cholesterol efflux and the expression of cholesterol metabolism genes were also assessed. Results Monocytes from HIV+ individuals showed increased foam cell formation compared to controls (18.9% vs 0% respectively, p=0.004) and serum from virologically suppressed HIV+ individuals potentiated foam cell formation by monocytes from both uninfected and HIV+ donors. Plasma TNF levels were increased in HIV+ vs control donors (5.9 vs 3.5 pg/ml, p=0.02) and foam cell formation was inhibited by blocking antibodies to TNF receptors, suggesting a direct effect on monocyte differentiation to foam cells. Monocytes from virologically suppressed HIV+ donors showed impaired cholesterol efflux and decreased expression of key genes regulating cholesterol metabolism, including the cholesterol transporter ABCA1 (p=0.02). Conclusions Monocytes from HIV+ individuals show impaired cholesterol efflux and are primed for foam cell formation following trans-endothelial migration. Factors present in HIV+ serum, including elevated TNF levels, further enhance foam cell formation. The pro-atherogenic phenotype of monocytes persists in virologically suppressed HIV+ individuals and may contribute mechanistically to increased atherosclerosis in this population. PMID:26244384

  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.

  1. Curcumin enhanced cholesterol efflux by upregulating ABCA1 expression through AMPK-SIRT1-LXRα signaling in THP-1 macrophage-derived foam cells.

    PubMed

    Lin, Xiao-long; Liu, Mi-Hua; Hu, Hui-Jun; Feng, Hong-ru; Fan, Xiao-Juan; Zou, Wei-wen; Pan, Yong-quan; Hu, Xue-mei; Wang, Zuo

    2015-09-01

    Curcumin, a traditional Chinese derivative from the rhizomes of Curcuma longa, is beneficial to health by modulating lipid metabolism and suppressing atherogenesis. A key part of atherosclerosis is the failure of macrophages to restore their cellular cholesterol homeostasis and the formation of foam cells. In this study, results showed that curcumin dramatically increased the expression of ATP-binding cassette transporter 1 (ABCA1), promoted cholesterol efflux from THP-1 macrophage-derived foam cells, and reduced cellular cholesterol levels. Curcumin activated AMP-activated protein kinase (AMPK) and SIRT1, and then activated LXRα in THP-1 macrophage-derived foam cells. Inhibiting AMPK/SIRT1 activity by its specific inhibitor or by small interfering RNA could inhibit LXRα activation and abolish curcumin-induced ABCA1 expression and cholesterol efflux. Thus, curcumin enhanced cholesterol efflux by upregulating ABCA1 expression through activating AMPK-SIRT1-LXRα signaling in THP-1 macrophage-derived foam cells. This study describes a possible mechanism for understanding the antiatherogenic effects of curcumin on attenuating the progression of atherosclerosis.

  2. 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. PMID:27045078

  3. Lactate and metabolic H+ transport and distribution after exercise in rainbow trout white muscle.

    PubMed

    Wang, Y; Heigenhauser, G J; Wood, C M

    1996-11-01

    An isolated-perfused tail-trunk preparation was employed to study the influence of transmembrane pH gradient and membrane potential on the transport and distribution of L(+)-lactate (Lac), metabolic H+ (delta Hm+), and related parameters in rainbow trout white muscle after exhaustive exercise. One resting [arterial pH (pHa) approximately 7.9] and four postexercise treatments (pHa approximately 7.4, 7.9, 8.4, and, high K+, pHa approximately 7.9, partially depolarized by 15 mM K+) were examined. Variations in HCO3- concentration (2-18 mM) at a constant PCO2 approximately 2 Torr were used to alter pHa. The elevated intracellular Lac (approximately 50 mM) remained unchanged after 60 min of perfusion because of very low rates of lactate efflux and oxidation. H+, HCO3-, and Lac- distributions were all well out of electrochemical equilibrium. Total CO2 efflux was reduced at high extracellular pH (pHe); alterations in the net driving force on HCO3- may have overshadowed the influence of PCO2 gradients in driving total CO2 efflux. Lac efflux and delta Hm+ flux were completely uncoupled. delta Hm+ flux reacted to both acid-base and electrochemical gradients as delta Hm+ efflux dropped and even reversed when pHe decreased, whereas partial depolarization in conjunction with depressed intracellular pH resulted in elevated delta Hm+ efflux. Lac efflux did not respond to changes in pHe. Changes in Lac efflux corresponded more closely to changes in the Lac- concentration gradient than in the lactic acid gradient. This study provides circumstantial evidence for the involvement of electroneutral mechanisms (i.e., Lac(-)-H+ cotransport and/or Lac-/anion exchange) in lactate efflux, but does not eliminate the possibility of an active transport mechanism contributing to the retention of Lac. PMID:8945959

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

    PubMed

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

    2014-11-01

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

  5. Effect of External Electric Field on Substrate Transport of a Secondary Active Transporter.

    PubMed

    Zhang, Ji-Long; Zheng, Qing-Chuan; Yu, Li-Ying; Li, Zheng-Qiang; Zhang, Hong-Xing

    2016-08-22

    Substrate transport across a membrane accomplished by a secondary active transporter (SAT) is essential to the normal physiological function of living cells. In the present research, a series of all-atom molecular dynamics (MD) simulations under different electric field (EF) strengths was performed to investigate the effect of an external EF on the substrate transport of an SAT. The results show that EF both affects the interaction between substrate and related protein's residues by changing their conformations and tunes the timeline of the transport event, which collectively reduces the height of energy barrier for substrate transport and results in the appearance of two intermediate conformations under the existence of an external EF. Our work spotlights the crucial influence of external EFs on the substrate transport of SATs and could provide a more penetrating understanding of the substrate transport mechanism of SATs. PMID:27472561

  6. 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. PMID:17214741

  7. 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.

  8. 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

  9. Promiscuous partnering and independent activity of MexB, the multidrug transporter protein from Pseudomonas aeruginosa.

    PubMed

    Welch, Alexander; Awah, Chidiebere U; Jing, Shiheng; van Veen, Hendrik W; Venter, Henrietta

    2010-09-01

    The MexAB-OprM drug efflux pump is central to multidrug resistance of Pseudomonas aeruginosa. The ability of the tripartite protein to confer drug resistance on the pathogen is crucially dependent on the presence of all three proteins of the complex. However, the role of each protein in the formation of the intact functional complex is not well understood. One of the key questions relates to the (in)ability of MexB to act independently of its cognitive partners, MexA and OprM. In the present study, we have demonstrated that, in the absence of MexA and OprM, MexB can: (i) recruit AcrA and TolC from Escherichia coli to form a functional drug-efflux complex; (ii) transport the toxic compound ethidium bromide in a Gram-positive organism where the periplasmic space and outer membrane are absent; and (iii) catalyse transmembrane chemical proton gradient (DeltapH)-dependent drug transport when purified and reconstituted into proteoliposomes. Our results represent the first evidence of drug transport by an isolated RND (resistance-nodulation-cell division)-type multidrug transporter, and provide a basis for further studies into the energetics of RND-type transporters and their assembly into multiprotein complexes.

  10. MEK1/2 inhibitors activate macrophage ABCG1 expression and reverse cholesterol transport-An anti-atherogenic function of ERK1/2 inhibition.

    PubMed

    Zhang, Ling; Chen, Yuanli; Yang, Xiaoxiao; Yang, Jie; Cao, Xingyue; Li, Xiaoju; Li, Luyuan; Miao, Qing Robert; Hajjar, David P; Duan, Yajun; Han, Jihong

    2016-09-01

    Expression of ATP-binding cassette transporter G1 (ABCG1), a molecule facilitating cholesterol efflux to HDL, is activated by liver X receptor (LXR). In this study, we investigated if inhibition of ERK1/2 can activate macrophage ABCG1 expression and functions. MEK1/2 inhibitors, PD98059 and U0126, increased ABCG1 mRNA and protein expression, and activated the natural ABCG1 promoter but not the promoter with the LXR responsive element (LXRE) deletion. Inhibition of ABCG1 expression by ABCG1 siRNA did enhance the formation of macrophage/foam cells and it attenuated the inhibitory effect of MEK1/2 inhibitors on foam cell formation. MEK1/2 inhibitors activated macrophage cholesterol efflux to HDL in vitro, and they enhanced reverse cholesterol transport (RCT) in vivo. ApoE deficient (apoE(-/-)) mice receiving U0126 treatment had reduced sinus lesions in the aortic root which was associated with activated macrophage ABCG1 expression in the lesion areas. MEK1/2 inhibitors coordinated the RXR agonist, but not the LXR agonist, to induce ABCG1 expression. Furthermore, induction of ABCG1 expression by MEK1/2 inhibitors was associated with activation of SIRT1, a positive regulator of LXR activity, and inactivation of SULT2B1 and RIP140, two negative regulators of LXR activity. Taken together, our study suggests that MEK1/2 inhibitors activate macrophage ABCG1 expression/RCT, and inhibit foam cell formation and lesion development by multiple mechanisms, supporting the concept that ERK1/2 inhibition is anti-atherogenic. PMID:27365310

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

    SciTech Connect

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

    1988-04-01

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

  12. Efflux in Fungi: La Pièce de Résistance

    PubMed Central

    Coleman, Jeffrey J.; Mylonakis, Eleftherios

    2009-01-01

    Pathogens must be able to overcome both host defenses and antimicrobial treatment in order to successfully infect and maintain colonization of the host. One way fungi accomplish this feat and overcome intercellular toxin accumulation is efflux pumps, in particular ATP-binding cassette transporters and transporters of the major facilitator superfamily. Members of these two superfamilies remove many toxic compounds by coupling transport with ATP hydrolysis or a proton gradient, respectively. Fungal genomes encode a plethora of members of these families of transporters compared to other organisms. In this review we discuss the role these two fungal superfamilies of transporters play in virulence and resistance to antifungal agents. These efflux transporters are responsible not only for export of compounds involved in pathogenesis such as secondary metabolites, but also export of host-derived antimicrobial compounds. In addition, we examine the current knowledge of these transporters in resistance of pathogens to clinically relevant antifungal agents. PMID:19557154

  13. HIV-1 Protein Nef Inhibits Activity of ATP-binding Cassette Transporter A1 by Targeting Endoplasmic Reticulum Chaperone Calnexin*

    PubMed Central

    Jennelle, Lucas; Hunegnaw, Ruth; Dubrovsky, Larisa; Pushkarsky, Tatiana; Fitzgerald, Michael L.; Sviridov, Dmitri; Popratiloff, Anastas; Brichacek, Beda; Bukrinsky, Michael

    2014-01-01

    HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism. PMID:25170080

  14. Monosaccharide absorption activity of Arabidopsis roots depends on expression profiles of transporter genes under high salinity conditions.

    PubMed

    Yamada, Kohji; Kanai, Motoki; Osakabe, Yuriko; Ohiraki, Haruka; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2011-12-16

    Plant roots are able to absorb sugars from the rhizosphere but also release sugars and other metabolites that are critical for growth and environmental signaling. Reabsorption of released sugar molecules could help reduce the loss of photosynthetically fixed carbon through the roots. Although biochemical analyses have revealed monosaccharide uptake mechanisms in roots, the transporters that are involved in this process have not yet been fully characterized. In the present study we demonstrate that Arabidopsis STP1 and STP13 play important roles in roots during the absorption of monosaccharides from the rhizosphere. Among 14 STP transporter genes, we found that STP1 had the highest transcript level and that STP1 was a major contributor for monosaccharide uptake under normal conditions. In contrast, STP13 was found to be induced by abiotic stress, with low expression under normal conditions. We analyzed the role of STP13 in roots under high salinity conditions where membranes of the epidermal cells were damaged, and we detected an increase in the amount of STP13-dependent glucose uptake. Furthermore, the amount of glucose efflux from stp13 mutants was higher than that from wild type plants under high salinity conditions. These results indicate that STP13 can reabsorb the monosaccharides that are released by damaged cells under high salinity conditions. Overall, our data indicate that sugar uptake capacity in Arabidopsis roots changes in response to environmental stresses and that this activity is dependent on the expression pattern of sugar transporters. PMID:22041897

  15. An Abiotic Glass-Bead Collector Exhibiting Active Transport

    PubMed Central

    Goto, Youhei; Kanda, Masato; Yamamoto, Daigo; Shioi, Akihisa

    2015-01-01

    Animals relocate objects as needed by active motion. Active transport is ubiquitous in living organisms but has been difficult to realize in abiotic systems. Here we show that a self-propelled droplet can gather scattered beads toward one place on a floor and sweep it clean. This is a biomimetic active transport with loadings and unloadings, because the transport was performed by a carrier and the motion of the carrier was maintained by the energy of the chemical reaction. The oil droplet produced fluctuation of the local number density of the beads on the floor, followed by its autocatalytic growth. This mechanism may inspire the technologies based on active transport wherein chemical and physical substances migrate as in living organisms. PMID:26387743

  16. An Abiotic Glass-Bead Collector Exhibiting Active Transport

    NASA Astrophysics Data System (ADS)

    Goto, Youhei; Kanda, Masato; Yamamoto, Daigo; Shioi, Akihisa

    2015-09-01

    Animals relocate objects as needed by active motion. Active transport is ubiquitous in living organisms but has been difficult to realize in abiotic systems. Here we show that a self-propelled droplet can gather scattered beads toward one place on a floor and sweep it clean. This is a biomimetic active transport with loadings and unloadings, because the transport was performed by a carrier and the motion of the carrier was maintained by the energy of the chemical reaction. The oil droplet produced fluctuation of the local number density of the beads on the floor, followed by its autocatalytic growth. This mechanism may inspire the technologies based on active transport wherein chemical and physical substances migrate as in living organisms.

  17. Space transportation activities in the United States

    NASA Technical Reports Server (NTRS)

    Gabris, Edward A.

    1994-01-01

    The status of the existing space transportation systems in the U.S. and options for increased capability is being examined in the context of mission requirements, options for new vehicles, cost to operate the existing vehicles, cost to develop new vehicles, and the capabilities and plans of other suppliers. This assessment is addressing the need to build and resupply the space station, to maintain necessary military assets in a rapidly changing world, and to continue a competitive commercial space transportation industry. The Department of Defense (DOD) and NASA each conducted an 'access to space' study using a common mission model but with the emphasis on their unique requirements. Both studies considered three options: maintain and improve the existing capability, build a new launch vehicle using contemporary technology, and build a new launch vehicle using advanced technology. While no decisions have been made on a course of action, it will be influenced by the availability of funds in the U.S. budget, the changing need for military space assets, the increasing competition among space launch suppliers, and the emerging opportunity for an advanced technology, low cost system and international partnerships to develop it.

  18. Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain.

    PubMed

    Soares, Ricardo V; Do, Tuan M; Mabondzo, Aloïse; Pons, Gérard; Chhun, Stéphanie

    2016-04-01

    The blood-brain barrier (BBB) is responsible for the control of solutes' concentration in the brain. Tight junctions and multiple ATP-binding cassette (ABC) and SoLute Carrier (SLC) efflux transporters protect brain cells from xenobiotics, therefore reducing brain exposure to intentionally administered drugs. In epilepsy, polymorphisms and overexpression of efflux transporters genes could be associated with pharmacoresistance. The ontogeny of these efflux transporters should also be addressed because their expression during development may be related to different brain exposure to antiepileptic drugs in the immature brain. We detected statistically significant higher expression of Abcb1b and Slc16a1 genes, and lower expression of Abcb1a and Abcg2 genes between the post-natal day 14 (P14) and the adult rat microvessels. P-gP efflux activity was also shown to be lower in P14 rats when compared with the adults. The P-gP proteins coded by rodent genes Abcb1a and Abcb1b are known to have different substrate affinities. The role of the Abcg2 gene is less clear in pharmacoresistance in epilepsy, nonetheless the coded protein Bcrp is frequently associated with drug resistance. Finally, we observed a higher expression of the Mct1 transporter gene in the P14 rat brain microvessels. Accordingly to our results, we suppose that age may be another factor influencing brain exposure to antiepileptics as a consequence of different expression patterns of efflux transporters between the adult and immature BBB.

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

    PubMed

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

    2016-01-01

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

  20. Transpiration alters the contribution of autotrophic and heterotrophic components of soil CO2 efflux.

    PubMed

    Grossiord, Charlotte; Mareschal, Louis; Epron, Daniel

    2012-05-01

    • An unbiased partitioning of autotrophic and heterotrophic components of soil CO(2) efflux is important to estimate forest carbon budgets and soil carbon sequestration. The contribution of autotrophic sources to soil CO(2) efflux (F(A)) may be underestimated during the daytime as a result of internal transport of CO(2) produced by root respiration through the transpiration stream. • Here, we tested the hypothesis that carbon isotope composition of soil CO(2) efflux (δ(FS)) in a Eucalyptus plantation grown on a C(4) soil is enriched during the daytime, which will indicate a decrease in F(A) during the periods of high transpiration. • Mean δ(FS) of soil CO(2) efflux decreased to -25.7‰ during the night and increased to -24.7‰ between 11:00 and 15:00 h when the xylem sap flux density was at its maximum. • Our results indicate a decrease in the contribution of root respiration to soil CO(2) efflux during the day that may be interpreted as a departure of root-produced CO(2) in the transpiration stream, leading to a 17% underestimation of autotrophic contribution to soil CO(2) efflux on a daily timescale. PMID:22356353

  1. Transpiration alters the contribution of autotrophic and heterotrophic components of soil CO2 efflux.

    PubMed

    Grossiord, Charlotte; Mareschal, Louis; Epron, Daniel

    2012-05-01

    • An unbiased partitioning of autotrophic and heterotrophic components of soil CO(2) efflux is important to estimate forest carbon budgets and soil carbon sequestration. The contribution of autotrophic sources to soil CO(2) efflux (F(A)) may be underestimated during the daytime as a result of internal transport of CO(2) produced by root respiration through the transpiration stream. • Here, we tested the hypothesis that carbon isotope composition of soil CO(2) efflux (δ(FS)) in a Eucalyptus plantation grown on a C(4) soil is enriched during the daytime, which will indicate a decrease in F(A) during the periods of high transpiration. • Mean δ(FS) of soil CO(2) efflux decreased to -25.7‰ during the night and increased to -24.7‰ between 11:00 and 15:00 h when the xylem sap flux density was at its maximum. • Our results indicate a decrease in the contribution of root respiration to soil CO(2) efflux during the day that may be interpreted as a departure of root-produced CO(2) in the transpiration stream, leading to a 17% underestimation of autotrophic contribution to soil CO(2) efflux on a daily timescale.

  2. Multidrug efflux pumps as main players in intrinsic and acquired resistance to antimicrobials.

    PubMed

    Hernando-Amado, Sara; Blanco, Paula; Alcalde-Rico, Manuel; Corona, Fernando; Reales-Calderón, Jose A; Sánchez, María B; Martínez, José L

    2016-09-01

    Multidrug efflux pumps constitute a group of transporters that are ubiquitously found in any organism. In addition to other functions with relevance for the cell physiology, efflux pumps contribute to the resistance to compounds used for treating different diseases, including resistance to anticancer drugs, antibiotics or antifungal compounds. In the case of antimicrobials, efflux pumps are major players in both intrinsic and acquired resistance to drugs currently in use for the treatment of infectious diseases. One important aspect not fully explored of efflux pumps consists on the identification of effectors able to induce their expression. Indeed, whereas the analysis of clinical isolates have shown that mutants overexpressing these resistance elements are frequently found, less is known on the conditions that may trigger expression of efflux pumps, hence leading to transient induction of resistance in vivo, a situation that is barely detectable using classical susceptibility tests. In the current article we review the structure and mechanisms of regulation of the expression of bacterial and fungal efflux pumps, with a particular focus in those for which a role in clinically relevant resistance has been reported. PMID:27620952

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

    PubMed Central

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

    2016-01-01

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

  4. Sucrose transporters of higher plants.

    PubMed

    Kühn, Christina; Grof, Christopher P L

    2010-06-01

    Recent advances have provided new insights into how sucrose is moved from sites of synthesis to sites of utilisation or storage in sink organs. Sucrose transporters play a central role, as they orchestrate sucrose allocation both intracellularly and at the whole plant level. Sucrose produced in mesophyll cells of leaves may be effluxed into the apoplasm of mesophyll or phloem parenchyma cells by a mechanism that remains elusive, but experimentally consistent with facilitated transport or energy-dependent sucrose/H(+) antiport. From the apoplasm, sucrose/H(+) symporters transport sucrose across the plasma membrane of cells making up the sieve element/companion cell (SE/CC) complex, the long distance conduits of the phloem. Phloem unloading of sucrose in key sinks such as developing seeds involves two sequential transport steps, sucrose efflux followed by sucrose influx. Besides plasma membrane specific sucrose transporters, sucrose transporters on the tonoplast contribute to the capacity for elevated sucrose accumulation in storage organs such as sugar beet roots or sugarcane culms. Except for several sucrose facilitators from seed coats of some leguminous plants all sucrose transporters cloned to date, including recently identified vacuolar sucrose transporters, have been characterised as sucrose/H(+) symporters. Transporters functioning to efflux sucrose into source or sink apoplasms as well as those supporting sucrose/H(+) antiport on tonoplasts, remain to be identified. Sucrose transporter expression and activity is tightly regulated at the transcriptional, post-transcriptional as well as post-translational levels. Light quality and phytohormones play essential regulatory roles and the sucrose molecule itself functions as a signal.

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  6. The energy requirements for the basal efflux of 3H-noradrenaline from sympathetically innervated organs.

    PubMed

    Russ, H; Schömig, E; Trendelenburg, U

    1991-09-01

    In the rat vas deferens (preloaded with 3H-noradrenaline, catechol-O-methyl transferase inhibited, calcium-free solution) ouabain, glucose deprivation or the combination of hypoxia plus presence of lactate were found to induce a carrier-mediated (desipramine-sensitive) outward transport of the 3H-amine. Glucose deprivation additionally increased the efflux of deaminated 3H-metabolites, as a consequence of an increased net leakage of vesicular 3H-noradrenaline; moreover, 3H-dihydroxymandelic acid then became the predominant neuronal metabolite. The simultaneous lack of oxygen and glucose resulted in a very pronounced release of the 3H-amine. Moreover, during spontaneous efflux more outward transport of 3H-noradrenaline was observed in the absence than in the presence of extracellular calcium. In rat atria (under the same experimental conditions) the contribution by carrier-mediated outward transport to the spontaneous efflux of tritium exceeded that in vasa deferentia. Moreover, the efflux of lactate (as an index of hypoxia of the tissue) exceeded that observed in vasa deferentia, under aerobic and anaerobic conditions. It is proposed that the greater contribution by outward transport of 3H-noradrenaline to spontaneous efflux in atria than in vasa deferentia does not reflect any basic difference between the varicosities in two different organs. It is likely that the less heterogeneous distribution of the 3H-amine in atria than in vasa deferentia is responsible for storage of the exogenous amine in atrial varicosities that are subject to some hypoxia, to an increased extracellular lactate level and to perhaps a minor degree of glucose deficiency; these factors may well be responsible for the difference with regard to outward transport of 3H-noradrenaline during spontaneous efflux. Thus, in addition to the heterogeneity of the distribution of 3H-noradrenaline, an additional heterogeneity with regard to the energy supply must be expected for incubated organs.

  7. Entropic Ratchet transport of interacting active Brownian particles

    SciTech Connect

    Ai, Bao-Quan; He, Ya-Feng; Zhong, Wei-Rong

    2014-11-21

    Directed transport of interacting active (self-propelled) Brownian particles is numerically investigated in confined geometries (entropic barriers). The self-propelled velocity can break thermodynamical equilibrium and induce the directed transport. It is found that the interaction between active particles can greatly affect the ratchet transport. For attractive particles, on increasing the interaction strength, the average velocity first decreases to its minima, then increases, and finally decreases to zero. For repulsive particles, when the interaction is very weak, there exists a critical interaction at which the average velocity is minimal, nearly tends to zero, however, for the strong interaction, the average velocity is independent of the interaction.

  8. Quinine dimers are potent inhibitors of the Plasmodium falciparum chloroquine resistance transporter and are active against quinoline-resistant P. falciparum.

    PubMed

    Hrycyna, Christine A; Summers, Robert L; Lehane, Adele M; Pires, Marcos M; Namanja, Hilda; Bohn, Kelsey; Kuriakose, Jerrin; Ferdig, Michael; Henrich, Philipp P; Fidock, David A; Kirk, Kiaran; Chmielewski, Jean; Martin, Rowena E

    2014-03-21

    Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the "chloroquine resistance transporter" (PfCRT). The resistance-conferring form of PfCRT (PfCRT(CQR)) mediates CQ resistance by effluxing the drug from the parasite's digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRT(CQR) can also decrease the parasite's susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRT(CQR) and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H(+) ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRT(CQR) reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRT(CQR) from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRT(CQR). This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum.

  9. Heterocyclic cyclohexanone monocarbonyl analogs of curcumin can inhibit the activity of ATP-binding cassette transporters in cancer multidrug resistance.

    PubMed

    Revalde, Jezrael L; Li, Yan; Hawkins, Bill C; Rosengren, Rhonda J; Paxton, James W

    2015-02-01

    Curcumin (CUR) is a phytochemical that inhibits the xenobiotic ABC efflux transporters implicated in cancer multidrug resistance (MDR), such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins 1 and 5 (MRP1 and MRP5). The use of CUR in the clinic however, is complicated by its instability and poor pharmacokinetic profile. Monocarbonyl analogs of CUR (MACs) are compounds without CUR's unstable β-diketone moiety and were reported to have improved stability and in vivo disposition. Whether the MACs can be used as MDR reversal agents is less clear, as the absence of a β-diketone may negatively impact transporter inhibition. In this study, we investigated 23 heterocyclic cyclohexanone MACs for inhibitory effects against P-gp, BCRP, MRP1 and MRP5. Using flow cytometry and resistance reversal assays, we found that many of these compounds inhibited the transport activity of the ABC transporters investigated, often with much greater potency than CUR. Overall the analogs were most effective at inhibiting BCRP and we identified three compounds, A12 (2,6-bis((E)-2,5-dimethoxy-benzylidene)cyclohexanone), A13 (2,6-bis((E)-4-hydroxyl-3-methoxybenzylidene)-cyclohexanone) and B11 (3,5-bis((E)-2-fluoro-4,5-dimethoxybenzylidene)-1-methylpiperidin-4-one), as the most promising BCRP inhibitors. These compounds inhibited BCRP activity in a non-cell line, non-substrate-specific manner. Their inhibition occurred by direct transporter interaction rather than modulating protein or cell surface expression. From these results, we concluded that MACs, such as the heterocyclic cyclohexanone analogs in this study, also have potential as MDR reversal agents and may be superior alternatives to the unstable parent compound, CUR.

  10. Nitrite Transport Activity of the ABC-Type Cyanate Transporter of the Cyanobacterium Synechococcus elongatus▿

    PubMed Central

    Maeda, Shin-ichi; Omata, Tatsuo

    2009-01-01

    In addition to the ATP-binding cassette (ABC)-type nitrate/nitrite-bispecific transporter, which has a high affinity for both substrates (Km, ∼1 μM), Synechococcus elongatus has an active nitrite transport system with an apparent Km (NO2−) value of 20 μM. We found that this activity depends on the cynABD genes, which encode a putative cyanate (NCO−) ABC-type transporter. Accordingly, nitrite transport by CynABD was competitively inhibited by NCO− with a Ki value of 0.025 μM. The transporter was induced under conditions of nitrogen deficiency, and the induced cells showed a Vmax value of 11 to 13 μmol/mg of chlorophyll per h for cyanate or nitrite, which could supply ∼30% of the amount of nitrogen required for optimum growth. Its relative specificity for the substrates and regulation at transcriptional and posttranslational levels suggested that the physiological role of the bispecific cyanate/nitrite transporter in S. elongatus is to allow nitrogen-deficient cells to assimilate low concentrations of cyanate in the medium. Its contribution to nitrite assimilation was significant in a mutant lacking the ABC-type nitrate/nitrite transporter, suggesting a possible role for CynABD in nitrite assimilation by cyanobacterial species that lack another high-affinity mechanism(s) for nitrite transport. PMID:19286804

  11. Detergent-Mediated Phospholipidation of Plasma Lipoproteins Increases HDL Cholesterophilicity and Cholesterol Efflux Via SR-BI†

    PubMed Central

    Pownall, Henry J.

    2008-01-01

    Cellular cholesterol efflux is an early, obligatory step in reverse cholesterol transport, the putative antiatherogenic mechanism by which human plasma high density lipoproteins (HDL) transport cholesterol from peripheral tissue to the liver for recycling or disposal. HDL-phospholipid content is the essential cholesterol-binding component of lipoproteins and therefore a major determinant of cholesterol efflux. Thus, increased phospholipidation of lipoproteins, particularly HDL, is one strategy for increasing cholesterol efflux. This study validates a simple, new detergent perturbation method for the phospholipidation of plasma lipoproteins; we have quantified the cholesterophilicity of human plasma lipoproteins and the effects of lipoprotein phospholipidation on cholesterophilicity and cellular cholesterol efflux mediated by the class B type I scavenger receptor (SR-BI). We determined that low density lipoproteins (LDL) are more cholesterophilic than HDL and that LDL has a higher affinity for phospholipids than HDL whereas HDL has a higher phospholipid capacity than LDL. Phospholipidation of total human plasma lipoproteins enhances cholesterol efflux, an effect that occurs largely through the preferential phospholipidation of HDL. We conclude that increasing HDL phospholipid increases its cholesterophilicity thereby making it a better acceptor of cellular cholesterol efflux. Phospholipidation of lipoproteins by detergent perturbation is a simple way to increase HDL cholesterophilicity and cholesterol efflux in a way that may be clinically useful. PMID:16981711

  12. Sphingomyelin Depletion Impairs Anionic Phospholipid Inward Translocation and Induces Cholesterol Efflux*

    PubMed Central

    Gulshan, Kailash; Brubaker, Gregory; Wang, Shuhui; Hazen, Stanley L.; Smith, Jonathan D.

    2013-01-01

    The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners. PMID:24220029

  13. Classroom Activities in Transportation: Technology Education.

    ERIC Educational Resources Information Center

    Wisconsin State Dept. of Public Instruction, Madison.

    This curriculum supplement was designed to correlate directly with "A Guide to Curriculum Planning in Technology Education," published by the Wisconsin Department of Public Instruction. It is also a companion book to three other classroom activity compilations, one in each of the other three major systems of technology--manufacturing,…

  14. 9-cis β-Carotene Increased Cholesterol Efflux to HDL in Macrophages.

    PubMed

    Bechor, Sapir; Zolberg Relevy, Noa; Harari, Ayelet; Almog, Tal; Kamari, Yehuda; Ben-Amotz, Ami; Harats, Dror; Shaish, Aviv

    2016-01-01

    Cholesterol efflux from macrophages is a key process in reverse cholesterol transport and, therefore, might inhibit atherogenesis. 9-cis-β-carotene (9-cis-βc) is a precursor for 9-cis-retinoic-acid (9-cis-RA), which regulates macrophage cholesterol efflux. Our objective was to assess whether 9-cis-βc increases macrophage cholesterol efflux and induces the expression of cholesterol transporters. Enrichment of a mouse diet with βc from the alga Dunaliella led to βc accumulation in peritoneal macrophages. 9-cis-βc increased the mRNA levels of CYP26B1, an enzyme that regulates RA cellular levels, indicating the formation of RA from βc in RAW264.7 macrophages. Furthermore, 9-cis-βc, as well as all-trans-βc, significantly increased cholesterol efflux to high-density lipoprotein (HDL) by 50% in RAW264.7 macrophages. Likewise, food fortification with 9-cis-βc augmented cholesterol efflux from macrophages ex vivo. 9-cis-βc increased both the mRNA and protein levels of ABCA1 and apolipoprotein E (APOE) and the mRNA level of ABCG1. Our study shows, for the first time, that 9-cis-βc from the diet accumulates in peritoneal macrophages and increases cholesterol efflux to HDL. These effects might be ascribed to transcriptional induction of ABCA1, ABCG1, and APOE. These results highlight the beneficial effect of βc in inhibition of atherosclerosis by improving cholesterol efflux from macrophages. PMID:27447665

  15. 9-cis β-Carotene Increased Cholesterol Efflux to HDL in Macrophages

    PubMed Central

    Bechor, Sapir; Zolberg Relevy, Noa; Harari, Ayelet; Almog, Tal; Kamari, Yehuda; Ben-Amotz, Ami; Harats, Dror; Shaish, Aviv

    2016-01-01

    Cholesterol efflux from macrophages is a key process in reverse cholesterol transport and, therefore, might inhibit atherogenesis. 9-cis-β-carotene (9-cis-βc) is a precursor for 9-cis-retinoic-acid (9-cis-RA), which regulates macrophage cholesterol efflux. Our objective was to assess whether 9-cis-βc increases macrophage cholesterol efflux and induces the expression of cholesterol transporters. Enrichment of a mouse diet with βc from the alga Dunaliella led to βc accumulation in peritoneal macrophages. 9-cis-βc increased the mRNA levels of CYP26B1, an enzyme that regulates RA cellular levels, indicating the formation of RA from βc in RAW264.7 macrophages. Furthermore, 9-cis-βc, as well as all-trans-βc, significantly increased cholesterol efflux to high-density lipoprotein (HDL) by 50% in RAW264.7 macrophages. Likewise, food fortification with 9-cis-βc augmented cholesterol efflux from macrophages ex vivo. 9-cis-βc increased both the mRNA and protein levels of ABCA1 and apolipoprotein E (APOE) and the mRNA level of ABCG1. Our study shows, for the first time, that 9-cis-βc from the diet accumulates in peritoneal macrophages and increases cholesterol efflux to HDL. These effects might be ascribed to transcriptional induction of ABCA1, ABCG1, and APOE. These results highlight the beneficial effect of βc in inhibition of atherosclerosis by improving cholesterol efflux from macrophages. PMID:27447665

  16. Caulis Sinomenii extracts activate DA/NE transporter and inhibit 5HT transporter.

    PubMed

    Zhao, Gang; Bi, Cheng; Qin, Guo-Wei; Guo, Li-He

    2009-08-01

    Caulis Sinomenii (QFT) has analgesic, sedative, and anxiolytic-like actions, and is proven effective for improving drug dependence that is known to be associated with abnormal monoaminergic transmission. We assessed whether QFT would be biologically active in functionally regulating monoamine transporters using CHO cells expressing dopamine transporter (DAT), norepinephrine transporter (NET), or serotonin transporter (SERT) (i.e. D8, N1, or S6 cells, respectively). Here, we showed that its primary extracts, such as QA, QC, QE, QD, and QB (QFT ethanol, chloroform, ethyl acetate, alkaloid-free chloroform, and alkaloid-containing chloroform extract, respectively), and secondary extracts, such as QE-2, - 3, - 5, - 7, QD-1, - 2, - 3, - 4, - 5, and QB-1, - 2, - 3, - 4, - 5 (fractioned from QE, QD, and QB, respectively), in differing degrees, either increased DA/ NE uptake by corresponding D8/N1 cells or decreased 5HT uptake by S6 cells; wherein, QE-2, QD-3, and QE-7 were potent DA/NE uptake activators while both QE-7 and QB-5 were potent 5HT uptake inhibitors. Furthermore, the enhancement of DA/NE uptake was dependent of DAT/NET activity, and the inhibition of 5HT uptake was typical of competition. Thus, QFT extracts, especially QE-2 and QE-7 (both with stronger potencies), are novel monoamine transporter modulators functioning as DAT/ NET activators and/or SERT inhibitors, and would likely improve neuropsychological disorders through regulating monoamine transporters.

  17. Girls' perception of physical environmental factors and transportation: reliability and association with physical activity and active transport to school

    PubMed Central

    Evenson, Kelly R; Birnbaum, Amanda S; Bedimo-Rung, Ariane L; Sallis, James F; Voorhees, Carolyn C; Ring, Kimberly; Elder, John P

    2006-01-01

    Background Preliminary evidence suggests that the physical environment and transportation are associated with youth physical activity levels. Only a few studies have examined the association of physical environmental factors on walking and bicycling to school. Therefore, the purpose of this study was (1) to examine the test-retest reliability of a survey designed for youth to assess perceptions of physical environmental factors (e.g. safety, aesthetics, facilities near the home) and transportation, and (2) to describe the associations of these perceptions with both physical activity and active transport to school. Methods Test and retest surveys, administered a median of 12 days later, were conducted with 480 sixth- and eighth-grade girls in or near six U.S. communities. The instrument consisted of 24 questions on safety and aesthetics of the perceived environment and transportation and related facilities. Additionally, girls were asked if they were aware of 14 different recreational facilities offering structured and unstructured activities, and if so, whether they would visit these facilities and the ease with which they could access them. Test-retest reliability was determined using kappa coefficients, overall and separately by grade. Associations with physical activity and active transport to school were examined using mixed model logistic regression (n = 610), adjusting for grade, race/ethnicity, and site. Results Item-specific reliabilities for questions assessing perceived safety and aesthetics of the neighborhood ranged from 0.31 to 0.52. Reliabilities of items assessing awareness of and interest in going to the 14 recreational facilities ranged from 0.47 to 0.64. Reliabilities of items assessing transportation ranged from 0.34 to 0.58. Some items on girls' perceptions of perceived safety, aesthetics of the environment, facilities, and transportation were important correlates of physical activity and, in some cases, active transport to school. Conclusion

  18. Active sodium transport and fluid secretion in the gall-bladder epithelium of Necturus.

    PubMed Central

    Giraldez, F

    1984-01-01

    Intracellular Na, K and Cl activities (acNa, acK and acCl) and membrane potentials were measured in Necturus gall-bladder epithelium using double-barrelled ion-sensitive micro-electrodes. Mucosal membrane potential was about -55 mV and the mean control activities were acNa = 14.7 mM, acK = 91.6 mM and acCl = 20.3 mM. Replacing mucosal Na by K caused a fall in acNa that followed an exponential time course. The rate of change in acNa was linearly related to acNa above a certain value (congruent to 3 mM). acK and acCl both increased in K Ringer solution. From the change in all three ions the cell was estimated to swell at an initial rate of 0.13% s-1. From the initial rate of change in acNa, a net cell efflux of Na of 405 pmol cm-2 s-1 was calculated. Replacement of Na by Tris or choline led to a similar result. The transepithelial Na transport rate was for this group of animals 346 pmol cm-2 s-1. Ouabain (10(-3) M) produced an increase in acNa and acCl, whereas acK decreased. The cells were estimated to swell at an initial rate of 0.06% s-1. The initial Na influx after Na-pump inhibition was calculated to be 162 pmol cm-2 s-1. The parallel measure of the transepithelial rate of transport of Na gave a value of 189 pmol cm-2 s-1. Ouabain inhibited the decrease in acNa after replacement of Na by K by about 80%. A fast depolarization, ranging from 2 to 7 mV, occurred after the perfusion with ouabain. Em then slowly decreased from about 53 to 32 mV in 1 h. It is concluded that (a) the major fraction of the transepithelial transport of Na is transcellular and mediated by the Na pump, (b) the pumping rate is linearly dependent on internal Na within a certain range and (c) the Na pump is electrogenic under normal circumstances. PMID:6716291

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

    PubMed Central

    2015-01-01

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

  20. Macrophage-activating lipopeptide-2 downregulates the expression of ATP-binding cassette transporter A1 by activating the TLR2/NF-кB/ZNF202 pathway in THP-1 macrophages.

    PubMed

    Peng, Liangjie; Zhang, Zizhen; Zhang, Min; Yu, Xiaohua; Yao, Feng; Tan, Yulin; Liu, Dan; Gong, Duo; Chong, Huang; Liu, Xiaoyan; Zheng, Xilong; Tian, Guoping; Tang, Chaoke

    2016-04-01

    Macrophage-activating lipopeptide-2 (MALP-2) has been shown to promote the development of atherosclerosis. ATP-binding cassette transporter A1 (ABCA1), a transmembrane protein, plays a critical role in mediating cholesterol export from macrophages to apolipoprotein A-I (apoA-I). However, whether MALP-2 can regulate the expression of ABCA1 is still largely unknown. The aim of this study was to explore the effects of MALP-2 on ABCA1 expression in THP-1 macrophages and the underlying mechanisms. Our results showed that the treatment of cells with MALP-2 decreased ABCA1 level and suppressed cholesterol efflux in both concentration- and time-dependent manners. The contents of intracellular cholesterol were significantly increased in the presence of MALP-2. Moreover, MALP-2-mediated inhibition of ABCA1 expression was abolished by siRNA of either Toll-like receptor 2 (TLR2) or nuclear factor κB (NF-κB). A similar effect was produced by treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate. In addition, MALP-2-induced activation of NF-κB markedly increased zinc finger protein 202 (ZNF202) level, and ZNF202 siRNA impaired the effects of MALP-2 on ABCA1 expression. Taken together, these results suggest that MALP-2 can decrease ABCA1 expression and subsequent cholesterol efflux through activation of the TLR2/NF-κB/ZNF202 signaling pathway in THP-1 macrophages. PMID:26922321

  1. Active Transportation to School: Findings from a National Survey

    ERIC Educational Resources Information Center

    Fulton, Janet E.; Shisler, Jessica L.; Yore, Michelle M.; Caspersen, Carl J.

    2005-01-01

    In the past, active transportation to school offered an important source of daily physical activity for youth; more recently, however, factors related to distance, safety, or physical or social environments may have contributed to the proportion of children who travel to school by motorized vehicle. The authors examine the characteristics of…

  2. Coupling of active motion and advection shapes intracellular cargo transport.

    PubMed

    Khuc Trong, Philipp; Guck, Jochen; Goldstein, Raymond E

    2012-07-13

    Intracellular cargo transport can arise from passive diffusion, active motor-driven transport along cytoskeletal filament networks, and passive advection by fluid flows entrained by such cargo-motor motion. Active and advective transport are thus intrinsically coupled as related, yet different representations of the same underlying network structure. A reaction-advection-diffusion system is used here to show that this coupling affects the transport and localization of a passive tracer in a confined geometry. For sufficiently low diffusion, cargo localization to a target zone is optimized either by low reaction kinetics and decoupling of bound and unbound states, or by a mostly disordered cytoskeletal network with only weak directional bias. These generic results may help to rationalize subtle features of cytoskeletal networks, for example as observed for microtubules in fly oocytes.

  3. Roles of changes in active glutamine transport in brain edema development during hepatic encephalopathy: an emerging concept.

    PubMed

    Zielińska, Magdalena; Popek, Mariusz; Albrecht, Jan

    2014-01-01

    Excessive glutamine (Gln) synthesis in ammonia-overloaded astrocytes contributes to astrocytic swelling and brain edema, the major complication of hepatic encephalopathy (HE). Much of the newly formed Gln is believed to enter mitochondria, where it is recycled to ammonia, which causes mitochondrial dysfunction (a "Trojan horse" mode of action). A portion of Gln may increase osmotic pressure in astrocytes and the interstitial space, directly and independently contributing to brain tissue swelling. Here we discuss the possibility that altered functioning of Gln transport proteins located in the cellular or mitochondrial membranes, modulates the effects of increased Gln synthesis. Accumulation of excess Gln in mitochondria involves a carrier-mediated transport which is activated by ammonia. Studies on the expression of the cell membrane N-system transporters SN1 (SNAT3) and SN2 (SNAT5), which mediate Gln efflux from astrocytes rendered HE model-dependent effects. HE lowered the expression of SN1 at the RNA and protein level in the cerebral cortex (cc) in the thioacetamide (TAA) model of HE and the effect paralleled induction of cerebral cortical edema. Neither SN1 nor SN2 expression was affected by simple hyperammonemia, which produces no cc edema. TAA-induced HE is also associated with decreased expression of mRNA coding for the system A carriers SAT1 and SAT2, which stimulate Gln influx to neurons. Taken together, changes in the expression of Gln transporters during HE appear to favor retention of Gln in astrocytes and/or the interstitial space of the brain. HE may also affect arginine (Arg)/Gln exchange across the astrocytic cell membrane due to changes in the expression of the hybrid Arg/Gln transporter y(+)LAT2. Gln export from brain across the blood-brain barrier may be stimulated by HE via its increased exchange with peripheral tryptophan. PMID:24072671

  4. Phosphorylation at S384 regulates the activity of the TaALMT1 malate transporter that underlies aluminum resistance in wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study we examined the role of protein phosphorylation & dephosphorylation in the transport properties of the wheat root malate efflux transporter underlying Al resistance, TaALMT1. Preincubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurospo...

  5. Red blood cell sodium transport in patients with cirrhosis.

    PubMed

    Henriksen, Ulrik Lütken; Kiszka-Kanowitz, Marianne; Bendtsen, Flemming; Henriksen, Jens H

    2016-09-01

    Patients with advanced cirrhosis have abnormal sodium homoeostasis. The study was undertaken to quantify the sodium transport across the plasma membrane of red blood cells (RBC) in patients with cirrhosis. RBC efflux and influx of sodium were studied in vitro with tracer (22) Na(+) according to linear kinetics in 24 patients with cirrhosis and 14 healthy controls. The sodium efflux was modified by ouabain (O), furosemide (F) and a combination of O and F (O + F). RBC sodium was significantly decreased (4·6 versus control 6·3 mmol l(-1) , P<0·001) and directly related to serum sodium (r = 0·57, P<0·05). The RBC fractional sodium efflux was higher in patients with cirrhosis (+46%, P<0·01) compared to controls. Inhibition in both high (145 mmol l(-1) )- and low (120 mmol l(-1) )-sodium buffers showed that the F-insensitive sodium efflux was twice as high in cirrhosis as in controls (P = 0·03-0·007), especially the O-sensitive, F-insensitive efflux was increased (+ 225%, P = 0·01-0·006). Fractional F-sensitive transport was normal in cirrhosis. RBC sodium influx was largely normal in cirrhosis. In conclusion, RBC sodium content is reduced in patients with cirrhosis with a direct relation to serum sodium. Increased RBC sodium efflux is especially related to ouabain-sensitive, furosemide-insensitive transport and thus most likely due to upregulated activity of the sodium-potassium pump. The study gives no evidence to an altered intracellular/extracellular sodium ratio or to a reduced fractional furosemide-sensitive sodium transport in cirrhosis.

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

    PubMed

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

    2013-02-15

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

  7. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Noel, Gregory; Stieger, Bruno; Fardel, Olivier

    2015-08-01

    Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies.

  8. Seasonal Variations in CO2 Efflux, Vadose Zone Gas Concentrations, and Natural Attenuation Rates at a Crude Oil Spill Site

    NASA Astrophysics Data System (ADS)

    Trost, J.; Sihota, N.; Delin, G. N.; Warren, E.

    2014-12-01

    Accurate estimates of hydrocarbon source zone natural attenuation (SZNA) rates are important for managing contaminated sites but are difficult to measure. Moreover, SZNA rates may vary seasonally in response to climatic conditions. Previous research at a crude oil spill site near Bemidji, Minnesota, USA showed that SZNA rates in the summer can be estimated by subtracting background soil CO2 efflux from the total soil CO2 efflux above the contaminated source. In this study, seasonal variations in surficial CO2 efflux were evaluated with measurements of gas concentrations (including 14CO2), temperature, and volumetric water content in the vadose zone at the site during a 2-year period. Soil CO2 effluxes in the source zone were consistently greater than background CO2 effluxes, and the magnitude and areal extent of the increased efflux varied seasonally. In the source zone, the 14CO2 and the CO2 efflux data showed a larger proportion of soil CO2 was derived from SZNA in fall and winter (October - February) compared to the summer (June - August). Surficial CO2 effluxes and vadose zone CO2 and CH4 concentrations in the source (2 - 7 meters below land surface) were positively correlated with soil temperature, indicating seasonal variability in SZNA rates. However, peak surficial CO2 effluxes did not correspond with periods of highest CO2 or CH4 concentrations at the 2 - 7 meter depth, demonstrating the effects of physical attributes (such as soil depth, frost, and volumetric water content) on gas transport. Overall, results showed that SZNA rates, background soil respiration rates, and gas transport varied seasonally, and that biological and physical factors are important to consider for accurately estimating SZNA rates.

  9. Microcalorimetric determination of energy expenditure due to active sodium-potassium transport in the soleus muscle and brown adipose tissue of the rat.

    PubMed

    Chinet, A; Clausen, T; Girardier, L

    1977-02-01

    1. The resting heat production rate (E) of soleus muscles from young rats and brown adipose tissue from adult rats was measured by means of a perfusable heat flux microcalorimeter in the absence and presence of ouabain. In the soleus muscle, the acute response of E to ouabain was compared with the ouabain-suppressible components of 22Na-efflux and 42K-influx. 2. In standard Krebs-Ringer bicarbonate buffer, ouabain (10(-3)M) induced an immediate but transient decrease in E of around 5%. Both in muscle and adipose tissue this was followed by a progressive rise in heat production rate. 3. When the medium was enriched with Mg (10 mM), ouabain produced a sustained decrease in E of the same magnitude as in the standard medium and the secondary rise was less marked or abolished. Under these conditions, in the soleus muscle, ouabain inhibited E by 5% (i.e. by 1-76 +/- 0-22 mcal.g wet wt.-1.min-1), 22Na-efflux by 58% (0-187 +/- 0-013 micronmole. g wet wt.-1.min-1) and 42K-influx by 34% (0-132 +/- 0-028 micronmole. g wet wt.-1.min-1). 4. When the muscles were loaded with Na by pre-incubation in K-free Mg-enriched medium, the addition of K (3mM) induced an immediate ouabain-suppressible increase in E of 2-98 +/- 0-33 mcal. g wet wt.-1.min-1 and a concomitant stimulation of 22Na-efflux of 0-388 +/- 0-136 micronmole. g wet wt.-1.min-1. 5. Maximum Na/ATP ratios for the active Na-K transport process were computed, with no assumption as to the in vivo free energy of ATP hydrolysis. These were 2-1, 1-9 and 2-3 under the conditions described in paragraphs (2), (3) and (4) respectively. 6. The calculated reversible thermodynamic work associated with active Na-K transport corresponded to 34% of the measured ouabain-induced decrease in E. On the premise that the maximum efficiency of the cellular energy conservation processes is 65%, this estimate indicates that the minimum energetic efficiency of ATP utilization by the active Na-K transport process in mammalian muscle is 52%.

  10. Urinary Dopamine as a Potential Index of the Transport Activity of Multidrug and Toxin Extrusion in the Kidney

    PubMed Central

    Kajiwara, Moto; Ban, Tsuyoshi; Matsubara, Kazuo; Nakanishi, Yoichi; Masuda, Satohiro

    2016-01-01

    Dopamine is a cationic natriuretic catecholamine synthesized in proximal tubular cells (PTCs) of the kidney before secretion into the lumen, a key site of its action. However, the molecular mechanisms underlying dopamine secretion into the lumen remain unclear. Multidrug and toxin extrusion (MATE) is a H+/organic cation antiporter that is highly expressed in the brush border membrane of PTCs and mediates the efflux of organic cations, including metformin and cisplatin, from the epithelial cells into the urine. Therefore, we hypothesized that MATE mediates dopamine secretion, a cationic catecholamine, into the tubule lumen, thereby regulating natriuresis. Here, we show that [3H]dopamine uptake in human (h) MATE1-, hMATE-2K- and mouse (m) MATE-expressing cells exhibited saturable kinetics. Fluid retention and decreased urinary excretion of dopamine and Na+ were observed in Mate1-knockout mice compared to that in wild-type mice. Imatinib, a MATE inhibitor, inhibited [3H]dopamine uptake by hMATE1-, hMATE2-K- and mMATE1-expressing cells in a concentration-dependent manner. At clinically-relevant concentrations, imatinib inhibited [3H]dopamine uptake by hMATE1- and hMATE2-K-expressing cells. The urinary excretion of dopamine and Na+ decreased and fluid retention occurred in imatinib-treated mice. In conclusion, MATE transporters secrete renally-synthesized dopamine, and therefore, urinary dopamine has the potential to be an index of the MATE transporter activity. PMID:27483254

  11. Accumulation and efflux of polychlorinated biphenyls in Escherichia coli.

    PubMed

    Geng, Shen; Fang, Jun; Turner, Kendrick B; Daunert, Sylvia; Wei, Yinan

    2012-06-01

    Polychlorinated biphenyls (PCBs) are environmental pollutants that have been associated with numerous adverse health effects in human and animals. Hydroxylated PCBs (HPCBs) are the product of the oxidative metabolism of PCBs. The presence of hydroxyl groups in HPCBs makes these compounds more hydrophilic than the parent PCBs. One of the best approaches to break down and remove these contaminants is bioremediation; an environmentally friendly process that uses microorganisms to degrade hazardous chemicals into non-toxic ones. In this study, we investigated the cellular accumulation and toxicity of selected PCBs and HPCBs in Gram-negative bacteria, using Escherichia coli as a model organism. We found that none of the five PCBs tested were toxic to E. coli, presumably due to their limited bioavailability. Nevertheless, different HPCBs tested showed different levels of toxicity. Furthermore, we demonstrated that the primary multidrug efflux system in E. coli, AcrAB-TolC, facilitated the efflux of HPCBs out of the cell. Since AcrAB-TolC is constitutively expressed in E. coli and is conserved in all sequenced Gram-negative bacterial genomes, our results suggest that the efflux activities of multidrug resistant pumps may affect the accumulation and degradation of PCBs in Gram-negative bacteria.

  12. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  13. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  14. The active transport of carbohydrates by Escherichia coli.

    PubMed

    Henderson, P J; Kornberg, H L

    1975-01-01

    The active transport of carbohydrates by Escherichia coli is discussed with particular reference to (1) identification of an uptake process as 'active transport', (2) nature and control of transport proteins, and (3) mechanisms of energy transduction. (1) The use of substrate analogues, of mutants blocked in metabolism and of subcellular vesicles in the isolation of the transport process from interference by subsequent metabolic reactions is described. Criteria are outlined for establishing that the solute is taken up against a concentration gradient and that this is energy-dependent. Three types of poisons for energy systems that act primarily on respiration, on ATP formation and as uncoupling ('proton conducting') agents are considered. (2) Methods are described for the selection of mutants impaired in the active uptake of specific carbohydrates. (3) Results show that the uptake of galactose, D-fucose and arabinose by appropriate strains of E. coli is inducible, specific and accompanied by proton uptake. Such and other data support a model based on a chemiosmotic theory of active transport.

  15. Active water transport in unicellular algae: where, why, and how.

    PubMed

    Raven, John A; Doblin, Martina A

    2014-12-01

    The occurrence of active water transport (net transport against a free energy gradient) in photosynthetic organisms has been debated for several decades. Here, active water transport is considered in terms of its roles, where it is found, and the mechanisms by which it could occur. First there is a brief consideration of the possibility of active water transport into plant xylem in the generation of root pressure and the refilling of embolized xylem elements, and from an unsaturated atmosphere into terrestrial organisms living in habitats with limited availability of liquid water. There is then a more detailed consideration of volume and osmotic regulation in wall-less freshwater unicells, and the possibility of generation of buoyancy in marine phytoplankton such as large-celled diatoms. Calculations show that active water transport is a plausible mechanism to assist cells in upwards vertical movements, requires less energy than synthesis of low-density organic solutes, and potentially on a par with excluding certain ions from the vacuole.

  16. Monocarboxylate transporter 1 contributes to growth factor-induced tumor cell migration independent of transporter activity

    PubMed Central

    Gray, Alana L.; Coleman, David T.; Shi, Runhua; Cardelli, James A.

    2016-01-01

    Tumor progression to metastatic disease contributes to the vast majority of incurable cancer. Understanding the processes leading to advanced stage cancer is important for the development of future therapeutic strategies. Here, we establish a connection between tumor cell migration, a prerequisite to metastasis, and monocarboxylate transporter 1 (MCT1). MCT1 transporter activity is known to regulate aspects of tumor progression and, as such, is a clinically relevant target for treating cancer. Knockdown of MCT1 expression caused decreased hepatocyte growth factor (HGF)-induced as well as epidermal growth factor (EGF)-induced tumor cell scattering and wound healing. Western blot analysis suggested that MCT1 knockdown (KD) hinders signaling through the HGF receptor (c-Met) but not the EGF receptor. Exogenous, membrane-permeable MCT1 substrates were not able to rescue motility in MCT1 KD cells, nor was pharmacologic inhibition of MCT1 able to recapitulate decreased cell motility as seen with MCT1 KD cells, indicating transporter activity of MCT1 was dispensable for EGF- and HGF-induced motility. These results indicate MCT1 expression, independent of transporter activity, is required for growth factor-induced tumor cell motility. The findings presented herein suggest a novel function for MCT1 in tumor progression independent of its role as a monocarboxylate transporter. PMID:27127175

  17. APP anterograde transport requires Rab3A GTPase activity for assembly of the transport vesicle

    PubMed Central

    Szodorai, A; Kuan, Y-H; Hunzelmann, S; Engel, U; Sakane, A; Sasaki, T; Takai, Y; Kirsch, J; Müller, U; Beyreuther, K; Brady, S; Morfini, G; Kins, S

    2010-01-01

    The amyloid precursor protein (APP) may be sequentially cleaved by β- and γ-secretases leading to accumulation of Aβ peptides in brains of Alzheimer’s Disease patients. Cleavage by α-secretase prevents Aβ generation. APP is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochemical composition of such a vesicle as well as the specific kinesin-1 motor responsible for transport are poorly defined. Here, we demonstrate by time-lapse analysis and immunoisolations that APP is a cargo of a vesicle containing the kinesin heavy chain isoform kinesin-1C, the small GTPase Rab3A and a specific subset of presynaptic protein components. Moreover, we report that assembly of kinesin-1C and APP in this vesicle type requires Rab3A GTPase activity. Finally, we show cleavage of APP in the analyzed transport vesicles by α-secretase activity, likely mediated by ADAM10. Together, these data indicate for the first time that maturation of transport vesicles, including coupling of conventional kinesin, requires Rab GTPase activity. PMID:19923287

  18. Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP.

    PubMed

    Neuberger, Arthur; van Veen, Hendrik W

    2015-01-01

    Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden "Janus" amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of "Hoechst 33342-like" drugs to cells and tissues in which these transporters are expressed. PMID:26540112

  19. Top consumer abundance influences lake methane efflux

    PubMed Central

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

    2015-01-01

    Lakes are important habitats for biogeochemical cycling of carbon. The organization and structure of aquatic communities influences the biogeochemical interactions between lakes and the atmosphere. Understanding how trophic structure regulates ecosystem functions and influences greenhouse gas efflux from lakes is critical to understanding global carbon cycling and climate change. With a whole-lake experiment in which a previously fishless lake was divided into two treatment basins where fish abundance was manipulated, we show how a trophic cascade from fish to microbes affects methane efflux to the atmosphere. Here, fish exert high grazing pressure and remove nearly all zooplankton. This reduction in zooplankton density increases the abundance of methanotrophic bacteria, which in turn reduce CH4 efflux rates by roughly 10 times. Given that globally there are millions of lakes emitting methane, an important greenhouse gas, our findings that aquatic trophic interactions significantly influence the biogeochemical cycle of methane has important implications. PMID:26531291

  20. Top consumer abundance influences lake methane efflux.

    PubMed

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

    2015-01-01

    Lakes are important habitats for biogeochemical cycling of carbon. The organization and structure of aquatic communities influences the biogeochemical interactions between lakes and the atmosphere. Understanding how trophic structure regulates ecosystem functions and influences greenhouse gas efflux from lakes is critical to understanding global carbon cycling and climate change. With a whole-lake experiment in which a previously fishless lake was divided into two treatment basins where fish abundance was manipulated, we show how a trophic cascade from fish to microbes affects methane efflux to the atmosphere. Here, fish exert high grazing pressure and remove nearly all zooplankton. This reduction in zooplankton density increases the abundance of methanotrophic bacteria, which in turn reduce CH4 efflux rates by roughly 10 times. Given that globally there are millions of lakes emitting methane, an important greenhouse gas, our findings that aquatic trophic interactions significantly influence the biogeochemical cycle of methane has important implications. PMID:26531291