Development of novel strategies to combat multidrug resistance mediated by efflux transporters and intracellular bacteria

OpenAIRE

Kuriakose, Jerrin

2014-01-01

Multidrug resistance (MDR) is the condition where cancer cells or microorganisms cease to respond to multiple drugs. MDR conferred by efflux transporters, that deprive the bioavailability of drugs at their site of action, are a threat to cancer and malarial chemotherapy. Specifically, the mammalian ABC transporter Pglycoprotein (P-gp) has undermined many drugs in treatment of cancer and other disease states. Mutations in the parasitic transporter Plasmodium falciparum chloroquine resistance t...

Fluoroquinolone resistance protein NorA of Staphylococcus aureus is a multidrug efflux transporter.

OpenAIRE

Neyfakh, A A; Borsch, C M; Kaatz, G W

1993-01-01

The gene of the Staphylococcus aureus fluoroquinolone efflux transporter protein NorA confers resistance to a number of structurally dissimilar drugs, not just to fluoroquinolones, when it is expressed in Bacillus subtilis. NorA provides B. subtilis with resistance to the same drugs and to a similar extent as the B. subtilis multidrug transporter protein Bmr does. NorA and Bmr share 44% sequence similarity. Both the NorA- and Bmr-conferred resistances can be completely reversed by reserpine.

Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump.

Science.gov (United States)

Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malvacio, Ivana; Malloci, Giuliano; Kleinekathöfer, Ulrich; Ruggerone, Paolo

2018-04-01

Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

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

Science.gov (United States)

Zhao, Jian; Li, Penghui; Motes, Christy M; Park, Sunghun; Hirschi, Kendal D

2015-11-01

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 of CHX14 promoter::GUS transgenic plants indicated that CHX14 was expressed in xylem parenchyma of root and shoot vascular tissues of seedlings. CHX14 knockout (chx14) and CHX14 overexpression seedlings displayed different growth phenotypes during K(+) stress as compared with wild-type seedlings. Roots of mutant seedlings displayed higher K(+) uptake rates than wild-type roots. CHX14 expression in yeast cells deficient in K(+) uptake renders the mutant cells more sensitive to deficiencies of K(+) in the medium. CHX14 mediates K(+) efflux in yeast cells loaded with high K(+) . Uptake experiments using (86) Rb(+) as a tracer for K(+) with both yeast and plant mutants demonstrated that CHX14 expression in yeast and in planta mediated low-affinity K(+) efflux. Functional green fluorescent protein (GFP)-tagged versions of CHX14 were localized to both the yeast and plant plasma membranes. Taken together, we suggest that CHX14 is a plasma membrane K(+) efflux transporter involved in K(+) homeostasis and K(+) recirculation. © 2015 John Wiley & Sons Ltd.

In vivo assessment of the impact of efflux transporter on oral drug absorption using portal vein-cannulated rats.

Science.gov (United States)

Matsuda, Yoshiki; Konno, Yoshihiro; Hashimoto, Takashi; Nagai, Mika; Taguchi, Takayuki; Satsukawa, Masahiro; Yamashita, Shinji

2013-08-01

The purpose of this study was to evaluate the impact of intestinal efflux transporters on the in vivo oral absorption process. Three model drugs-fexofenadine (FEX), sulfasalazine (SASP), and topotecan (TPT)-were selected as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and P-gp and BCRP substrates, respectively. The drugs were orally administered to portal vein-cannulated rats after pretreatment with zosuquidar (ZSQ), P-gp inhibitor, and/or Ko143, BCRP inhibitor. Intestinal availability (Fa·Fg) of the drugs was calculated from the difference between portal and systemic plasma concentrations. When rats were orally pretreated with ZSQ, Fa·Fg of FEX increased 4-fold and systemic clearance decreased to 75% of the control. In contrast, intravenous pretreatment with ZSQ did not affect Fa·Fg of FEX, although systemic clearance decreased significantly. These data clearly show that the method presented herein using portal vein-cannulated rats can evaluate the effects of intestinal transporters on Fa·Fg of drugs independently of variable systemic clearance. In addition, it was revealed that 71% of FEX taken up into enterocytes underwent selective efflux via P-gp to the apical surface, while 79% of SASP was effluxed by Bcrp. In the case of TPT, both transporters were involved in its oral absorption. Quantitative analysis indicated a 3.5-fold higher contribution from Bcrp than P-gp. In conclusion, the use of portal vein-cannulated rats enabled the assessment of the impact of efflux transporters on intestinal absorption of model drugs. This experimental system is useful for clarifying the cause of low bioavailability of various drugs.

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

Science.gov (United States)

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

2017-04-01

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

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

Science.gov (United States)

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

2017-05-01

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

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

Science.gov (United States)

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

2009-06-01

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

Single gold nanoparticle plasmonic spectroscopy for study of chemical-dependent efflux function of single ABC transporters of single live Bacillus subtilis cells.

Science.gov (United States)

Browning, Lauren M; Lee, Kerry J; Cherukuri, Pavan K; Huang, Tao; Songkiatisak, Preeyaporn; Warren, Seth; Xu, Xiao-Hong Nancy

2018-03-26

ATP-binding cassette (ABC) membrane transporters serve as self-defense transport apparatus in many living organisms and they can selectively extrude a wide variety of substrates, leading to multidrug resistance (MDR). The detailed molecular mechanisms remain elusive. Single nanoparticle plasmonic spectroscopy highly depends upon their sizes, shapes, chemical and surface properties. In our previous studies, we have used the size-dependent plasmonic spectra of single silver nanoparticles (Ag NPs) to study the real-time efflux kinetics of the ABC (BmrA) transporter and MexAB-OprM transporter in single live cells (Gram-positive and Gram-negative bacterium), respectively. In this study, we prepared and used purified, biocompatible and stable (non-aggregated) gold nanoparticles (Au NPs) (12.4 ± 0.9 nm) to study the efflux kinetics of single BmrA membrane transporters of single live Bacillus subtillis cells, aiming to probe chemical dependent efflux functions of BmrA transporters and their potential chemical sensing capability. Similar to those observed using Ag NPs, accumulation of the intracellular Au NPs in single live cells (WT and ΔBmrA) highly depends upon the cellular expression of BmrA and the NP concentration (0.7 and 1.4 nM). The lower accumulation of intracellular Au NPs in WT (normal expression of BmrA) than ΔBmrA (deletion of bmrA) indicates that BmrA extrudes the Au NPs out of the WT cells. The accumulation of Au NPs in the cells increases with NP concentration, suggesting that the Au NPs most likely passively diffuse into the cells, similar to antibiotics. The result demonstrates that such small Au NPs can serve as imaging probes to study the efflux function of the BmrA membrane transporter in single live cells. Furthermore, the dependence of the accumulation rate of intracellular Au NPs in single live cells upon the expression of BmrA and the concentration of the NPs is about twice higher than that of the same sized Ag NPs. This interesting finding

Efflux as a mechanism of antimicrobial drug resistance in clinical relevant microorganisms: the role of efflux inhibitors.

Science.gov (United States)

Willers, Clarissa; Wentzel, Johannes Frederik; du Plessis, Lissinda Hester; Gouws, Chrisna; Hamman, Josias Hendrik

2017-01-01

Microbial resistance against antibiotics is a serious threat to the effective treatment of infectious diseases. Several mechanisms exist through which microorganisms can develop resistance against antimicrobial drugs, of which the overexpression of genes to produce efflux pumps is a major concern. Several efflux transporters have been identified in microorganisms, which infer resistance against specific antibiotics and even multidrug resistance. Areas covered: This paper focuses on microbial resistance against antibiotics by means of the mechanism of efflux and gives a critical overview of studies conducted to overcome this problem by combining efflux pump inhibitors with antibiotics. Information was obtained from a literature search done with MEDLINE, Pubmed, Scopus, ScienceDirect, OneSearch and EBSCO host. Expert opinion: Efflux as a mechanism of multidrug resistance has presented a platform for improved efficacy against resistant microorganisms by co-administration of efflux pump inhibitors with antimicrobial agents. Although proof of concept has been shown for this approach with in vitro experiments, further research is needed to develop more potent inhibitors with low toxicity which is clinically effective.

Substrate specificities and efflux efficiencies of RND efflux pumps of Acinetobacter baumannii.

Science.gov (United States)

Leus, Inga V; Weeks, Jon W; Bonifay, Vincent; Smith, Lauren; Richardson, Sophie; Zgurskaya, Helen I

2018-04-16

Antibiotic resistant Acinetobacter baumannii causes infections that are extremely difficult to treat. A significant role in these resistance profiles is attributed to multidrug efflux pumps, especially those belonging to Resistance-Nodulation-cell Division (RND) superfamily of transporters. In this study, we analyzed functions and properties of RND efflux pumps in A. baumannii ATCC 17978. This strain is susceptible to antibiotics and does not contain mutations that are commonly selected upon exposure to high concentrations of antibiotics. We constructed derivatives of ATCC 17978 lacking chromosomally encoded RND pumps and complemented these strains by the plasmid-borne genes. We analyzed the substrate selectivities and efficiencies of the individual pumps in the context of native outer membranes and their hyperporinated variants. Our results show that inactivation of AdeIJK provides the strongest potentiation of antibiotic activities, whereas inactivation of AdeFGH triggers the overexpression of AdeAB. The plasmid-borne overproduction complements the hypersusceptible phenotypes of the efflux deletion mutants to the levels of the parental ATCC 17978. Only a few antibiotics strongly benefitted from the overproduction of efflux pumps and antibacterial activities of some of those depended on the synergistic interaction with the low permeability barrier of the outer membrane. Either overproduction or inactivation of efflux pumps change dramatically the lipidome of ATCC 17978. We conclude that efflux pumps of A. baumannii are tightly integrated into physiology of this bacterium and that clinical levels of antibiotic resistance in A. baumannii isolates are unlikely to be reached solely due to overproduction of RND efflux pumps. Importance RND-type efflux pumps are important contributors in development of clinical antibiotic resistance in A. baumannii However, their specific roles and the extent of contribution to antibiotic resistance remain unclear. We analyzed

Brain barriers and functional interfaces with sequential appearance of ABC efflux transporters during human development

DEFF Research Database (Denmark)

Møllgård, Kjeld; Dziegielewska, Katarzyna M.; Holst, Camilla B.

2017-01-01

Adult brain is protected from entry of drugs and toxins by specific mechanisms such as ABC (ATP-binding Cassette) efflux transporters. Little is known when these appear in human brain during development. Cellular distribution of three main ABC transporters (ABCC1, ABCG2, ABCB1) was determined...... at blood-brain barriers and interfaces in human embryos and fetuses in first half of gestation. Antibodies against claudin-5 and-11 and antibodies to α-fetoprotein were used to describe morphological and functional aspects of brain barriers. First exchange interfaces to be established, probably at 4...... three transporters. Results provide evidence for sequential establishment of brain exchange interfaces and spatial and temporal timetable for three main ABC transporters in early human brain....

Reconstitution of the activity of RND efflux pumps: a "bottom-up" approach.

Science.gov (United States)

Puvanendran, Dhenesh; Cece, Quentin; Picard, Martin

2017-12-05

Efflux pumps are systems devoted to the extrusion of noxious compounds. In this review, we discuss the various strategies that have thus far been undertaken for the investigation of efflux pumps after reconstitution into liposomes. It is challenging to uncover mechanisms and dynamics of efflux pumps due to a number of characteristics: their function depends on the correct assembly of three components and they span two adjacent membranes whose lipid compositions are very different. In addition, efflux pumps are active transporters that need energy to work. We present possible lines of improvement for the study of such systems and provide insights into future goals and challenges of efflux pump reconstitution and transport. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Chloroquine transport in Plasmodium falciparum. 1. Influx and efflux kinetics for live trophozoite parasites using a novel fluorescent chloroquine probe.

Science.gov (United States)

Cabrera, Mynthia; Natarajan, Jayakumar; Paguio, Michelle F; Wolf, Christian; Urbach, Jeffrey S; Roepe, Paul D

2009-10-13

Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfortunately impossible to obtain with traditional radio-tracer methods. Thus, we have designed and synthesized fluorescent CQ analogues for drug transport studies. One probe places a NBD (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) group at the tertiary aliphatic N of CQ, via a flexible 6 C amide linker. This probe localizes to the malarial parasite digestive vacuole (DV) during initial perfusion under physiologic conditions and exhibits similar pharmacology relative to CQ, vs both CQ-sensitive (CQS) and CQ-resistant (CQR) parasites. Using live, synchronized intraerythrocytic parasites under continuous perfusion, we define NBD-CQ influx and efflux kinetics for CQS vs CQR parasites. Since this fluorescence approach provides data at much higher kinetic resolution relative to fast-filtration methods using (3)H-CQ, rate constants vs linear initial rates for CQ probe flux can be analyzed in detail. Importantly, we find that CQR parasites have a decreased rate constant for CQ influx into the DV and that this is due to mutation of PfCRT. Analysis of zero trans efflux for CQS and CQR parasites suggests that distinguishing between bound vs free pools of intra-DV drug probe is essential for proper kinetic analysis of efflux. The accompanying paper (DOI 10.1021/bi901035j ) further probes efflux kinetics for proteoliposomes containing purified, reconstituted PfCRT.

Glutamate Efflux at the Blood-Brain Barrier

DEFF Research Database (Denmark)

Cederberg-Helms, Hans Christian; Uhd-Nielsen, Carsten; Brodin, Birger

2014-01-01

is well known, however endothelial cells may also play an important role through mediating brain-to-blood L-glutamate efflux. Expression of excitatory amino acid transporters has been demonstrated in brain endothelial cells of bovine, human, murine, rat and porcine origin. These can account for high...... affinity concentrative uptake of L-glutamate from the brain interstitial fluid into the capillary endothelial cells. The mechanisms in between L-glutamate uptake in the endothelial cells and L-glutamate appearing in the blood are still unclear and may involve a luminal transporter for L......-glutamate, metabolism of L-glutamate and transport of metabolites or a combination of the two. However, both in vitro and in vivo studies have demonstrated blood-to-brain transport of L-glutamate, at least during pathological events. This review summarizes the current knowledge on the brain-to-blood L-glutamate efflux...

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

Directory of Open Access Journals (Sweden)

Takanari Nakano

Full Text Available 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

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

Science.gov (United States)

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

2014-09-19

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

Multidrug efflux pumps in Staphylococcus aureus and their clinical implications.

Science.gov (United States)

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.

ATP binding cassette G1-dependent cholesterol efflux during inflammation.

Science.gov (United States)

de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

2011-02-01

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

Identification of a novel topoisomerase inhibitor effective in cells overexpressing drug efflux transporters.

Science.gov (United States)

Fayad, Walid; Fryknäs, Mårten; Brnjic, Slavica; Olofsson, Maria Hägg; Larsson, Rolf; Linder, Stig

2009-10-02

Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology. A library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo. The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

Deciphering the role of RND efflux transporters in Burkholderia cenocepacia.

Directory of Open Access Journals (Sweden)

Silvia Bazzini

Full Text Available Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division efflux pumps are known to be among the mediators of multidrug resistance in gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16 has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9, and a double-mutant in both efflux pumps (named D4-D9, were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4-D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

MOLECULAR DYNAMICS COMPUTER SIMULATIONS OF MULTIDRUG RND EFFLUX PUMPS

Directory of Open Access Journals (Sweden)

Paolo Ruggerone

2013-02-01

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

Molecular Dynamics Computer Simulations of Multidrug RND Efflux Pumps

Directory of Open Access Journals (Sweden)

Paolo Ruggerone

2013-02-01

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

22Na and 86Rb effluxes from bull spermatozoa

International Nuclear Information System (INIS)

Petzoldt, R.; Steffens, T.; Bernhardt, I.

1986-01-01

Active transport of sodium and potassium has been postulated for bull sperms by various authors. In the present paper the sodium and rubidium efflux was determined by tracer kinetics. For uninhibited sodium and rubidium efflux a rate constant of 8.9 +- 2.13/min and 13.9 +- 3.89 min, respectively, was found for a bull sperm suspension (20 vol.%, 310 K). After ouabain treatment (0.1 mM), a reduction of the rate constant of sodium efflux to 5.1 +- 1.06/min was found. After cryopreserving (pelletizing process) the majority of samples investigated did not exhibit any inhibition of sodium and rubidium efflux as compared with fresh bull sperms. The inhibition of sodium efflux observed in some cases corresponds to the reduction of the rate constant of sodium efflux caused by ouabain. At storage in seminal plasma (24 h, 278 K) the rate constant of sodium efflux is reduced to 2.7 +- 0.25/min. Both after ouabain treatment and after cryopreserving of sperms having a reduced rate constant the motility of bull sperms is reduced. It is concluded from the results that in bull sperms there exists an ouabain-sensitive sodium efflux, the inhibition of which reduces the rate constant of sodium efflux by 42%. The ouabain-sensitive sodium efflux is related to the motility of the bull sperm cell. (author)

Androgen regulation of the androgen receptor coregulators

International Nuclear Information System (INIS)

Urbanucci, Alfonso; Waltering, Kati K; Suikki, Hanna E; Helenius, Merja A; Visakorpi, Tapio

2008-01-01

The critical role of the androgen receptor (AR) in the development of prostate cancer is well recognized. The transcriptional activity of AR is partly regulated by coregulatory proteins. It has been suggested that these coregulators could also be important in the progression of prostate cancer. The aim of this study was to identify coregulators whose expression is regulated by either the androgens and/or by the expression level of AR. We used empty vector and AR cDNA-transfected LNCaP cells (LNCaP-pcDNA3.1, and LNCaP-ARhi, respectively), and grew them for 4 and 24 hours in the presence of dihydrotestosterone (DHT) at various concentrations. The expression of 25 AR coregulators (SRC1, TIF2, PIAS1, PIASx, ARIP4, BRCA1, β-catenin, AIB3, AIB1, CBP, STAT1, NCoR1, AES, cyclin D1, p300, ARA24, LSD1, BAG1L, gelsolin, prohibitin, JMJD2C, JMJD1A, MAK, PAK6 and MAGE11) was then measured by using real-time quantitative RT-PCR (Q-RT-PCR). Five of the coregulators (AIB1, CBP, MAK, BRCA1 and β-catenin) showed more than 2-fold induction and 5 others (cyclin D1, gelsolin, prohibitin, JMJD1A, and JMJD2C) less than 2-fold induction. Overexpression of AR did not affect the expression of the coregulators alone. However, overexpression of AR enhanced the DHT-stimulated expression of MAK, BRCA1, AIB1 and CBP and reduced the level of expression of β-catenin, cyclinD1 and gelsolin. In conclusion, we identified 5 coactivators whose expression was induced by androgens suggesting that they could potentiate AR signaling. Overexpression of AR seems to sensitize cells for low levels of androgens

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

Science.gov (United States)

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

2016-05-01

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

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

Directory of Open Access Journals (Sweden)

Beibei eWang

2015-04-01

Full Text Available 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→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.

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

Directory of Open Access Journals (Sweden)

Kati Juuti-Uusitalo

Full Text Available Retinal pigment epithelial (RPE cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP, the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC and RPE derived from the hESC (hESC-RPE. Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.

RND-type Drug Efflux Pumps from Gram-negative bacteria: Molecular Mechanism and Inhibition

Directory of Open Access Journals (Sweden)

Henrietta eVenter

2015-04-01

Full Text Available Drug efflux protein complexes confer multidrug resistance on bacteria by transporting a wide spectrum of structurally diverse antibiotics. Moreover, organisms can only acquire resistance in the presence of an active efflux pump. The substrate range of drug efflux pumps is not limited to antibiotics, but it also includes toxins, dyes, detergents, lipids and molecules involved in quorum sensing; hence efflux pumps are also associated with virulence and biofilm formation. Inhibitors of efflux pumps are therefore attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. Recent successes on the structure determination and functional analysis of the AcrB and MexB components of the AcrAB-TolC and MexAB-OprM drug efflux systems as well as the structure of the fully assembled, functional triparted AcrAB-TolC complex significantly contributed to our understanding of the mechanism of substrate transport and the options for inhibition of efflux. These data, combined with the well-developed methodologies for measuring efflux pump inhibition, could allow the rational design and subsequent experimental verification of potential efflux pump inhibitors. In this review we will explore how the available biochemical and structural information can be translated into the discovery and development of new compounds that could reverse drug resistance in Gram-negative pathogens. The current literature on efflux pump inhibitors will also be analysed and the reasons why no compounds have yet progressed into clinical use will be explored.

Identification of a novel topoisomerase inhibitor effective in cells overexpressing drug efflux transporters.

Directory of Open Access Journals (Sweden)

Walid Fayad

Full Text Available BACKGROUND: Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology. METHOD AND FINDINGS: A library of natural products (NCI Natural Product set was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine, an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo. CONCLUSIONS: The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

MicroRNA-20a/b regulates cholesterol efflux through post-transcriptional repression of ATP-binding cassette transporter A1.

Science.gov (United States)

Liang, Bin; Wang, Xin; Song, Xiaosu; Bai, Rui; Yang, Huiyu; Yang, Zhiming; Xiao, Chuanshi; Bian, Yunfei

2017-09-01

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in reverse cholesterol transport and exhibits anti-atherosclerosis effects. Some microRNAs (miRs) regulate ABCA1 expression, and recent studies have shown that miR-20a/b might play a critical role in atherosclerotic diseases. Here, we attempted to clarify the potential contribution of miR-20a/b in post-transcriptional regulation of ABCA1, cholesterol efflux, and atherosclerosis. We performed bioinformatics analysis and found that miR-20a/b was highly conserved and directly bound to ABCA1 mRNA with low binding free energy. Luciferase-reporter assay also confirmed that miR-20a/b significantly reduced luciferase activity associated with the ABCA1 3' untranslated region reporter construct. Additionally, miR-20a/b decreased ABCA1 expression, which, in turn, decreased cholesterol efflux and increased cholesterol content in THP-1 and RAW 264.7 macrophage-derived foam cells. In contrast, miR-20a/b inhibitors increased ABCA1 expression and cholesterol efflux, decreased cholesterol content, and inhibited foam-cell formation. Consistent with our in vitro results, miR-20a/b-treated ApoE -/- mice showed decreased ABCA1expression in the liver and reductions of reverse cholesterol transport in vivo. Furthermore, miR-20a/b regulated the formation of nascent high-density lipoprotein and promoted atherosclerotic development, whereas miR-20a/b knockdown attenuated atherosclerotic formation. miR-20 is a new miRNA capable of targeting ABCA1 and regulating ABCA1 expression. Therefore, miR-20 inhibition constitutes a new strategy for ABCA1-based treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Voluntary exercise increases cholesterol efflux but not macrophage reverse cholesterol transport in vivo in mice

Directory of Open Access Journals (Sweden)

Kuipers Folkert

2010-07-01

Full Text Available Abstract Physical exercise beneficially impacts on the plasma lipoprotein profile as well as on the incidence of cardiovascular events and is therefore recommended in primary and secondary prevention strategies against atherosclerotic cardiovascular disease. However, the underlying mechanisms of the protective effect of exercise remain largely unknown. Therefore, the present study tested the hypothesis that voluntary exercise in mice impacts on cholesterol efflux and in vivo reverse cholesterol transport (RCT. After two weeks of voluntary wheel running (average 10.1 ± 1.4 km/day plasma triglycerides were lower (p

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

Science.gov (United States)

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

The dynamics of nuclear receptors and nuclear receptor coregulators in the pathogenesis of endometriosis

Science.gov (United States)

Han, Sang Jun; O'Malley, Bert W.

2014-01-01

BACKGROUND Endometriosis is defined as the colonization and growth of endometrial tissue at anatomic sites outside the uterine cavity. Up to 15% of reproductive-aged women in the USA suffer from painful symptoms of endometriosis, such as infertility, pelvic pain, menstrual cycle abnormalities and increased risk of certain cancers. However, many of the current clinical treatments for endometriosis are not sufficiently effective and yield unacceptable side effects. There is clearly an urgent need to identify new molecular mechanisms that critically underpin the initiation and progression of endometriosis in order to develop more specific and effective therapeutics which lack the side effects of current therapies. The aim of this review is to discuss how nuclear receptors (NRs) and their coregulators promote the progression of endometriosis. Understanding the pathogenic molecular mechanisms for the genesis and maintenance of endometriosis as modulated by NRs and coregulators can reveal new therapeutic targets for alternative endometriosis treatments. METHODS This review was prepared using published gene expression microarray data sets obtained from patients with endometriosis and published literature on NRs and their coregulators that deal with endometriosis progression. Using the above observations, our current understanding of how NRs and NR coregulators are involved in the progression of endometriosis is summarized. RESULTS Aberrant levels of NRs and NR coregulators in ectopic endometriosis lesions are associated with the progression of endometriosis. As an example, endometriotic cell-specific alterations in gene expression are correlated with a differential methylation status of the genome compared with the normal endometrium. These differential epigenetic regulations can generate favorable cell-specific NR and coregulator milieus for endometriosis progression. Genetic alterations, such as single nucleotide polymorphisms and insertion/deletion polymorphisms of NR

Structural and functional aspects of the multidrug efflux pump AcrB.

Science.gov (United States)

Eicher, Thomas; Brandstätter, Lorenz; Pos, Klaas M

2009-08-01

The tripartite efflux system AcrA/AcrB/TolC is the main pump in Escherichia coli for the efflux of multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB is central to substrate recognition and energy transduction and acts as a proton/drug antiporter. Recent structural studies show that homotrimeric AcrB can adopt different monomer conformations representing consecutive states in an allosteric functional rotation transport cycle. The conformational changes create an alternate access drug transport tunnel including a hydrophobic substrate binding pocket in one of the cycle intermediates.

Effects of extracellular pH on UV-induced K+ efflux from cultured rose cells

International Nuclear Information System (INIS)

Huerta, A.J.; Murphy, T.M.

1989-01-01

Ultraviolet (UV) light causes a specific leakage of K + from cultured rose cells (Rosa damascena). During K + efflux, there is also an increase in extracellular HCO 3 - and acidification of the cell interior. We hypothesized that the HCO 3 - originated from intracellular hydration of respiratory CO 2 and served as a charge balancing mechanism during K + efflux, the K + and HCO 3 - being co transported out of the cell through specific channels. An alternative hypothesis which would yield similar results would be the counter transport of K + and H + . To test these hypotheses, we studied the effect of a range of external pH values (pH 5-9), regulated by various methods (pH-stat, 100 millimolar Tris-Mes buffer, or CO 2 partial pressure), on the UV-induced K + efflux. Both UV-C (less than 290 nanometers) and UV-B (290-310 nanometers) induced K + efflux with a minimum at about pH 6 to 7, and greater efflux at pH values of 5, 8, and 9. Since pH values of 8 and 9 increased instead of reduced the efflux of K + , these data are not consistent with notion that the efflux of K + is dependent on an influx of H + , a process that would be sensitive to external H + concentration. We suggest that the effect of pH on K + efflux may be mediated through the titration of specific K + -transporting proteins or channels in the plasma membrane. Since we could not detect the presence of carbonic anhydrase activity in cell extracts, we could not use the location of this enzyme to aid in our interpretation regarding the site of hydration of CO 2 . (author)

Architecture and roles of periplasmic adaptor proteins in tripartite efflux assemblies.

Directory of Open Access Journals (Sweden)

Vassiliy N. Bavro

2015-05-01

Full Text Available Recent years have seen major advances in the structural understanding of the different components of tripartite efflux assemblies, which encompass the multidrug efflux (MDR pumps and type I secretion systems. The majority of these investigations have focused on the role played by the inner membrane transporters and the outer membrane factor (OMF, leaving the third component of the system – the Periplasmic Adaptor Proteins (PAPs - relatively understudied. Here we review the current state of knowledge of these versatile proteins which, far from being passive linkers between the OMF and the transporter, emerge as active architects of tripartite assemblies, and play diverse roles in the transport process. Recognition between the PAPs and OMFs is essential for pump assembly and function, and targeting this interaction may provide a novel avenue for combating multidrug resistance. With the recent advances elucidating the drug-efflux and energetics of the tripartite assemblies, the understanding of the interaction between the OMFs and PAPs is the last piece remaining in the complete structure of the tripartite pump assembly puzzle.

Efflux in fungi: la pièce de résistance.

Directory of Open Access Journals (Sweden)

Jeffrey J Coleman

2009-06-01

Full Text Available 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.

Excessive Cellular S-nitrosothiol Impairs Endocytosis of Auxin Efflux Transporter PIN2

Directory of Open Access Journals (Sweden)

Min Ni

2017-11-01

Full Text Available S-nitrosoglutathione reductase (GSNOR1 is the key enzyme that regulates cellular levels of S-nitrosylation across kingdoms. We have previously reported that loss of GSNOR1 resulted in impaired auxin signaling and compromised auxin transport in Arabidopsis, leading to the auxin-related morphological phenotypes. However, the molecular mechanism underpinning the compromised auxin transport in gsnor1-3 mutant is still unknown. Endocytosis of plasma-membrane (PM-localized efflux PIN proteins play critical roles in auxin transport. Therefore, we investigate whether loss of GSNOR1 function has any effects on the endocytosis of PIN-FORMED (PIN proteins. It was found that the endocytosis of either the endogenous PIN2 or the transgenically expressed PIN2-GFP was compromised in the root cells of gsnor1-3 seedlings relative to Col-0. The internalization of PM-associated PIN2 or PIN2-GFP into Brefeldin A (BFA bodies was significantly reduced in gsnor1-3 upon BFA treatment in a manner independent of de novo protein synthesis. In addition, the exogenously applied GSNO not only compromised the endocytosis of PIN2-GFP but also inhibited the root elongation in a concentration-dependent manner. Taken together, our results indicate that, besides the reduced PIN2 level, one or more compromised components in the endocytosis pathway could account for the reduced endocytosis of PIN2 in gsnor1-3.

Localization of the placental BCRP/ABCG2 transporter to lipid rafts: Role for cholesterol in mediating efflux activity.

Science.gov (United States)

Szilagyi, John T; Vetrano, Anna M; Laskin, Jeffrey D; Aleksunes, Lauren M

2017-07-01

The breast cancer resistance protein (BCRP/ABCG2) is an efflux transporter in the placental barrier. By transporting chemicals from the fetal to the maternal circulation, BCRP limits fetal exposure to a range of drugs, toxicants, and endobiotics such as bile acids and hormones. The purpose of the present studies was to 1) determine whether BCRP localizes to highly-ordered, cholesterol-rich lipid raft microdomains in placenta microvillous membranes, and 2) determine the impact of cholesterol on BCRP-mediated placental transport in vitro. BCRP expression was analyzed in lipid rafts isolated from placentas from healthy, term pregnancies and BeWo trophoblasts by density gradient ultracentrifugation. BeWo cells were also tested for their ability to efflux BCRP substrates after treatment with the cholesterol sequestrant methyl-β-cyclodextrin (MβCD, 5 mM, 1 h) or the cholesterol synthesis inhibitor pravastatin (200 μM, 48 h). BCRP was found to co-localize with lipid raft proteins in detergent-resistant, lipid raft-containing fractions from placental microvillous membranes and BeWo cells. Treatment of BeWo cells with MβCD redistributed BCRP protein into higher density non-lipid raft fractions. Repletion of the cells with cholesterol restored BCRP localization to lipid raft-containing fractions. Treatment of BeWo cells with MβCD or pravastatin increased cellular retention of two BCRP substrates, the fluorescent dye Hoechst 33342 and the mycotoxin zearalenone. Repletion with cholesterol restored BCRP transporter activity. Taken together, these data demonstrate that cholesterol may play a critical role in the post-translational regulation of BCRP in placental lipid rafts. Copyright © 2017 Elsevier Ltd. All rights reserved.

Drug efflux proteins in multidrug resistant bacteria

NARCIS (Netherlands)

vanVeen, HW; Konings, WN

Bacteria contain an array of transport proteins in their cytoplasmic membrane. Many of these proteins play an important role in conferring resistance to toxic compounds. The multidrug efflux systems encountered in prokaryotic cells are very similar to those observed in eukaryotic cells. Therefore, a

Atypical dopamine efflux caused by 3,4-methylenedioxypyrovalerone (MDPV) via the human dopamine transporter.

Science.gov (United States)

Shekar, Aparna; Aguilar, Jenny I; Galli, Greta; Cozzi, Nicholas V; Brandt, Simon D; Ruoho, Arnold E; Baumann, Michael H; Matthies, Heinrich J G; Galli, Aurelio

2017-10-01

Synthetic cathinones are similar in chemical structure to amphetamines, and their behavioral effects are associated with enhanced dopaminergic signaling. The past ten years of research on the common constituent of bath salts, MDPV (the synthetic cathinone 3,4-methylenedioxypyrovalerone), has aided the understanding of how synthetic cathinones act at the dopamine (DA) transporter (DAT). Several groups have described the ability of MDPV to block the DAT with high-affinity. In this study, we demonstrate for the first time a new mode of action of MDPV, namely its ability to promote DAT-mediated DA efflux. Using single cell amperometric assays, we determined that low concentrations of MDPV (1nM) can cause reverse transport of DA via DAT. Notably, administration of MDPV leads to hyperlocomotion in Drosophila melanogaster. These data describe further how MDPV acts at the DAT, possibly paving the way for novel treatment strategies for individuals who abuse bath salts. Copyright © 2017 Elsevier B.V. All rights reserved.

Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells

Science.gov (United States)

Slomiany, Mark G.; Grass, G. Daniel; Robertson, Angela D.; Yang, Xiao Y.; Maria, Bernard L.; Beeson, Craig; Toole, Bryan P.

2013-01-01

Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells. PMID:19176383

Placental Drug Transport-on-a-Chip: A Microengineered In Vitro Model of Transporter-Mediated Drug Efflux in the Human Placental Barrier.

Science.gov (United States)

Blundell, Cassidy; Yi, Yoon-Suk; Ma, Lin; Tess, Emily R; Farrell, Megan J; Georgescu, Andrei; Aleksunes, Lauren M; Huh, Dongeun

2018-01-01

The current lack of knowledge about the effect of maternally administered drugs on the developing fetus is a major public health concern worldwide. The first critical step toward predicting the safety of medications in pregnancy is to screen drug compounds for their ability to cross the placenta. However, this type of preclinical study has been hampered by the limited capacity of existing in vitro and ex vivo models to mimic physiological drug transport across the maternal-fetal interface in the human placenta. Here the proof-of-principle for utilizing a microengineered model of the human placental barrier to simulate and investigate drug transfer from the maternal to the fetal circulation is demonstrated. Using the gestational diabetes drug glyburide as a model compound, it is shown that the microphysiological system is capable of reconstituting efflux transporter-mediated active transport function of the human placental barrier to limit fetal exposure to maternally administered drugs. The data provide evidence that the placenta-on-a-chip may serve as a new screening platform to enable more accurate prediction of drug transport in the human placenta. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Energy Technology Data Exchange (ETDEWEB)

Faria, Melissa [Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18, 08034 Barcelona (Spain); CESAM & Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro (Portugal); Pavlichenko, Vasiliy [Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch of Russian Academy of Sciences (SIPPB SB RAS), Lermontov Str. 132, 664033, Irkutsk (Russian Federation); Department of Bioanalytical Ecotoxicology, UFZ — Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig (Germany); Burkhardt-Medicke, Kathleen [Department of Bioanalytical Ecotoxicology, UFZ — Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig (Germany); Institute of Hydrobiology, Dresden University of Technology, D-01062 Dresden (Germany); Soares, Amadeu M.V.M. [CESAM & Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro (Portugal); Altenburger, Rolf [Department of Bioanalytical Ecotoxicology, UFZ — Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig (Germany); Barata, Carlos [Department of Environmental Chemistry, IDAEA-CSIC, Jordi Girona 18, 08034 Barcelona (Spain); Luckenbach, Till, E-mail: till.luckenbach@ufz.de [Department of Bioanalytical Ecotoxicology, UFZ — Helmholtz Centre for Environmental Research, Permoserstr. 15, D-04318 Leipzig (Germany)

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. - Highlights: • Sequences and function of ABC efflux transporters in bivalve gills were explored. • Full length Dreissena polymorpha abcb1 and abcc1 cDNA sequences were identified. • A mixture effect

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

International Nuclear Information System (INIS)

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

2016-01-01

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. - Highlights: • Sequences and function of ABC efflux transporters in bivalve gills were explored. • Full length Dreissena polymorpha abcb1 and abcc1 cDNA sequences were identified. • A mixture effect

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

Science.gov (United States)

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

2005-12-01

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

Role of efflux pumps and intracellular thiols in natural antimony resistant isolates of Leishmania donovani.

Directory of Open Access Journals (Sweden)

Smita Rai

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

Efflux-mediated antimicrobial resistance.

Science.gov (United States)

Poole, Keith

2005-07-01

Antibiotic resistance continues to plague antimicrobial chemotherapy of infectious disease. And while true biocide resistance is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are common and the history of antibiotic resistance should not be ignored in the development and use of biocidal agents. Efflux mechanisms of resistance, both drug specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials, with some accommodating both antibiotics and biocides. This latter raises the spectre (as yet generally unrealized) of biocide selection of multiple antibiotic-resistant organisms. Multidrug efflux mechanisms are broadly conserved in bacteria, are almost invariably chromosome-encoded and their expression in many instances results from mutations in regulatory genes. In contrast, drug-specific efflux mechanisms are generally encoded by plasmids and/or other mobile genetic elements (transposons, integrons) that carry additional resistance genes, and so their ready acquisition is compounded by their association with multidrug resistance. While there is some support for the latter efflux systems arising from efflux determinants of self-protection in antibiotic-producing Streptomyces spp. and, thus, intended as drug exporters, increasingly, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, appear not to be intended as drug exporters but as exporters with, perhaps, a variety of other roles in bacterial cells. Still, given the clinical significance of multidrug (and drug-specific) exporters, efflux must be considered in formulating strategies/approaches to treating drug-resistant infections, both in the development of new agents, for example, less impacted by efflux and in targeting efflux directly with efflux inhibitors.

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

Science.gov (United States)

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

2016-01-01

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

Computer simulation and interpretation of 45Ca efflux profile patterns

International Nuclear Information System (INIS)

Borle, A.B.; Uchikawa, T.; Anderson, J.H.

1982-01-01

Stimulations or inhibitions by various agents of 45 Ca efflux from prelabeled cells or tissues display distinct and reproducible profile patterns when the results are plotted against time as fractional efflux ratios (FER). FER is the fractional efflux of 45 Ca from stimulated cells divided by the fractional efflux from a control unstimulated group. These profile patterns fall into three categories: peak patterns, exponential patterns, and mixed patterns. Each category can be positive (stimulation) or negative (inhibition). The interpretation of these profiles is difficult because 45 Ca efflux depends on three variables: the rate of calcium transport out of the cell, the specific activity of the cell compartment from which the calcium originates, and the concentration of free calcium in this compartment. A computer model based on data obtained by kinetic analyses of 45 Ca desaturation curves and consisting of two distinct intracellular pools was designed to follow the concentration of the traced substance ( 40 Ca), the tracer ( 45 Ca), and the specific activity of each compartment before, during, and after the stimulation or the inhibition of calcium fluxes at various pool boundaries. The computer model can reproduce all the FER profiles obtained experimentally and bring information which may be helpful to the interpretation of this type of data. Some predictions of the model were tested experimentally, and the results support the views that a peak pattern may reflect a sustained change in calcium transport across the plasma membrane, that an exponential pattern arises from calcium mobilization from an internal subcellular pool, and that a mixed pattern may be caused by a simultaneous change in calcium fluxes at both compartment boundaries

New methods for the identification of efflux mediated MDR bacteria, genetic assessment of regulators and efflux pump constituents, characterization of efflux systems and screening for inhibitors of efflux pumps

DEFF Research Database (Denmark)

Viveiros, M; Martins, M; Couto, I

2008-01-01

We have developed a number of methods that identify efflux pump mediated multi-drug resistant bacteria, characterize efflux systems and screen for inhibitors of efflux pumps. These approaches were complemented by the quantification of the expression of genes that regulate and code for constituents...

Proposing a Model of Co-Regulated Learning for Graduate Medical Education.

Science.gov (United States)

Rich, Jessica V

2017-08-01

Primarily grounded in Zimmerman's social cognitive model of self-regulation, graduate medical education is guided by principles that self-regulated learning takes place within social context and influence, and that the social context and physical environment reciprocally influence persons and their cognition, behavior, and development. However, contemporary perspectives on self-regulation are moving beyond Zimmerman's triadic reciprocal orientation to models that consider social transactions as the central core of regulated learning. Such co-regulated learning models emphasize shared control of learning and the role more advanced others play in scaffolding novices' metacognitive engagement.Models of co-regulated learning describe social transactions as periods of distributed regulation among individuals, which instrumentally promote or inhibit the capacity for individuals to independently self-regulate. Social transactions with other regulators, including attending physicians, more experienced residents, and allied health care professionals, are known to mediate residents' learning and to support or hamper the development of their self-regulated learning competence. Given that social transactions are at the heart of learning-oriented assessment and entrustment decisions, an appreciation for co-regulated learning is likely important for advancing medical education research and practice-especially given the momentum of new innovations such as entrustable professional activities.In this article, the author explains why graduate medical educators should consider adopting a model of co-regulated learning to complement and extend Zimmerman's models of self-regulated learning. In doing so, the author suggests a model of co-regulated learning and provides practical examples of how the model is relevant to graduate medical education research and practice.

Multidrug resistance transporters Snq2p and Pdr5p mediate caffeine efflux in Saccharomyces cerevisiae.

Science.gov (United States)

Tsujimoto, Yoshiyuki; Shimizu, Yoshihiro; Otake, Kazuya; Nakamura, Tatsuya; Okada, Ryutaro; Miyazaki, Toshitaka; Watanabe, Kunihiko

2015-01-01

SNQ2 was identified as a caffeine-resistance gene by screening a genomic library of Saccharomyces cerevisiae in a multicopy vector YEp24. SNQ2 encodes an ATP-binding cassette transporter and is highly homologous to PDR5. Multicopy of PDR5 also conferred resistance to caffeine, while its resistance was smaller than that of SNQ2. Residual caffeine contents were analyzed after transiently exposing cells to caffeine. The ratios of caffeine contents were 21.3 ± 8.8% (YEp24-SNQ2) and 81.9 ± 8.7% (YEp24-PDR5) relative to control (YEp24, 100%). In addition, multicopies of SNQ2 or PDR5 conferred resistance to rhodamine 6G (R6G), which was widely used as a substrate for transport assay. R6G was exported by both transporters, and their efflux activities were inhibited by caffeine with half-maximal inhibitory concentrations of 5.3 ± 1.9 (YEp24-SNQ2) and 17.2 ± 9.6 mM (YEp24-PDR5). These results demonstrate that Snq2p is a more functional transporter of caffeine than Pdr5p in yeast cells.

Computer simulations of the activity of RND efflux pumps.

Science.gov (United States)

Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malloci, Giuliano; Malvacio, Ivana; Atzori, Alessio; Ruggerone, Paolo

2018-01-31

The putative mechanism by which bacterial RND-type multidrug efflux pumps recognize and transport their substrates is a complex and fascinating enigma of structural biology. How a single protein can recognize a huge number of unrelated compounds and transport them through one or just a few mechanisms is an amazing feature not yet completely unveiled. The appearance of cooperativity further complicates the understanding of structure-dynamics-activity relationships in these complex machineries. Experimental techniques may have limited access to the molecular determinants and to the energetics of key processes regulating the activity of these pumps. Computer simulations are a complementary approach that can help unveil these features and inspire new experiments. Here we review recent computational studies that addressed the various molecular processes regulating the activity of RND efflux pumps. Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Mechanistic kinetic modeling generates system-independent P-glycoprotein mediated transport elementary rate constants for inhibition and, in combination with 3D SIM microscopy, elucidates the importance of microvilli morphology on P-glycoprotein mediated efflux activity.

Science.gov (United States)

Ellens, Harma; Meng, Zhou; Le Marchand, Sylvain J; Bentz, Joe

2018-06-01

In vitro transporter kinetics are typically analyzed by steady-state Michaelis-Menten approximations. However, no clear evidence exists that these approximations, applied to multiple transporters in biological membranes, yield system-independent mechanistic parameters needed for reliable in vivo hypothesis generation and testing. Areas covered: The classical mass action model has been developed for P-glycoprotein (P-gp) mediated transport across confluent polarized cell monolayers. Numerical integration of the mass action equations for transport using a stable global optimization program yields fitted elementary rate constants that are system-independent. The efflux active P-gp was defined by the rate at which P-gp delivers drugs to the apical chamber, since as much as 90% of drugs effluxed by P-gp partition back into nearby microvilli prior to reaching the apical chamber. The efflux active P-gp concentration was 10-fold smaller than the total expressed P-gp for Caco-2 cells, due to their microvilli membrane morphology. The mechanistic insights from this analysis are readily extrapolated to P-gp mediated transport in vivo. Expert opinion: In vitro system-independent elementary rate constants for transporters are essential for the generation and validation of robust mechanistic PBPK models. Our modeling approach and programs have broad application potential. They can be used for any drug transporter with minor adaptations.

Computational Study of Correlated Domain Motions in the AcrB Efflux Transporter

Directory of Open Access Journals (Sweden)

Robert Schulz

2015-01-01

Full Text Available As active part of the major efflux system in E. coli bacteria, AcrB is responsible for the uptake and pumping of toxic substrates from the periplasm toward the extracellular space. In combination with the channel protein TolC and membrane fusion protein AcrA, this efflux pump is able to help the bacterium to survive different kinds of noxious compounds. With the present study we intend to enhance the understanding of the interactions between the domains and monomers, for example, the transduction of mechanical energy from the transmembrane domain into the porter domain, correlated motions of different subdomains within monomers, and cooperative effects between monomers. To this end, targeted molecular dynamics simulations have been employed either steering the whole protein complex or specific parts thereof. By forcing only parts of the complex towards specific conformational states, the risk for transient artificial conformations during the simulations is reduced. Distinct cooperative effects between the monomers in AcrB have been observed. Possible allosteric couplings have been identified providing microscopic insights that might be exploited to design more efficient inhibitors of efflux systems.

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

Directory of Open Access Journals (Sweden)

Adam R Brown

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

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

Science.gov (United States)

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

2015-04-01

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

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias).

Science.gov (United States)

Koomoa, D L; Musch, M W; MacLean, A V; Goldstein, L

2001-09-01

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.

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.

Science.gov (United States)

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

Interaction of antibacterial compounds with RND efflux pumps in Pseudomonas aeruginosa

Directory of Open Access Journals (Sweden)

Juerg eDreier

2015-07-01

Full Text Available Pseudomonas aeruginosa infections are becoming increasingly difficult to treat due to intrinsic antibiotic resistance and the propensity of this pathogen to accumulate diverse resistance mechanisms. Hyperexpression of efflux pumps of the Resistance-Nodulation-Division-type multidrug efflux pumps (e.g. MexAB-OprM, chromosomally encoded by mexAB-oprM, mexCD-oprJ, mexEF-oprN, and mexXY (-oprA is often detected in clinical isolates and contributes to worrying multi-drug resistance phenotypes.Not all antibiotics are affected to the same extent by the aforementioned RND efflux pumps. The impact of efflux on antibiotic activity varies not only between different classes of antibiotics but also between members of the same family of antibiotics. Subtle differences in physicochemical features of compound-pump and compound-solvent interactions largely determine how compounds are affected by efflux activity.The combination of different high-resolution techniques helps to gain insight into the functioning of these molecular machineries. This review discusses substrate recognition patterns based on experimental evidence and computer simulations with a focus on MexB, the pump subunit of the main RND transporter in P. aeruginosa.

Lapatinib potentiates cytotoxicity of YM155 in neuroblastoma via inhibition of the ABCB1 efflux transporter

DEFF Research Database (Denmark)

Radic-Sarikas, Branka; Halasz, Melinda; Huber, Kilian V. M.

2017-01-01

and simultaneously help to overcome drug resistance. Neuroblastoma is the most common cancer in infancy and extremely heterogeneous in clinical presentation and features. Applying a systematic pairwise drug combination screen we observed a highly potent synergy in neuroblastoma cells between the EGFR kinase...... inhibitor lapatinib and the anticancer compound YM155 that is preserved across several neuroblastoma variants. Mechanistically, the synergy was based on a lapatinib induced inhibition of the multidrug-resistance efflux transporter ABCB1, which is frequently expressed in resistant neuroblastoma cells, which...... allowed prolonged and elevated cytotoxicity of YM155. In addition, the drug combination (i.e. lapatinib plus YM155) decreased neuroblastoma tumor size in an in vivo model....

Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

Science.gov (United States)

Damgaard, Christian Kroun; Lykke-Andersen, Jens

2011-01-01

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5′-terminal oligopyrimidine tracts (5′TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5′TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5′TOP mRNA regulation, which upon amino acid starvation assemble onto the 5′ end of 5′TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5′TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5′TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment. PMID:21979918

Optimized efflux assay for the NorA multidrug efflux pump in Staphylococcus aureus.

Science.gov (United States)

Zimmermann, Saskia; Tuchscherr, Lorena; Rödel, Jürgen; Löffler, Bettina; Bohnert, Jürgen A

2017-11-01

Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC 3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible. Copyright © 2017 Elsevier B.V. All rights reserved.

Efflux pumps as antimicrobial resistance mechanisms.

Science.gov (United States)

Poole, Keith

2007-01-01

Antibiotic resistance continues to hamper antimicrobial chemotherapy of infectious disease, and while biocide resistance outside of the laboratory is as yet unrealized, in vitro and in vivo episodes of reduced biocide susceptibility are not uncommon. Efflux mechanisms, both drug-specific and multidrug, are important determinants of intrinsic and/or acquired resistance to these antimicrobials in important human pathogens. Multidrug efflux mechanisms are generally chromosome-encoded, with their expression typically resultant from mutations in regulatory genes, while drug-specific efflux mechanisms are encoded by mobile genetic elements whose acquisition is sufficient for resistance. While it has been suggested that drug-specific efflux systems originated from efflux determinants of self-protection in antibiotic-producing Actinomycetes, chromosomal multidrug efflux determinants, at least in Gram-negative bacteria, are appreciated as having an intended housekeeping function unrelated to drug export and resistance. Thus, it will be important to elucidate the intended natural function of these efflux mechanisms in order, for example, to anticipate environmental conditions or circumstances that might promote their expression and, so, compromise antimicrobial chemotherapy. Given the clinical significance of antimicrobial exporters, it is clear that efflux must be considered in formulating strategies for treatment of drug-resistant infections, both in the development of new agents, for example, less impacted by efflux or in targeting efflux directly with efflux inhibitors.

Effect of vildagliptin and pravastatin combination on cholesterol efflux in adipocytes.

Science.gov (United States)

Mostafa, Ahmed M; Hamdy, Nadia M; Abdel-Rahman, Sherif Z; El-Mesallamy, Hala O

2016-07-01

Many reports suggested that some statins are almost ineffective in reducing triglycerides or enhancing HDL-C plasma levels, although statin treatment was still efficacious in reducing LDL-C. In diabetic dyslipidemic patients, it may therefore be necessary to use a combination therapy with other drugs to achieve either LDL-C- and triglyceride-lowering or HDL-C-enhancing goals. Such ineffectiveness of statins can be attributed to their effect on the liver X receptor (LXR) which regulates the expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. A decrease in the expression of these transporters eventually leads to decreased cholesterol efflux from peripheral tissues leading to low levels of HDL-C. Although manipulating the LXR pathway may complement the effects of statins, LXR synthetic ligands as T091317 have shown significant hypertriglyceridemic action which limits their use. We recently found that the antidiabetic drug vildagliptin stimulates LXR expression leading to increased ABCB1/ABCG1 expression which improves cholesterol efflux from adipocytes. Therefore, a combination of vildagliptin and statin may provide a solution without the hypertriglyceridemic action observed with LXR agonist. We hypothesize that a combination of vildagliptin and pravastatin will improve cholesterol efflux in adipocytes. Statin-treated 3T3-L1 adipocytes were treated with vildagliptin, and the expression of LXR-ABCA1/ABCG1 cascade and the cholesterol efflux were then determined. Our data indicate that a combination of vildagliptin and pravastatin significantly induces the expression of LXR-ABCA1/ABCG1 cascade and improves cholesterol efflux (P > 0.05) in adipocytes. Our data may explain, at least in part, the improvement in HDL-C levels observed in patients receiving both medications. © 2016 IUBMB Life, 68(7):535-543, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

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

Directory of Open Access Journals (Sweden)

Maricla Galetti

Full Text Available 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.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.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.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.

Uncovering packaging features of co-regulated modules based on human protein interaction and transcriptional regulatory networks

Directory of Open Access Journals (Sweden)

He Weiming

2010-07-01

Full Text Available Abstract Background Network co-regulated modules are believed to have the functionality of packaging multiple biological entities, and can thus be assumed to coordinate many biological functions in their network neighbouring regions. Results Here, we weighted edges of a human protein interaction network and a transcriptional regulatory network to construct an integrated network, and introduce a probabilistic model and a bipartite graph framework to exploit human co-regulated modules and uncover their specific features in packaging different biological entities (genes, protein complexes or metabolic pathways. Finally, we identified 96 human co-regulated modules based on this method, and evaluate its effectiveness by comparing it with four other methods. Conclusions Dysfunctions in co-regulated interactions often occur in the development of cancer. Therefore, we focussed on an example co-regulated module and found that it could integrate a number of cancer-related genes. This was extended to causal dysfunctions of some complexes maintained by several physically interacting proteins, thus coordinating several metabolic pathways that directly underlie cancer.

Current Advances in Developing Inhibitors of Bacterial Multidrug 
Efflux Pumps

Science.gov (United States)

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

Contribution of vesicular and cytosolic dopamine to the increased striatal dopamine efflux elicited by intrastriatal injection of SKF38393.

NARCIS (Netherlands)

Saigusa, T.; Aono, Y.; Sekino, R.; Uchida, T.; Takada, K.; Oi, Y.; Koshikawa, N.; Cools, A.R.

2009-01-01

Like dexamphetamine, SKF38393 induces an increase in striatal dopamine efflux which is insensitive for tetrodotoxin, Ca(2+) independent and prevented by a dopamine transporter inhibitor. The dexamphetamine-induced striatal dopamine efflux originates from both the reserpine-sensitive vesicular

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

International Nuclear Information System (INIS)

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

2014-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Yang, Yanbo; Thyagarajan, Narmadaa; Coady, Breanne M.; Brown, Robert J., E-mail: rbrown@mun.ca

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.

Efflux of inorganic substances from young barley roots. I. Efflux in water culture under various conditions

Energy Technology Data Exchange (ETDEWEB)

Fujimoto, H; Kojima, S [Radiation Center of Osaka Prefecture, Sakai (Japan)

1977-09-01

The efflux of elements from the roots of hydropomically grown young barley plants was studied. The effects of different mutrient compositions and pH values of the solutions was also studied using /sup 22/Na and /sup 45/Ca as the indexes. In all culture conditions, there was efflux of both elements. In two media with dilute hydrochloric acid and AlCl/sub 3/, respectively, the tendencies of Na and Ca efflux were similar in both media at first, but after 72 hr, the Na efflux in AlCl/sub 3/ decreased and that in dilute hydrochloric acid medium increased. The Ca efflux was high in AlCl/sub 3/ medium,however. The efflux of both Na and Ca was higher in the standard medium than in the media with some bases of high concentrations.

Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1

NARCIS (Netherlands)

Beulens, J.W.J.; Sierksma, A.; Tol, A. van; Fournier, N.; Gent, T. van; Paul, J.L.; Hendriks, H.F.J.

2004-01-01

Moderate alcohol consumption increases HDL cholesterol, which is involved in reverse cholesterol transport (RCT). The aim of this study was to investigate the effect of moderate alcohol consumption on cholesterol efflux, using J774 mouse macrophages and Fu5AH cells, and on other parameters in the

[Efflux systems in Serratia marcescens].

Science.gov (United States)

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.

Phytosterols Differentially Influence ABC transporter Expression, Cholesterol Efflux and Inflammatory Cytokine Secretion in Macrophage Foam Cells

Science.gov (United States)

Sabeva, Nadezhda S; McPhaul, Christopher M; Li, Xiangan; Cory, Theodore J.; Feola, David J.; Graf, Gregory A

2010-01-01

Phytosterol supplements lower low density lipoprotein (LDL) cholesterol, but accumulate in vascular lesions of patients and limit the anti-atherosclerotic effects of LDL lowering in apolipoprotein E deficient mice, suggesting that the cholesterol lowering benefit of phytosterol supplementation may not be fully realized. Individual phytosterols have cell-type specific effects that may either be beneficial or deleterious with respect to atherosclerosis, but little is known concerning their effects on macrophage function. The effects of phytosterols on ABCA1 and ABCG1 abundance, cholesterol efflux, and inflammatory cytokine secretion were determined in cultured macrophage foam cells. Among the commonly consumed phytosterols, stigmasterol increased expression of ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein (Apo) AI and high density lipoprotein (HDL). Campesterol and sitosterol had no effect on ABCA1 or ABCG1 levels. Sitosterol had no effect of cholesterol efflux to Apo AI or HDL, whereas campesterol had a modest, but significant reduction in cholesterol efflux to HDL in THP-1 macrophages. Whereas stigmasterol blunted aggregated LDL-induced increases in tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β secretion, sitosterol exacerbated these effects. The presence of campesterol had no effect on agLDL-induced inflammatory cytokine secretion from THP-1 macrophages. In conclusion, the presence of stigmasterol in modified lipoproteins promoted cholesterol efflux and suppressed inflammatory cytokine secretion in response to lipid loading in macrophage foam cells. While campesterol was largely inert, the presence of sitosterol increased the proinflammatory cytokine secretion. PMID:21111593

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

International Nuclear Information System (INIS)

Dissing, S.; Hoffman, J.F.

1990-01-01

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o

Tripartite assembly of RND multidrug efflux pumps.

Science.gov (United States)

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

2016-02-12

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.

In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma.

Directory of Open Access Journals (Sweden)

Susanna J E Veringa

Full Text Available 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.

Excipient-drug pharmacokinetic interactions: Effect of disintegrants on efflux across excised pig intestinal tissues

Directory of Open Access Journals (Sweden)

Werner Gerber

2018-04-01

Full Text Available Pharmaceutical excipients were designed originally to be pharmacologically inert. However, certain excipients were found to have altering effects on drug pharmacodynamics and/or pharmacokinetics. Pharmacokinetic interactions may be caused by modulation of efflux transporter proteins, intercellular tight junctions and/or metabolic enzyme amongst others. In this study, five disintegrants from different chemical classes were evaluated for P-glycoprotein (P-gp related inhibition and tight junction modulation effects. Bi-directional transport studies of the model compound, Rhodamine 123 (R123 were conducted in the absence (control group and presence (experimental groups of four concentrations of each selected disintegrant across excised pig jejunum tissue. The results showed that some of the selected disintegrants (e.g. Ac-di-sol® and Kollidon® CL-M increased R123 absorptive transport due to inhibition of P-gp related efflux, while another disintegrant (e.g. sodium alginate changed R123 transport due to inhibition of P-gp in conjunction with a transient opening of the tight junctions in a concentration dependent way. It may be concluded that the co-application of some disintegrants to the intestinal epithelium may lead to pharmacokinetic interactions with drugs that are susceptible to P-gp related efflux. However, the clinical significance of these in vitro permeation findings should be confirmed by means of in vivo studies. Keywords: Disintegrants, Excipient, Ex vivo, P-glycoprotein, Pharmacokinetic interactions, Rhodamine 123

Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport.

Science.gov (United States)

Naramoto, Satoshi

2017-12-01

Directional cell-to-cell transport of functional molecules, called polar transport, enables plants to sense and respond to developmental and environmental signals. Transporters that localize to plasma membranes (PMs) in a polar manner are key components of these systems. PIN-FORMED (PIN) auxin efflux carriers, which are the most studied polar-localized PM proteins, are implicated in the polar transport of auxin that in turn regulates plant development and tropic growth. In this review, the regulatory mechanisms underlying polar localization of PINs, control of auxin efflux activity, and PIN abundance at PMs are considered. Up to date information on polar-localized nutrient transporters that regulate directional nutrient movement from soil into the root vasculature is also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2012-07-15

A library of 117 chalcones was screened for efflux pump inhibitory (EPI) activity against NorA mediated ethidium bromide efflux. Five of the chalcones (5-7, 9, and 10) were active and two chalcones (9 and 10) were equipotent to reserpine with IC(50)-values of 9.0 and 7.7 μM, respectively. Twenty chalcones were subsequently proved to be inhibitors of the NorA efflux pump in everted membrane vesicles. Compounds 5, 7, and 9 synergistically increased the effect of ciprofloxacin on Staphylococcus aureus. Our results suggest that chalcones might be developed into drugs for overcoming multidrug resistance based on efflux transporters of microorganisms. Copyright © 2012 Elsevier Ltd. All rights reserved.

AN APPROACH TO SELF- AND CO-REGULATION OF ELECTRONIC COMMERCE IN BUILDING TRUST OVER THE INTERNET

Directory of Open Access Journals (Sweden)

R. Serbu

2016-10-01

Full Text Available The goal of this paper is to highlights some thoughts about regulation and self-regulation for e-commerce, to present cases and the importance of co- and self-regulation in e-commerce. There are a couple of question that rise in this area and we will try to answer with this paper: What are the benefits and disadvantages of self- and co-regulation, how can online industry be a cause and effect for self and co-regulation, should our web portal and search engines be a subject to any kind of regulation, what is the impact of the Internet and self and co-regulation on the e-commerce.

IMB2026791, a Xanthone, Stimulates Cholesterol Efflux by Increasing the Binding of Apolipoprotein A-I to ATP-Binding Cassette Transporter A1

Directory of Open Access Journals (Sweden)

Zijian Xie

2012-03-01

Full Text Available It is known that the ATP-binding cassette transporter A1 (ABCA1 plays a major role in cholesterol homeostasis and high density lipoprotein (HDL metabolism. Several laboratories have demonstrated that ABCA1 binding to lipid-poor apolipoprotein A-I (apoA-I will mediate the assembly of nascent HDL and cellular cholesterol efflux, which suggests a possible receptor-ligand interaction between ABCA1 and apoA-I. In this study, a cell-based-ELISA-like high-throughput screening (HTS method was developed to identify the synthetic and natural compounds that can regulate binding activity of ABCA1 to apoA-I. The cell-based-ELISA-like high-throughput screen was conducted in a 96-well format using Chinese hamster ovary (CHO cells stably transfected with ABCA1 pIRE2-EGFP (Enhanced Green Fluorecence Protein expression vector and the known ABCA1 inhibitor glibenclamide as the antagonist control. From 2,600 compounds, a xanthone compound (IMB 2026791 was selected using this HTS assay, and it was proved as an apoA-I binding agonist to ABCA1 by a flow cytometry assay and western blot analysis. The [3H] cholesterol efflux assay of IMB2026791 treated ABCA1-CHO cells and PMA induced THP-1 macrophages (human acute monocytic leukemia cell further confirmed the compound as an accelerator of cholesterol efflux in a dose-dependent manner with an EC50 of 25.23 μM.

Improving the Way State and Federal Co-Regulators Communicate about Risk

International Nuclear Information System (INIS)

Easton, E.; Janairo, L.R.

2009-01-01

This paper explores risk communications concepts that could be used by Federal and state governments to help the public understand how government officials rely on risk analysis and management to ensure that shipments of spent fuel and other radioactive wastes take place in a safe, secure manner that merits public confidence. A key focus in the communication concepts put forward in the paper is the relationship between understanding and validating the public's concerns and explaining how those concerns are being addressed by current safety requirements and practices. The authors will recommend best practices to state and Federal officials that have the responsibility for communicating with the public about radioactive waste transportation. The paper will also suggest ways to bring these state and federal co-regulators together to communicate more effectively and to speak with one voice on the issue of shipment safety. (authors)

The AcrB efflux pump: conformational cycling and peristalsis lead to multidrug resistance.

Science.gov (United States)

Seeger, Markus A; Diederichs, Kay; Eicher, Thomas; Brandstätter, Lorenz; Schiefner, André; Verrey, François; Pos, Klaas M

2008-09-01

Antimicrobial resistance of human pathogenic bacteria is an emerging problem for global public health. This resistance is often associated with the overproduction of membrane transport proteins that are capable to pump chemotherapeutics, antibiotics, detergents, dyes and organic solvents out of the cell. In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude a large variety of cytotoxic substances from the cell membrane directly into the medium bypassing the periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump in charge of the efflux of multiple antibiotics, dyes, bile salts and detergents. The trimeric outer membrane factor (OMF) TolC forms a beta-barrel pore in the outer membrane and exhibits a long periplasmic alpha-helical conduit. The periplasmic membrane fusion protein (MFP) AcrA serves as a linker between TolC and the trimeric resistance nodulation cell division (RND) pump AcrB, located in the inner membrane acting as a proton/drug antiporter. The newly elucidated asymmetric structure of trimeric AcrB reveals three different monomer conformations representing consecutive states in a transport cycle. The monomers show tunnels with occlusions at different sites leading from the lateral side through the periplasmic porter (pore) domains towards the funnel of the trimer and TolC. The structural changes create a hydrophobic pocket in one monomer, which is not present in the other two monomers. Minocyclin and doxorubicin, both AcrB substrates, specifically bind to this pocket substantiating its role as drug binding pocket. The energy transduction from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (Asp407/Asp408/Lys940) residing in the hydrophobic transmembrane domain appear to be transduced by

CO2 efflux from soils with seasonal water repellency

Science.gov (United States)

Urbanek, Emilia; Doerr, Stefan H.

2017-10-01

Soil carbon dioxide (CO2) emissions are strongly dependent on pore water distribution, which in turn can be modified by reduced wettability. Many soils around the world are affected by soil water repellency (SWR), which reduces infiltration and results in diverse moisture distribution. SWR is temporally variable and soils can change from wettable to water-repellent and vice versa throughout the year. Effects of SWR on soil carbon (C) dynamics, and specifically on CO2 efflux, have only been studied in a few laboratory experiments and hence remain poorly understood. Existing studies suggest soil respiration is reduced with increasing severity of SWR, but the responses of soil CO2 efflux to varying water distribution created by SWR are not yet known.Here we report on the first field-based study that tests whether SWR indeed reduces soil CO2 efflux, based on in situ measurements carried out over three consecutive years at a grassland and pine forest sites under the humid temperate climate of the UK.Soil CO2 efflux was indeed very low on occasions when soil exhibited consistently high SWR and low soil moisture following long dry spells. Low CO2 efflux was also observed when SWR was absent, in spring and late autumn when soil temperatures were low, but also in summer when SWR was reduced by frequent rainfall events. The highest CO2 efflux occurred not when soil was wettable, but when SWR, and thus soil moisture, was spatially patchy, a pattern observed for the majority of the measurement period. Patchiness of SWR is likely to have created zones with two different characteristics related to CO2 production and transport. Zones with wettable soil or low persistence of SWR with higher proportion of water-filled pores are expected to provide water with high nutrient concentration resulting in higher microbial activity and CO2 production. Soil zones with high SWR persistence, on the other hand, are dominated by air-filled pores with low microbial activity, but facilitating O2

Coregulation of FANCA and BRCA1 in human cells.

Science.gov (United States)

Haitjema, Anneke; Mol, Berber M; Kooi, Irsan E; Massink, Maarten Pg; Jørgensen, Jens Al; Rockx, Davy Ap; Rooimans, Martin A; de Winter, Johan P; Meijers-Heijboer, Hanne; Joenje, Hans; Dorsman, Josephine C

2014-01-01

Fanconi anemia (FA) is a genetically heterogeneous syndrome associated with increased cancer predisposition. The underlying genes govern the FA pathway which functions to protect the genome during the S-phase of the cell cycle. While upregulation of FA genes has been linked to chemotherapy resistance, little is known about their regulation in response to proliferative stimuli. The purpose of this study was to examine how FA genes are regulated, especially in relation to the cell cycle, in order to reveal their possible participation in biochemical networks. Expression of 14 FA genes was monitored in two human cell-cycle models and in two RB1/E2F pathway-associated primary cancers, retinoblastoma and basal breast cancer. In silico studies were performed to further evaluate coregulation and identify connected networks and diseases. Only FANCA was consistently induced over 2-fold; FANCF failed to exhibit any regulatory fluctuations. Two tools exploiting public data sets indicated coregulation of FANCA with BRCA1. Upregulation of FANCA and BRCA1 correlated with upregulation of E2F3. Genes coregulated with both FANCA and BRCA1 were enriched for MeSH-Term id(s) genomic instability, microcephaly, and Bloom syndrome, and enriched for the cellular component centrosome. The regulation of FA genes appears highly divergent. In RB1-linked tumors, upregulation of FA network genes was associated with reduced expression of FANCF. FANCA and BRCA1 may jointly act in a subnetwork - supporting vital function(s) at the subcellular level (centrosome) as well as at the level of embryonic development (mechanisms controlling head circumference).

Nose-to-Brain Delivery of Antiviral Drugs: A Way to Overcome Their Active Efflux?

Directory of Open Access Journals (Sweden)

Alessandro Dalpiaz

2018-03-01

Full Text Available Although several viruses can easily infect the central nervous system (CNS, antiviral drugs often show dramatic difficulties in penetrating the brain from the bloodstream since they are substrates of active efflux transporters (AETs. These transporters, located in the physiological barriers between blood and the CNS and in macrophage membranes, are able to recognize their substrates and actively efflux them into the bloodstream. The active transporters currently known to efflux antiviral drugs are P-glycoprotein (ABCB1 or P-gp or MDR1, multidrug resistance-associated proteins (ABCC1 or MRP1, ABCC4 or MRP4, ABCC5 or MRP5, and breast cancer resistance protein (ABCG2 or BCRP. Inhibitors of AETs may be considered, but their co-administration causes serious unwanted effects. Nasal administration of antiviral drugs is therefore proposed in order to overcome the aforementioned problems, but innovative devices, formulations (thermoreversible gels, polymeric micro- and nano-particles, solid lipid microparticles, nanoemulsions, absorption enhancers (chitosan, papaverine, and mucoadhesive agents (chitosan, polyvinilpyrrolidone are required in order to selectively target the antiviral drugs and, possibly, the AET inhibitors in the CNS. Moreover, several prodrugs of antiretroviral agents can inhibit or elude the AET systems, appearing as interesting substrates for innovative nasal formulations able to target anti-Human Immunodeficiency Virus (HIV agents into macrophages of the CNS, which are one of the most important HIV Sanctuaries of the body.

Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

Science.gov (United States)

Ramesh, Sunita A; Kamran, Muhammad; Sullivan, Wendy; Chirkova, Larissa; Okamoto, Mamoru; Degryse, Fien; McLaughlin, Michael; Gilliham, Matthew; Tyerman, Stephen D

2018-05-01

Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABA A receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA] i ) in both wheat ( Triticum aestivum ) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA] i because TaALMT1 facilitates GABA efflux but GABA does not complex Al 3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14 C-GABA uptake into TaALMT1 -expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1 F213C ) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA] i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status. © 2018 American Society of Plant Biologists. All rights reserved.

Direct action of aldosterone on transmembrane 22Na efflux from arterial smooth muscle. Rapid and delayed effects

International Nuclear Information System (INIS)

Moura, A.M.; Worcel, M.

1984-01-01

Acute subcutaneous (s.c.) administration of aldosterone increases ex vivo 22 Na efflux from rat tail artery smooth muscle, which appears to be due to a specific action on mineralocorticoid receptors. Indeed, this effect is blocked by the antimineralocorticoid compounds RU 28318 [17 beta-hydroxy-3-oxo,7 alpha-propyl(17 alpha)-pregn 4-ene, 21 potassium carboxylate] and spironolactone. The specific glucocorticoid receptor agonist RU 26988 does not modify 22 Na efflux. The authors show here that aldosterone has, at physiological concentrations, a mineralocorticoid specific stimulating effect on passive and sodium pump dependent transmembrane movements of sodium from the rat tail artery smooth muscle. Aldosterone exerts two types of action on sodium transport: 1) a delayed stimulation of ouabain-dependent 22 Na efflux and ouabain-independent 22 Na efflux, which are completely blocked by actinomycin D; and 2) a very rapid increase of passive 22 Na efflux, which is insensitive to actinomycin D and therefore does not seem to depend on transcription of genomic information

Internal respiration of Amazon tree stems greatly exceeds external CO2 efflux

Directory of Open Access Journals (Sweden)

J. Q. Chambers

2012-12-01

Full Text Available Respiration in tree stems is an important component of forest carbon balance. The rate of CO2 efflux from the stem has often been assumed to be a measure of stem respiration. However, recent work in temperate forests has demonstrated that stem CO2 efflux can either overestimate or underestimate respiration rate because of emission or removal of CO2 by transport in xylem water. Here, we studied gas exchange from stems of tropical forest trees using a new approach to better understand respiration in an ecosystem that plays a key role in the global carbon cycle. Our main questions were (1 is internal CO2 transport important in tropical trees, and, if so, (2 does this transport result in net release of CO2 respired in the roots at the stem, or does it cause the opposite effect of net removal of stem-respired CO2? To answer these questions, we measured the ratio of stem CO2 efflux to O2 influx. This ratio, defined here as apparent respiratory quotient (ARQ, is expected to equal 1.0 if carbohydrates are the substrate for respiration, and the net transport of CO2 in the xylem water is negligible. Using a stem chamber approach to quantifying ARQ, we found values of 0.66 ± 0.18. These low ARQ values indicate that a large portion of respired CO2 (~ 35% is not emitted locally, and is probably transported upward in the stem. ARQ values of 0.21 ± 0.10 were found for the steady-state gas concentration within the stem, sampled by in-stem equilibration probes. These lower values may result from the proximity to the xylem water stream. In contrast, we found ARQ values of 1.00 ± 0.13 for soil respiration. Our results indicate the existence of a considerable internal flux of CO2 in the stems of tropical trees. If the transported CO2 is used in the canopy as a substrate for photosynthesis, it could account for up to 10% of the C fixed by the tree, and perhaps serve as a mechanism that buffers the response of the tree to changing CO2 levels. Our results also

Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo

International Nuclear Information System (INIS)

Bošnjak, Ivana; Borra, Marco; Iamunno, Franco; Benvenuto, Giovanna; Ujević, Ivana; Bušelić, Ivana; Roje-Busatto, Romana; Mladineo, Ivona

2014-01-01

Highlights: • Effects of BPA on embryonic development of Paracentrotus lividus were determined. • Transport assay, intracellular BPA measurements and gene expression surveys were made. • Multidrug efflux transporter P-gp/ABCB1 is involved in BPA elimination. • Endocrine disruption is inferred by orphan steroid hormone receptor (shr2) upregulation. • BPA delayed mitosis, inducing aberrant karyokinesis and dysfunctional microfilaments. - Abstract: Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation

Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo

Energy Technology Data Exchange (ETDEWEB)

Bošnjak, Ivana [Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, Zagreb (Croatia); Borra, Marco [Molecular Biology Service, Stazione Zoologica Anton Dohrn, Villa Comunale 80121, Napoli (Italy); Iamunno, Franco; Benvenuto, Giovanna [Electron Microscopy Service, Stazione Zoologica Anton Dohrn, Villa Comunale 80121, Napoli (Italy); Ujević, Ivana [Laboratory of Plankton and Shellfish Toxicity, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Bušelić, Ivana [Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Roje-Busatto, Romana [Laboratory of Plankton and Shellfish Toxicity, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Mladineo, Ivona, E-mail: mladineo@izor.hr [Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000 Split (Croatia); Assemble Marine Laboratory, Stazione Zoological Anton Dohrn, Villa Comunale, Naples (Italy)

2014-11-15

Highlights: • Effects of BPA on embryonic development of Paracentrotus lividus were determined. • Transport assay, intracellular BPA measurements and gene expression surveys were made. • Multidrug efflux transporter P-gp/ABCB1 is involved in BPA elimination. • Endocrine disruption is inferred by orphan steroid hormone receptor (shr2) upregulation. • BPA delayed mitosis, inducing aberrant karyokinesis and dysfunctional microfilaments. - Abstract: Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100 nM and 4 μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation.

Colocalization of coregulated genes: a steered molecular dynamics study of human chromosome 19.

Directory of Open Access Journals (Sweden)

Marco Di Stefano

Full Text Available The connection between chromatin nuclear organization and gene activity is vividly illustrated by the observation that transcriptional coregulation of certain genes appears to be directly influenced by their spatial proximity. This fact poses the more general question of whether it is at all feasible that the numerous genes that are coregulated on a given chromosome, especially those at large genomic distances, might become proximate inside the nucleus. This problem is studied here using steered molecular dynamics simulations in order to enforce the colocalization of thousands of knowledge-based gene sequences on a model for the gene-rich human chromosome 19. Remarkably, it is found that most (≈ 88% gene pairs can be brought simultaneously into contact. This is made possible by the low degree of intra-chromosome entanglement and the large number of cliques in the gene coregulatory network. A clique is a set of genes coregulated all together as a group. The constrained conformations for the model chromosome 19 are further shown to be organized in spatial macrodomains that are similar to those inferred from recent HiC measurements. The findings indicate that gene coregulation and colocalization are largely compatible and that this relationship can be exploited to draft the overall spatial organization of the chromosome in vivo. The more general validity and implications of these findings could be investigated by applying to other eukaryotic chromosomes the general and transferable computational strategy introduced here.

Conservation of gene co-regulation in prokaryotes and eukaryotes.

NARCIS (Netherlands)

Snel, B.; Bork, P.; Huynen, M.A.

2002-01-01

We raise some issues in detecting the conservation (or absence thereof) of co-regulation using gene order; how we think the variations in the cellular network in various species can be studied; and how to determine and interpret the higher order structure in networks of functional relations.

Mercury toxicokinetics of the healthy human term placenta involve amino acid transporters and ABC transporters

International Nuclear Information System (INIS)

Straka, Elisabeth; Ellinger, Isabella; Balthasar, Christina; Scheinast, Matthias; Schatz, Jasmin; Szattler, Tamara; Bleichert, Sonja; Saleh, Leila; Knöfler, Martin; Zeisler, Harald; Hengstschläger, Markus; Rosner, Margit; Salzer, Hans; Gundacker, Claudia

2016-01-01

Highlights: • It is known that MeHg is able to pass the placenta and to affect fetal brain development. • Uptake and efflux transporters were examined in human primary trophoblast cells and BeWo cells. • Involvement in mercury transfer was assessed by measurement of cellular mercury content upon siRNA mediated gene knockdown. • Localization of transporters was determined by immunofluorescence microscopy. • LAT1 and rBAT at the apical membrane of the syncytiotrophoblast (STB) are involved in MeHg uptake. • MRP1 located at basal membrane of STB mediates mercury efflux. - Abstract: Background: The capacity of the human placenta to handle exogenous stressors is poorly understood. The heavy metal mercury is well-known to pass the placenta and to affect brain development. An active transport across the placenta has been assumed. The underlying mechanisms however are virtually unknown. Objectives: Uptake and efflux transporters (17 candidate proteins) assumed to play a key role in placental mercury transfer were examined for expression, localization and function in human primary trophoblast cells and the trophoblast-derived choriocarcinoma cell line BeWo. Methods: To prove involvement of the transporters, we used small interfering RNA (siRNA) and exposed cells to methylmercury (MeHg). Total mercury contents of cells were analyzed by Cold vapor-atomic fluorescence spectrometry (CV-AFS). Localization of the proteins in human term placenta sections was determined via immunofluorescence microscopy. Results: We found the amino acid transporter subunits L-type amino acid transporter (LAT)1 and rBAT (related to b 0,+ type amino acid transporter) as well as the efflux transporter multidrug resistance associated protein (MRP)1 to be involved in mercury kinetics of trophoblast cells (t-test P < 0.05). Conclusion: The amino acid transporters located at the apical side of the syncytiotrophoblast (STB) manage uptake of MeHg. Mercury conjugated to glutathione (GSH) is

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

Science.gov (United States)

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

2014-09-05

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. Copyright © 2014 Elsevier Inc. All rights reserved.

Extra-Renal Elimination of Uric Acid via Intestinal Efflux Transporter BCRP/ABCG2

Science.gov (United States)

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. PMID:22348008

Divide and conquer: processive transport enables multidrug transporters to tackle challenging drugs

Directory of Open Access Journals (Sweden)

Nir Fluman

2014-09-01

Full Text Available Multidrug transporters are membrane proteins that catalyze efflux of antibiotics and other toxic compounds from cells, thereby conferring drug resistance on various organisms. Unlike most solute transporters that transport a single type of compound or similar analogues, multidrug transporters are extremely promiscuous. They transport a broad spectrum of dissimilar drugs and represent a serious obstacle to antimicrobial or anticancer chemotherapy. Many challenging aspects of multidrug transporters, which are unique, have been studied in detail, including their ability to interact with chemically unrelated drugs, and how they utilize energy to drive efflux of compounds that are not only structurally but electrically different. A new and surprising dimension of the promiscuous nature of multidrug transporters has been described recently: they can move long molecules through the membrane in a processive manner.

MTA family of coregulators in nuclear receptor biology and pathology

Science.gov (United States)

Manavathi, Bramanandam; Singh, Kamini; Kumar, Rakesh

2007-01-01

Nuclear receptors (NRs) rely on coregulators (coactivators and corepressors) to modulate the transcription of target genes. By interacting with nucleosome remodeling complexes, NR coactivators potentiate transcription, whereas corepressors inhibit transcription of the target genes. Metastasis-associated proteins (MTA) represent an emerging family of novel NR coregulators. In general, MTA family members form independent nucleosome remodeling and deacetylation (NuRD) complexes and repress the transcription of different genes by recruiting histone deacetylases onto their target genes. However, MTA1 also acts as a coactivator in a promoter-context dependent manner. Recent findings that repression of estrogen receptor transactivation functions by MTA1, MTA1s, and MTA2 and regulation of MTA3 by estrogen signaling have indicated the significance of these proteins in NR signaling. Here, we highlight the action of MTA proteins on NR signaling and their roles in pathophysiological conditions. PMID:18174918

Multiple efflux pumps are involved in the transepithelial transport of colchicine: combined effect of p-glycoprotein and multidrug resistance-associated protein 2 leads to decreased intestinal absorption throughout the entire small intestine.

Science.gov (United States)

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

2009-10-01

The purpose of this study was to thoroughly characterize the efflux transporters involved in the intestinal permeability of the oral microtubule polymerization inhibitor colchicine and to evaluate the role of these transporters in limiting its oral absorption. The effects of P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on colchicine bidirectional permeability were studied across Caco-2 cell monolayers, inhibiting one versus multiple transporters simultaneously. Colchicine permeability was then investigated in different regions of the rat small intestine by in situ single-pass perfusion. Correlation with the P-gp/MRP2 expression level throughout different intestinal segments was investigated by immunoblotting. P-gp inhibitors [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), verapamil, and quinidine], and MRP2 inhibitors [3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), indomethacin, and p-aminohippuric acid (p-AH)] significantly increased apical (AP)-basolateral (BL) and decreased BL-AP Caco-2 transport in a concentration-dependent manner. No effect was obtained by the BCRP inhibitors fumitremorgin C (FTC) and pantoprazole. P-gp/MRP2 inhibitors combinations greatly reduced colchicine mucosal secretion, including complete abolishment of efflux (GF120918/MK571). Colchicine displayed low (versus metoprolol) and constant permeability along the rat small-intestine. GF120918 significantly increased colchicine permeability in the ileum with no effect in the jejunum, whereas MK571 augmented jejunal permeability without changing the ileal transport. The GF120918/MK571 combination caused an effect similar to that of MK571 alone in the jejunum and to that of GF120918 alone in the ileum. P-gp expression followed a gradient increasing from

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

Science.gov (United States)

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. Copyright © 2013 Elsevier Inc. All rights reserved.

The Co-regulation Data Harvester: Automating gene annotation starting from a transcriptome database

Science.gov (United States)

Tsypin, Lev M.; Turkewitz, Aaron P.

Identifying co-regulated genes provides a useful approach for defining pathway-specific machinery in an organism. To be efficient, this approach relies on thorough genome annotation, a process much slower than genome sequencing per se. Tetrahymena thermophila, a unicellular eukaryote, has been a useful model organism and has a fully sequenced but sparsely annotated genome. One important resource for studying this organism has been an online transcriptomic database. We have developed an automated approach to gene annotation in the context of transcriptome data in T. thermophila, called the Co-regulation Data Harvester (CDH). Beginning with a gene of interest, the CDH identifies co-regulated genes by accessing the Tetrahymena transcriptome database. It then identifies their closely related genes (orthologs) in other organisms by using reciprocal BLAST searches. Finally, it collates the annotations of those orthologs' functions, which provides the user with information to help predict the cellular role of the initial query. The CDH, which is freely available, represents a powerful new tool for analyzing cell biological pathways in Tetrahymena. Moreover, to the extent that genes and pathways are conserved between organisms, the inferences obtained via the CDH should be relevant, and can be explored, in many other systems.

Pharmacokinetic interplay of phase II metabolism and transport: a theoretical study.

Science.gov (United States)

Wu, Baojian

2012-01-01

Understanding of the interdependence of cytochrome P450 enzymes and P-glycoprotein in disposition of drugs (also termed "transport-metabolism interplay") has been significantly advanced in recent years. However, whether such "interplay" exists between phase II metabolic enzymes and efflux transporters remains largely unknown. The objective of this article is to explore the role of efflux transporters (acting on the phase II metabolites) in disposition of the parent drug in Caco-2 cells, liver, and intestine via simulations utilizing a catenary model (for Caco-2 system) and physiologically based pharmacokinetic (PBPK) models (for the liver and intestine). In all three models, "transport-metabolism interplay" (i.e., inhibition of metabolite efflux decreases the metabolism) can be observed only when futile recycling (or deconjugation) occurred. Futile recycling appeared to bridge the two processes (i.e., metabolite formation and excretion) and enable the interplay thereof. Without futile recycling, metabolite formation was independent on its downstream process excretion, thus impact of metabolite excretion on its formation was impossible. Moreover, in liver PBPK model with futile recycling, impact of biliary metabolite excretion on the exposure of parent drug [(systemic (reservoir) area under the concentration-time curve (AUC(R1))] was limited; a complete inhibition of efflux resulted in AUC(R1) increases of less than 1-fold only. In intestine PBPK model with futile recycling, even though a complete inhibition of efflux could result in large elevations (e.g., 3.5-6.0-fold) in AUC(R1), an incomplete inhibition of efflux (e.g., with a residual activity of ≥ 20% metabolic clearance) saw negligible increases (interplay between phase II enzymes and efflux transporters. Those studying such "interplay" are encouraged to adequately consider potential consequences of inhibition of efflux transporters in humans. Copyright © 2011 Wiley-Liss, Inc.

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

Energy Technology Data Exchange (ETDEWEB)

Xu, Xiaolin [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China); Li, Qian [Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai (China); Pang, Liewen; Huang, Guoqian; Huang, Jiechun; Shi, Meng; Sun, Xiaotian [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China); Wang, Yiqing [Department of Cardiothoracic Surgery, Huashan Hospital, Fudan University, Shanghai 200032 (China)

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.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Enhanced Efflux Pump Activity in Old Candida glabrata Cells.

Science.gov (United States)

Bhattacharya, Somanon; Fries, Bettina C

2018-03-01

We investigated the effect of replicative aging on antifungal resistance in Candida glabrata Our studies demonstrate significantly increased transcription of ABC transporters and efflux pump activity in old versus young C. glabrata cells of a fluconazole-sensitive and -resistant strain. In addition, higher tolerance to killing by micafungin and amphotericin B was noted and is associated with higher transcription of glucan synthase gene FKS1 and lower ergosterol content in older cells. Copyright © 2018 American Society for Microbiology.

Binding of bisphenol A, bisphenol AF, and bisphenol S on the androgen receptor: Coregulator recruitment and stimulation of potential interaction sites.

Science.gov (United States)

Perera, Lalith; Li, Yin; Coons, Laurel A; Houtman, Rene; van Beuningen, Rinie; Goodwin, Bonnie; Auerbach, Scott S; Teng, Christina T

2017-10-01

Bisphenol A (BPA), bisphenol AF (BPAF), and bisphenol S (BPS) are well known endocrine disruptors. Previous in vitro studies showed that these compounds antagonize androgen receptor (AR) transcriptional activity; however, the mechanisms of action are unclear. In the present study, we investigated interactions of coregulator peptides with BPA, BPAF, or BPS at the AR complexes using Micro Array for Real-time Coregulator Nuclear Receptor Interaction (MARCoNI) assays and assessed the binding of these compounds on AR by molecular dynamics (MD) simulations. The set of coregulator peptides that are recruited by BPA-bound AR, either positively/or negatively, are different from those recruited by the agonist R1881-bound AR. Therefore, the data indicates that BPA shows no similarities to R1881 and suggests that it may recruit other coregulators to the AR complex. BPAF-bound AR recruits about 70-80% of the same coregulator peptides as BPA-bound AR. Meanwhile, BPS-bound AR interacts with only few peptides compared to BPA or BPAF-bound AR. MD results show that multiple binding sites with varying binding affinities are available on AR for BPA, BPAF, and BPS, indicating the availability of modified binding surfaces on AR for coregulator interactions. These findings help explain some of the distinct AR-related toxicities observed with bisphenol chemicals and raise concern for the use of substitutes for BPA in commercial products. Published by Elsevier Ltd.

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity.

Science.gov (United States)

Cukier, Alexandre M O; Therond, Patrice; Didichenko, Svetlana A; Guillas, Isabelle; Chapman, M John; Wright, Samuel D; Kontush, Anatol

2017-09-01

High-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised. Reconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL+LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages. rHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux. Increasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics. Non-standard abbreviations and acronyms: AAPH, 2,2'-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low

An Electrically Tight In Vitro Blood-Brain Barrier Model Displays Net Brain-to-Blood Efflux of Substrates for the ABC Transporters, P-gp, Bcrp and Mrp-1

DEFF Research Database (Denmark)

Helms, Hans Christian; Hersom, Maria; Kuhlmann, Louise Borella

2014-01-01

Efflux transporters of the ATP-binding cassette superfamily including breast cancer resistance protein (Bcrp/Abcg2), P-glycoprotein (P-gp/Abcb1) and multidrug resistance-associated proteins (Mrp's/Abcc's) are expressed in the blood-brain barrier (BBB). The aim of this study was to investigate......, zosuquidar, reversan and MK 571 alone or in combinations. Digoxin was mainly transported via P-gp, estrone-3-sulphate via Bcrp and Mrp's and etoposide via P-gp and Mrp's. The expression of P-gp, Bcrp and Mrp-1 was confirmed using immunocytochemistry. The findings indicate that P-gp, Bcrp and at least one...... isoform of Mrp are functionally expressed in our bovine/rat co-culture model and that the model is suitable for investigations of small molecule transport....

Intestinal SR-BI does not impact cholesterol absorption or transintestinal cholesterol efflux in mice

NARCIS (Netherlands)

Bura, Kanwardeep S.; Lord, Caleb; Marshall, Stephanie; McDaniel, Allison; Thomas, Gwyn; Warrier, Manya; Zhang, Jun; Davis, Matthew A.; Sawyer, Janet K.; Shah, Ramesh; Wilson, Martha D.; Dikkers, Arne; Tietge, Uwe J. F.; Collet, Xavier; Rudel, Lawrence L.; Temel, Ryan E.; Brown, J. Mark

Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the non-biliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI

Akt inhibition promotes ABCA1-mediated cholesterol efflux to ApoA-I through suppressing mTORC1.

Directory of Open Access Journals (Sweden)

Fumin Dong

Full Text Available ATP-binding cassette transporter A1 (ABCA1 plays an essential role in mediating cholesterol efflux to apolipoprotein A-I (apoA-I, a major housekeeping mechanism for cellular cholesterol homeostasis. After initial engagement with ABCA1, apoA-I directly interacts with the plasma membrane to acquire cholesterol. This apoA-I lipidation process is also known to require cellular signaling processes, presumably to support cholesterol trafficking to the plasma membrane. We report here that one of major signaling pathways in mammalian cells, Akt, is also involved. In several cell models that express ABCA1 including macrophages, pancreatic beta cells and hepatocytes, inhibition of Akt increases cholesterol efflux to apoA-I. Importantly, Akt inhibition has little effect on cells expressing non-functional mutant of ABCA1, implicating a specific role of Akt in ABCA1 function. Furthermore, we provide evidence that mTORC1, a major downstream target of Akt, is also a negative regulator of cholesterol efflux. In cells where mTORC1 is constitutively activated due to tuberous sclerosis complex 2 deletion, cholesterol efflux to apoA-I is no longer sensitive to Akt activity. This suggests that Akt suppresses cholesterol efflux through mTORC1 activation. Indeed, inhibition of mTORC1 by rapamycin or Torin-1 promotes cholesterol efflux. On the other hand, autophagy, one of the major pathways of cholesterol trafficking, is increased upon Akt inhibition. Furthermore, Akt inhibition disrupts lipid rafts, which is known to promote cholesterol efflux to apoA-I. We therefore conclude that Akt, through its downstream targets, mTORC1 and hence autophagy, negatively regulates cholesterol efflux to apoA-I.

Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella enterica Serovar Typhimurium

Directory of Open Access Journals (Sweden)

Xuan Wang-Kan

2017-07-01

Full Text Available AcrAB-TolC is the paradigm resistance-nodulation-division (RND multidrug resistance efflux system in Gram-negative bacteria, with AcrB being the pump protein in this complex. We constructed a nonfunctional AcrB mutant by replacing D408, a highly conserved residue essential for proton translocation. Western blotting confirmed that the AcrB D408A mutant had the same native level of expression of AcrB as the parental strain. The mutant had no growth deficiencies in rich or minimal medium. However, compared with wild-type SL1344, the mutant had increased accumulation of Hoechst 33342 dye and decreased efflux of ethidium bromide and was multidrug hypersusceptible. The D408A mutant was attenuated in vivo in mouse and Galleria mellonella models and showed significantly reduced invasion into intestinal epithelial cells and macrophages in vitro. A dose-dependent inhibition of invasion was also observed when two different efflux pump inhibitors were added to the wild-type strain during infection of epithelial cells. RNA sequencing (RNA-seq revealed downregulation of bacterial factors necessary for infection, including those in the Salmonella pathogenicity islands 1, 2, and 4; quorum sensing genes; and phoPQ. Several general stress response genes were upregulated, probably due to retention of noxious molecules inside the bacterium. Unlike loss of AcrB protein, loss of efflux function did not induce overexpression of other RND efflux pumps. Our data suggest that gene deletion mutants are unsuitable for studying membrane transporters and, importantly, that inhibitors of AcrB efflux function will not induce expression of other RND pumps.

Lapatinib potentiates cytotoxicity of  YM155 in neuroblastoma via inhibition of the ABCB1 efflux transporter.

Science.gov (United States)

Radic-Sarikas, Branka; Halasz, Melinda; Huber, Kilian V M; Winter, Georg E; Tsafou, Kalliopi P; Papamarkou, Theodore; Brunak, Søren; Kolch, Walter; Superti-Furga, Giulio

2017-06-08

Adverse side effects of cancer agents are of great concern in the context of childhood tumors where they can reduce the quality of life in young patients and cause life-long adverse effects. Synergistic drug combinations can lessen potential toxic side effects through lower dosing and simultaneously help to overcome drug resistance. Neuroblastoma is the most common cancer in infancy and extremely heterogeneous in clinical presentation and features. Applying a systematic pairwise drug combination screen we observed a highly potent synergy in neuroblastoma cells between the EGFR kinase inhibitor lapatinib and the anticancer compound YM155 that is preserved across several neuroblastoma variants. Mechanistically, the synergy was based on a lapatinib induced inhibition of the multidrug-resistance efflux transporter ABCB1, which is frequently expressed in resistant neuroblastoma cells, which allowed prolonged and elevated cytotoxicity of YM155. In addition, the drug combination (i.e. lapatinib plus YM155) decreased neuroblastoma tumor size in an in vivo model.

Role of bacterial efflux pumps in biofilm formation.

Science.gov (United States)

Alav, Ilyas; Sutton, J Mark; Rahman, Khondaker Miraz

2018-02-28

Efflux pumps are widely implicated in antibiotic resistance because they can extrude the majority of clinically relevant antibiotics from within cells to the extracellular environment. However, there is increasing evidence from many studies to suggest that the pumps also play a role in biofilm formation. These studies have involved investigating the effects of efflux pump gene mutagenesis and efflux pump inhibitors on biofilm formation, and measuring the levels of efflux pump gene expression in biofilms. In particular, several key pathogenic species associated with increasing multidrug resistance, such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, have been investigated, whilst other studies have focused on Salmonella enterica serovar Typhimurium as a model organism and problematic pathogen. Studies have shown that efflux pumps, including AcrAB-TolC of E. coli, MexAB-OprM of P. aeruginosa, AdeFGH of A. baumannii and AcrD of S. enterica, play important roles in biofilm formation. The substrates for such pumps, and whether changes in their efflux activity affect biofilm formation directly or indirectly, remain to be determined. By understanding the roles that efflux pumps play in biofilm formation, novel therapeutic strategies can be developed to inhibit their function, to help disrupt biofilms and improve the treatment of infections. This review will discuss and evaluate the evidence for the roles of efflux pumps in biofilm formation and the potential approaches to overcome the increasing problem of biofilm-based infections.

Efflux Pump-mediated Drug Resistance in Burkholderia

Directory of Open Access Journals (Sweden)

Nicole L Podnecky

2015-04-01

Full Text Available Several members of the genus Burkholderia are prominent pathogens. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. Virtually all Burkholderia species are also resistant to polymyxin, prohibiting use of drugs like colistin that are available for treatment of infections caused by most other drug resistant Gram-negative bacteria. Despite clinical significance and antibiotic resistance of Burkholderia species, characterization of efflux pumps lags behind other non-enteric Gram-negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa. Although efflux pumps have been described in several Burkholderia species, they have been best studied in B. cenocepacia and B. pseudomallei. As in other non-enteric Gram-negatives, efflux pumps of the resistance nodulation cell division (RND family are the clinically most significant efflux systems in these two species. Several efflux pumps were described in B. cenocepacia, which when expressed confer resistance to clinically significant antibiotics, including aminoglycosides, chloramphenicol, fluoroquinolones, and tetracyclines. Three RND pumps have been characterized in B. pseudomallei, two of which confer either intrinsic or acquired resistance to aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some instances trimethoprim+sulfamethoxazole. Several strains of the host-adapted B. mallei, a clone of B. pseudomallei, lack AmrAB-OprA and are therefore aminoglycoside and macrolide susceptible. B. thailandensis is closely related to B. pseudomallei, but non-pathogenic to humans. Its pump repertoire and ensuing drug resistance profile parallels that of B. pseudomallei. An efflux pump in B. vietnamiensis plays a significant role in acquired aminoglycoside resistance. Summarily, efflux pumps are significant players in Burkholderia drug resistance.

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

KAUST Repository

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

2014-01-01

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

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

KAUST Repository

Zourelidou, Melina

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.

Efflux-mediated resistance to a benzothiadiazol derivative effective against Burkholderia cenocepacia

Directory of Open Access Journals (Sweden)

Viola Camilla eScoffone

2015-08-01

Full Text Available Burkholderia cenocepacia is a major concern for people suffering from Cystic Fibrosis as it contributes to serious respiratory tract infections. The lack of drugs effective against this opportunistic pathogen, along with the high level of resistance to multiple antibiotics, render the treatment of these infections particularly difficult.Here a new compound, belonging to the 2,1,3-benzothiadiazol-5-yl family (10126109, with a bactericidal effect and a MIC of 8 µg/ml against B. cenocepacia, is described. The compound is not cytotoxic and effective against B. cenocepacia clinical isolates and members of all the known Burkholderia cepacia complex species.Spontaneous mutants resistant to 10126109 were isolated and mutations in the MerR transcriptional regulator BCAM1948 were identified. In this way, a mechanism of resistance to this new molecule was described, which relies on the overexpression of the RND-9 efflux pump. Indeed, rnd-9 overexpression was confirmed by qRT-PCR, and RND-9 was identified in the membrane fractions of the mutant strains. Moreover, the increase in the MIC values of different drugs in the mutant strains, together with complementation experiments, suggested the involvement of RND-9 in the efflux of 10126109, thus indicating again the central role of efflux transporters in B. cenocepacia drug resistance.

Fluconazole Resistance Associated with Drug Efflux and Increased Transcription of a Drug Transporter Gene, PDH1, in Candida glabrata

Science.gov (United States)

Miyazaki, Haruko; Miyazaki, Yoshitsugu; Geber, Antonia; Parkinson, Tanya; Hitchcock, Christopher; Falconer, Derek J.; Ward, Douglas J.; Marsden, Katherine; Bennett, John E.

1998-01-01

Sequential Candida glabrata isolates were obtained from the mouth of a patient infected with human immunodeficiency virus type 1 who was receiving high doses of fluconazole for oropharyngeal thrush. Fluconazole-susceptible colonies were replaced by resistant colonies that exhibited both increased fluconazole efflux and increased transcripts of a gene which codes for a protein with 72.5% identity to Pdr5p, an ABC multidrug transporter in Saccharomyces cerevisiae. The deduced protein had a molecular mass of 175 kDa and was composed of two homologous halves, each with six putative transmembrane domains and highly conserved sequences of ATP-binding domains. When the earliest and most azole-susceptible isolate of C. glabrata from this patient was exposed to fluconazole, increased transcripts of the PDR5 homolog appeared, linking azole exposure to regulation of this gene. PMID:9661006

Calmodulin kinase II interacts with the dopamine transporter C terminus to regulate amphetamine-induced reverse transport

DEFF Research Database (Denmark)

Fog, Jacob U; Khoshbouei, Habibeh; Holy, Marion

2006-01-01

Efflux of dopamine through the dopamine transporter (DAT) is critical for the psychostimulatory properties of amphetamines, but the underlying mechanism is unclear. Here we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) plays a key role in this efflux. CaMKIIalpha bound to the d...

An approach to self- and co-regulation of electronic Commerce in building trust over the Internet

OpenAIRE

SERBU R.

2016-01-01

The goal of this paper is to highlights some thoughts about regulation and self-regulation for e-commerce, to present cases and the importance of co- and self-regulation in e-commerce. There are a couple of question that rise in this area and we will try to answer with this paper: What are the benefits and disadvantages of self- and co-regulation, how can online industry be a cause and effect for self and co-regulation, should our web portal and search engines be a subject to any kind of regu...

Expression of multidrug resistance associated protein 5 (MRP5) on cornea and its role in drug efflux.

Science.gov (United States)

Karla, Pradeep K; Quinn, Tim L; Herndon, Betty L; Thomas, Priscilla; Pal, Dhananjay; Mitra, Ashim

2009-04-01

The purpose of this manuscript is to investigate the presence of nucleoside/nucleotide efflux transporter in cornea and to evaluate the role in ocular drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis and immunostaining were employed to establish molecular presence of multidrug resistance associated protein 5 (MRP5) on cornea. Corneal efflux by MRP5 was studied with bis(POM)-PMEA and acyclovir using rabbit and human corneal epithelial cells along with MRP5 over expressing cells (MDCKII-MRP5). Ex vivo studies using excised rabbit cornea and in vivo ocular microdialysis in male New Zealand white rabbits were used to further evaluate the role of MRP5 in conferring ocular drug resistance. RT-PCR confirms the expression of MRP5 in both rabbit and human corneal epithelial cells along with MDCKII-MRP5 cells. Immunoprecipitation followed by Western blot analysis using a rat (M511-54) monoclonal antibody that reacts with human epitope confirms the expression of MRP5 protein in human corneal epithelial cells and MDCKII-MRP5 cells. Immunostaining performed on human cornea indicates the localization of this efflux pump on both epithelium and endothelium. Efflux studies reveal that depletion of ATP decreased PMEA efflux significantly. MRP5 inhibitors also diminished PMEA and acyclovir efflux. However, depletion of glutathione did not alter efflux. MDR1 and MRP2 did not contribute to PMEA efflux. However, MRP2 is involved in acyclovir efflux while MDR1 do not participate in this process. TLC/autoradiography suggested the conversion of bis(POM)-PMEA to PMEA in rabbit and human corneal epithelial cells. Two well known antiglaucoma drugs, bimatoprost and latanoprost were rapidly effluxed by MRP5. Ex vivo study on intact rabbit corneas demonstrated accumulation of PMEA in cornea in the presence of ATP-depleting medium. In vivo ocular pharmacokinetics also revealed a significant increase in maximum aqueous humor concentration (C(max)) and area under the

Cellular cholesterol efflux to plasma from proteinuric patients is elevated and remains unaffected by antiproteinuric treatment

NARCIS (Netherlands)

Vogt, L; Laverman, GD; van Tol, A; Groen, AK; Navis, G; Dullaart, RPF

Background. Lipid derangements are assumed to contribute to the elevated cardiovascular risk in proteinuric patients. The impact of proteinuria on reverse cholesterol transport (RCT) is unknown. The first step in RCT, cellular cholesterol efflux to plasma, may be altered in proteinuria, consequent

Multidrug Efflux Systems in Microaerobic and Anaerobic Bacteria

OpenAIRE

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 i...

Effects of a series of acidic drugs on L-lactic acid transport by the monocarboxylate transporters MCT1 and MCT4.

Science.gov (United States)

Leung, Yat Hei; Belanger, Francois; Lu, Jennifer; Turgeon, Jacques; Michaud, Veronique

2018-03-07

Drug-induced myopathy is a serious side effect that often requires removal of a medication from a drug regimen. For most drugs, the underlying mechanism of drug-induced myopathy remains unclear. Monocarboxylate transporters (MCTs) mediate L-lactic acid transport, and inhibition of MCTs may potentially lead to perturbation of L-lactic acid accumulation and muscular disorders. Therefore, we hypothesized that L-lactic acid transport may be involved in the development of drug-induced myopathy. The aim of this study was to assess the inhibitory potential of 24 acidic drugs on L-lactic acid transport using breast cancer cell lines Hs578T and MDA-MB-231, which selectively express MCT1 and MCT4, respectively. The influx transport of L-lactic acid was minimally inhibited by all drugs tested. The efflux transport was next examined: loratadine (IC50: 10 and 61 µM) and atorvastatin (IC50: 78 and 41 µM) demonstrated the greatest potency for inhibition of L-lactic acid efflux by MCT1 and MCT4, respectively. Acidic drugs including fluvastatin, cerivastatin, simvastatin acid, lovastatin acid, irbesartan and losartan exhibited weak inhibitory potency on L-lactic acid efflux. Our results suggest that some acidic drugs, such as loratadine and atorvastatin, can inhibit the efflux transport of L-lactic acid. This inhibition may cause an accumulation of intracellular L-lactic acid leading to acidification and muscular disorders. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Efflux of inorganic substances from young barley roots. II. Movement in roots and efflux of sodium in plants with divided root systems

Energy Technology Data Exchange (ETDEWEB)

Fujimoto, H; Kojima, S [Radiation Center of Osaka Prefecture, Sakai (Japan)

1977-09-01

The root system of young barley was almost halved, and the two portions were planted in culture grounds with different composition after severing the capillary connection between both root groups. With one portion in the acid medium solution of various compositions and the other in the /sup 22/Na-absorbing medium solution, the sodium absorbed from one root group moved to and flowed out from the other root group, and this state was observed. Also, the efflux of potassium from the root was observed. (1) The Na efflux was small in the culture ground with dilute hydrochloric acid, and larger in that with AlCl/sub 3/ or phosphate. (2) The K efflux was large under short-day condition. (3) Under short-day condition, in the culture ground with soluble Al, the K efflux was promoted by nitrogen-source addition, but the Na efflux was suppressed.

Efflux of rhodamine from CD56+ cells as a surrogate marker for reversal of P-glycoprotein-mediated drug efflux by PSC 833

DEFF Research Database (Denmark)

Robey, R; Bakke, S; Stein, W

1999-01-01

minutes. A dose-response relationship was shown between the concentration of PSC 833 in the blood and the inhibition of rhodamine efflux, with an apparent plateau of the inhibition of rhodamine efflux at approximately 1,000 ng/mL. The Ki, defined as the concentration required for half-maximal inhibition...... of Pgp-mediated rhodamine efflux, was determined to be in the range of 29 to 181 ng/mL; although results in two patients were distinctly different, with Ki values of 914 and 916 ng/mL. MRK-16 staining was similar among all patients. We conclude that measurement of rhodamine efflux from CD56(+) cells...

Metabolic targeting of lactate efflux by malignant glioma inhibits invasiveness and induces necrosis: an in vivo study.

Science.gov (United States)

Colen, Chaim B; Shen, Yimin; Ghoddoussi, Farhad; Yu, Pingyang; Francis, Todd B; Koch, Brandon J; Monterey, Michael D; Galloway, Matthew P; Sloan, Andrew E; Mathupala, Saroj P

2011-07-01

Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity.

Metabolic Targeting of Lactate Efflux by Malignant Glioma Inhibits Invasiveness and Induces Necrosis: An In Vivo Study

Directory of Open Access Journals (Sweden)

Chaim B Colen

2011-07-01

Full Text Available Glioblastoma multiforme (GBM are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs. We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA, a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion. Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity.

The N terminus of monoamine transporters is a lever required for the action of amphetamines

DEFF Research Database (Denmark)

Sucic, Sonja; Dallinger, Stefan; Zdrazil, Barbara

2010-01-01

The serotonin transporter (SERT) terminates neurotransmission by removing serotonin from the synaptic cleft. In addition, it is the site of action of antidepressants (which block the transporter) and of amphetamines (which induce substrate efflux). We explored the functional importance of the N......(+) entry and accumulation of SERT(T81A) in the inward facing conformation ought to favor amphetamine-induced efflux. Thus, we surmised that the N terminus must play a direct role in driving the transporter into a state that supports amphetamine-induced efflux. This hypothesis was verified by truncating...

Culture as Mediator: Co-Regulation, Self-Regulation, and Middle School Mathematics Achievement

Science.gov (United States)

Hinnant-Crawford, Brandi Nicole; Faison, Morgan Z.; Chang, Mei-Lin

2016-01-01

Purpose: Self-regulation is defined as strategic, metacognitive behavior, motivation and cognition aimed at a goal (Zimmmerman and Schunk, 2011). Co-regulation, arguably more aligned with norms in communal cultures, is the process of learners sharing "a common problem-solving plane" through which self-regulatory strategies are learned…

Inactivation of Efflux Pumps Abolishes Bacterial Biofilm Formation

DEFF Research Database (Denmark)

Kvist, Malin; Hancock, Viktoria; Klemm, Per

2008-01-01

Bacterial biofilms cause numerous problems in health care and industry; notably, biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Bacteria rely on efflux pumps...... to get rid of toxic substances. We discovered that efflux pumps are highly active in bacterial biofilms, thus making efflux pumps attractive targets for antibiofilm measures. A number of efflux pump inhibitors (EPIs) are known. EPIs were shown to reduce biofilm formation, and in combination they could...... abolish biofilm formation completely. Also, EPIs were able to block the antibiotic tolerance of biofilms. The results of this feasibility study might pave the way for new treatments for biofilm-related infections and may be exploited for prevention of biofilms in general....

Interaction of Drug or Food with Drug Transporters in Intestine and Liver.

Science.gov (United States)

Nakanishi, Takeo; Tamai, Ikumi

2015-01-01

Oral bioavailability (F) is determined as fraction of the drug dose absorbed through the gastrointestinal membranes (Fa), the unmetabolized fraction of the absorbed dose that passes through the gut into the portal blood (Fg), and the hepatic first pass availability (Fh), namely F is expressed as the product of Fa, Fg and Fh (F = Fa.Fg.Fh). Current evidence suggests that transporter proteins play a role in intestinal absorption and hepatobiliary clearance of drugs. Among those transporters, this review will focus on PEPT1 and OATP2B1 as influx transporter and p-glycoprotein (P-gp) and BCRP as efflux transporter in intestinal epithelial cells, and on OATP1B1 and 1B3 as influx transporter and MRP2 as efflux transporter in hepatocytes, respectively, because drug-drug (DDI) and -food (DFI) interactions on these transporter are considered to affect bioavailability of their substrate drugs. DDI and DFI may reduce systemic exposure to drug by blocking influx transporters in intestine, but increase it by modulating influx and efflux transporters in liver and efflux transporters in intestines. Namely, drug disposition and efficacy are likely affected by DDI and DFI, resulting in treatment failures or increase in adverse effect. Therefore, it is of significantly importance to understand precise mechanism of DDI and DFI. This review will present information about transporter-based DDI and DFI in the processes of intestinal absorption and hepatic clearance of drugs, and discuss about their clinical implication.

Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

Science.gov (United States)

Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

2016-05-01

P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP. Copyright © 2016 Elsevier B.V. All rights reserved.

Efflux of inorganic substances from young barley roots, (2)

International Nuclear Information System (INIS)

Fujimoto, Hiroshi; Kojima, Shigeru

1977-01-01

The root system of young barley was almost halved, and the two portions were planted in culture grounds with different composition after severing the capillary connection between both root groups. With one portion in the acid medium solution of various compositions and the other in the 22 Na-absorbing medium solution, the sodium absorbed from one root group moved to and flowed out from the other root group, and this state was observed. Also, the efflux of potassium from the root was observed. (1) The Na efflux was small in the culture ground with dilute hydrochloric acid, and larger in that with AlCl 3 or phosphate. (2) The K efflux was large under short-day condition. (3) Under short-day condition, in the culture ground with soluble Al, the K efflux was promoted by nitrogen-source addition, but the Na efflux was suppressed. (Mori, K.)

TLX1 and NOTCH coregulate transcription in T cell acute lymphoblastic leukemia cells

Directory of Open Access Journals (Sweden)

Lee Norman H

2010-07-01

Full Text Available Abstract Background The homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11 is inappropriately expressed in a major subgroup of T cell acute lymphoblastic leukemia (T-ALL where it is strongly associated with activating NOTCH1 mutations. Despite the recognition that these genetic lesions cooperate in leukemogenesis, there have been no mechanistic studies addressing how TLX1 and NOTCH1 functionally interact to promote the leukemic phenotype. Results Global gene expression profiling after downregulation of TLX1 and inhibition of the NOTCH pathway in ALL-SIL cells revealed that TLX1 synergistically regulated more than 60% of the NOTCH-responsive genes. Structure-function analysis demonstrated that TLX1 binding to Groucho-related TLE corepressors was necessary for maximal transcriptional regulation of the NOTCH-responsive genes tested, implicating TLX1 modulation of the NOTCH-TLE regulatory network. Comparison of the dataset to publicly available biological databases indicated that the TLX1/NOTCH-coregulated genes are frequently targeted by MYC. Gain- and loss-of-function experiments confirmed that MYC was an essential mediator of TLX1/NOTCH transcriptional output and growth promotion in ALL-SIL cells, with TLX1 contributing to the NOTCH-MYC regulatory axis by posttranscriptional enhancement of MYC protein levels. Functional classification of the TLX1/NOTCH-coregulated targets also showed enrichment for genes associated with other human cancers as well as those involved in developmental processes. In particular, we found that TLX1, NOTCH and MYC coregulate CD1B and RAG1, characteristic markers of early cortical thymocytes, and that concerted downregulation of the TLX1 and NOTCH pathways resulted in their irreversible repression. Conclusions We found that TLX1 and NOTCH synergistically regulate transcription in T-ALL, at least in part via the sharing of a TLE corepressor and by augmenting expression of MYC. We conclude that

Salvianolic acid B accelerated ABCA1-dependent cholesterol efflux by targeting PPAR-γ and LXRα

Energy Technology Data Exchange (ETDEWEB)

Yue, Jianmei [Department of Endocrinology, Shandong Province Hospital Affiliated to Shandong University, 324# Jing 5 Road, Jinan 255021 (China); Department of Endocrinology, The First Hospital of Zibo, 4# E Mei Shan Dong Road, Zibo 255200 (China); Li, Bo, E-mail: libosubmit@163.com [Department of Cardiology, Central Hospital of Zibo, 54# Gong Qing Tuan Xi Road, Zibo, Shandong Province (China); Jing, Qingping [Department of Endocrinology, The First Hospital of Zibo, 4# E Mei Shan Dong Road, Zibo 255200 (China); Guan, Qingbo, E-mail: guanqingbosubmit@163.com [Department of Endocrinology, Shandong Province Hospital Affiliated to Shandong University, 324# Jing 5 Road, Jinan 255021 (China)

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.

Evaluation of a series of 2-napthamide derivatives as inhibitors of the drug efflux pump AcrB for the reversal of antimicrobial resistance.

Science.gov (United States)

Wang, Yinhu; Mowla, Rumana; Guo, Liwei; Ogunniyi, Abiodun D; Rahman, Taufiq; De Barros Lopes, Miguel A; Ma, Shutao; Venter, Henrietta

2017-02-15

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biocide Selective TolC-Independent Efflux Pumps in Enterobacteriaceae.

Science.gov (United States)

Slipski, Carmine J; Zhanel, George G; Bay, Denice C

2018-02-01

Bacterial resistance to biocides used as antiseptics, dyes, and disinfectants is a growing concern in food preparation, agricultural, consumer manufacturing, and health care industries, particularly among Gram-negative Enterobacteriaceae, some of the most common community and healthcare-acquired bacterial pathogens. Biocide resistance is frequently associated with antimicrobial cross-resistance leading to reduced activity and efficacy of both antimicrobials and antiseptics. Multidrug resistant efflux pumps represent an important biocide resistance mechanism in Enterobacteriaceae. An assortment of structurally diverse efflux pumps frequently co-exist in these species and confer both unique and overlapping biocide and antimicrobial selectivity. TolC-dependent multicomponent systems that span both the plasma and outer membranes have been shown to confer clinically significant resistance to most antimicrobials including many biocides, however, a growing number of single component TolC-independent multidrug resistant efflux pumps are specifically associated with biocide resistance: small multidrug resistance (SMR), major facilitator superfamily (MFS), multidrug and toxin extruder (MATE), cation diffusion facilitator (CDF), and proteobacterial antimicrobial compound efflux (PACE) families. These efflux systems are a growing concern as they are rapidly spread between members of Enterobacteriaceae on conjugative plasmids and mobile genetic elements, emphasizing their importance to antimicrobial resistance. In this review, we will summarize the known biocide substrates of these efflux pumps, compare their structural relatedness, Enterobacteriaceae distribution, and significance. Knowledge gaps will be highlighted in an effort to unravel the role that these apparent "lone wolves" of the efflux-mediated resistome may offer.

Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations.

Science.gov (United States)

Sun, Jingjing; Deng, Ziqing; Yan, Aixin

2014-10-17

Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

CO2 efflux from cleared mangrove peat.

Directory of Open Access Journals (Sweden)

Catherine E Lovelock

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

Interplay of drug metabolizing enzymes with cellular transporters.

Science.gov (United States)

Böhmdorfer, Michaela; Maier-Salamon, Alexandra; Riha, Juliane; Brenner, Stefan; Höferl, Martina; Jäger, Walter

2014-11-01

Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.

Translational co-regulation of a ligand and inhibitor by a conserved RNA element

DEFF Research Database (Denmark)

Zaucker, Andreas; Nagorska, Agnieszka; Kumari, Pooja

2018-01-01

In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element wh...

Metabolic Targeting of Lactate Efflux by Malignant Glioma Inhibits Invasiveness and Induces Necrosis: An In Vivo Study1

Science.gov (United States)

Colen, Chaim B; Shen, Yimin; Ghoddoussi, Farhad; Yu, Pingyang; Francis, Todd B; Koch, Brandon J; Monterey, Michael D; Galloway, Matthew P; Sloan, Andrew E; Mathupala, Saroj P

2011-01-01

Glioblastoma multiforme (GBM) are the most malignant among brain tumors. They are frequently refractory to chemotherapy and radiotherapy with mean patient survival of approximately 6 months, despite surgical intervention. The highly glycolytic nature of glioblastomas describes their propensity to metabolize glucose to lactic acid at an elevated rate. To survive, GBMs efflux lactic acid to the tumor microenvironment through transmembrane transporters denoted monocarboxylate transporters (MCTs). We hypothesized that inhibition of MCT function would impair the glycolytic metabolism and affect both glioma invasiveness and survival. We examined the effect on invasiveness with α-cyano-4-hydroxy-cinnamic acid (ACCA, 4CIN, CHCA), a small-molecule inhibitor of lactate transport, through Matrigel-based and organotypic (brain) slice culture invasive assays using U87-MG and U251-MG glioma cells. We then conducted studies in immunodeficient rats by stereotaxic intracranial implantation of the glioma cells followed by programmed orthotopic application of ACCA through osmotic pumps. Effect on the implanted tumor was monitored by small-animal magnetic resonance imaging. Our assays indicated that glioma invasion was markedly impaired when lactate efflux was inhibited. Convection-enhanced delivery of inhibitor to the tumor bed caused tumor necrosis, with 50% of the animals surviving beyond the experimental end points (3 months after inhibitor exhaustion). Most importantly, control animals did not display any adverse neurologic effects during orthotopic administration of ACCA to brain through programmed delivery. These results indicate the clinical potential of targeting lactate efflux in glioma through delivery of small-molecule inhibitors of MCTs either to the tumor bed or to the postsurgical resection cavity. PMID:21750656

Natural and Synthetic Polymers as Inhibitors of Drug Efflux Pumps

Science.gov (United States)

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

Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

Science.gov (United States)

Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

2016-02-16

Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

Gene co-regulation is highly conserved in the evolution of eukaryotes and prokaryotes.

NARCIS (Netherlands)

Snel, B.; Noort, V. van; Huynen, M.A.

2004-01-01

Differences between species have been suggested to largely reside in the network of connections among the genes. Nevertheless, the rate at which these connections evolve has not been properly quantified. Here, we measure the extent to which co-regulation between pairs of genes is conserved over

Co-regulation of metabolic genes is better explained by flux coupling than by network distance.

Directory of Open Access Journals (Sweden)

Richard A Notebaart

2008-01-01

Full Text Available To what extent can modes of gene regulation be explained by systems-level properties of metabolic networks? Prior studies on co-regulation of metabolic genes have mainly focused on graph-theoretical features of metabolic networks and demonstrated a decreasing level of co-expression with increasing network distance, a naïve, but widely used, topological index. Others have suggested that static graph representations can poorly capture dynamic functional associations, e.g., in the form of dependence of metabolic fluxes across genes in the network. Here, we systematically tested the relative importance of metabolic flux coupling and network position on gene co-regulation, using a genome-scale metabolic model of Escherichia coli. After validating the computational method with empirical data on flux correlations, we confirm that genes coupled by their enzymatic fluxes not only show similar expression patterns, but also share transcriptional regulators and frequently reside in the same operon. In contrast, we demonstrate that network distance per se has relatively minor influence on gene co-regulation. Moreover, the type of flux coupling can explain refined properties of the regulatory network that are ignored by simple graph-theoretical indices. Our results underline the importance of studying functional states of cellular networks to define physiologically relevant associations between genes and should stimulate future developments of novel functional genomic tools.

Two Paralogous Genes Encoding Auxin Efflux Carrier Differentially Expressed in Bitter Gourd (Momordica charantia

Directory of Open Access Journals (Sweden)

Yi-Li Li

2017-11-01

Full Text Available The phytohormone auxin regulates various developmental programs in plants, including cell growth, cell division and cell differentiation. The auxin efflux carriers are essential for the auxin transport. To show an involvement of auxin transporters in the coordination of fruit development in bitter gourd, a juicy fruit, we isolated novel cDNAs (referred as McPIN encoding putative auxin efflux carriers, including McPIN1, McPIN2 (allele of McPIN1 and McPIN3, from developing fruits of bitter gourd. Both McPIN1 and McPIN3 genes possess six exons and five introns. Hydropathy analysis revealed that both polypeptides have two hydrophobic regions with five transmembrane segments and a predominantly hydrophilic core. Phylogenetic analyses revealed that McPIN1 shared the highest homology to the group of Arabidopsis, cucumber and tomato PIN1, while McPIN3 belonged to another group, including Arabidopsis and tomato PIN3 as well as PIN4. This suggests different roles for McPIN1 and McPIN3 in auxin transport involved in the fruit development of bitter gourd. Maximum mRNA levels for both genes were detected in staminate and pistillate flowers. McPIN1 is expressed in a particular period of early fruit development but McPIN3 continues to be expressed until the last stage of fruit ripening. Moreover, these two genes are auxin-inducible and qualified as early auxin-response genes. Their expression patterns suggest that these two auxin transporter genes play a pivotal role in fruit setting and development.

Effect of bisphenol A on P-glycoprotein-mediated efflux and ultrastructure of the sea urchin embryo.

Science.gov (United States)

Bošnjak, Ivana; Borra, Marco; Iamunno, Franco; Benvenuto, Giovanna; Ujević, Ivana; Bušelić, Ivana; Roje-Busatto, Romana; Mladineo, Ivona

2014-11-01

Usage of bisphenol A (BPA) in production of polycarbonate plastics has resulted in global distribution of BPA in the environment. These high concentrations cause numerous negative effects to the aquatic biota, among which the most known is the induction of endocrine disruption. The focus of this research was to determine the effects of two experimentally determined concentrations of BPA (100nM and 4μM) on cellular detoxification mechanisms during the embryonic development (2-cell, pluteus) of the rocky sea urchin (Paracentrotus lividus), primarily the potential involvement of multidrug efflux transport in the BPA intercellular efflux. The results of transport assay, measurements of the intracellular BPA and gene expression surveys, for the first time indicate the importance of P-glycoprotein (P-gp/ABCB1) in defense against BPA. Cytotoxic effects of BPA, validated by the immunohistochemistry (IHC) and the transmission electron microscopy (TEM), induced the aberrant karyokinesis, and consequently, the impairment of embryo development through the first cell division and retardation. Copyright © 2014 Elsevier B.V. All rights reserved.

LCGbase: A Comprehensive Database for Lineage-Based Co-regulated Genes.

Science.gov (United States)

Wang, Dapeng; Zhang, Yubin; Fan, Zhonghua; Liu, Guiming; Yu, Jun

2012-01-01

Animal genes of different lineages, such as vertebrates and arthropods, are well-organized and blended into dynamic chromosomal structures that represent a primary regulatory mechanism for body development and cellular differentiation. The majority of genes in a genome are actually clustered, which are evolutionarily stable to different extents and biologically meaningful when evaluated among genomes within and across lineages. Until now, many questions concerning gene organization, such as what is the minimal number of genes in a cluster and what is the driving force leading to gene co-regulation, remain to be addressed. Here, we provide a user-friendly database-LCGbase (a comprehensive database for lineage-based co-regulated genes)-hosting information on evolutionary dynamics of gene clustering and ordering within animal kingdoms in two different lineages: vertebrates and arthropods. The database is constructed on a web-based Linux-Apache-MySQL-PHP framework and effective interactive user-inquiry service. Compared to other gene annotation databases with similar purposes, our database has three comprehensible advantages. First, our database is inclusive, including all high-quality genome assemblies of vertebrates and representative arthropod species. Second, it is human-centric since we map all gene clusters from other genomes in an order of lineage-ranks (such as primates, mammals, warm-blooded, and reptiles) onto human genome and start the database from well-defined gene pairs (a minimal cluster where the two adjacent genes are oriented as co-directional, convergent, and divergent pairs) to large gene clusters. Furthermore, users can search for any adjacent genes and their detailed annotations. Third, the database provides flexible parameter definitions, such as the distance of transcription start sites between two adjacent genes, which is extendable to genes that flanking the cluster across species. We also provide useful tools for sequence alignment, gene

Mathematical Modeling and Experimental Validation of Nanoemulsion-Based Drug Transport across Cellular Barriers.

Science.gov (United States)

Kadakia, Ekta; Shah, Lipa; Amiji, Mansoor M

2017-07-01

Nanoemulsions have shown potential in delivering drug across epithelial and endothelial cell barriers, which express efflux transporters. However, their transport mechanisms are not entirely understood. Our goal was to investigate the cellular permeability of nanoemulsion-encapsulated drugs and apply mathematical modeling to elucidate transport mechanisms and sensitive nanoemulsion attributes. Transport studies were performed in Caco-2 cells, using fish oil nanoemulsions and a model substrate, rhodamine-123. Permeability data was modeled using a semi-mechanistic approach, capturing the following cellular processes: endocytotic uptake of the nanoemulsion, release of rhodamine-123 from the nanoemulsion, efflux and passive permeability of rhodamine-123 in aqueous solution. Nanoemulsions not only improved the permeability of rhodamine-123, but were also less sensitive to efflux transporters. The model captured bidirectional permeability results and identified sensitive processes, such as the release of the nanoemulsion-encapsulated drug and cellular uptake of the nanoemulsion. Mathematical description of cellular processes, improved our understanding of transport mechanisms, such as nanoemulsions don't inhibit efflux to improve drug permeability. Instead, their endocytotic uptake, results in higher intracellular drug concentrations, thereby increasing the concentration gradient and transcellular permeability across biological barriers. Modeling results indicated optimizing nanoemulsion attributes like the droplet size and intracellular drug release rate, may further improve drug permeability.

Soil efflux and total emission rates of magmatic CO2 at the horseshoe lake tree kill, mammoth mountain, California, 1995-1999

Science.gov (United States)

Gerlach, T.M.; Doukas, M.P.; McGee, K.A.; Kessler, R.

2001-01-01

We report the results of eight soil CO2 efflux surveys by the closed circulation chamber method at the Horseshoe Lake tree kill (HLTK) - the largest tree kill on Mammoth Mountain. The surveys were undertaken from 1995 to 1999 to constrain total HLTK CO2 emissions and to evaluate occasional efflux surveys as a surveillance tool for the tree kills. HLTK effluxes range from 1 to > 10,000 g m -2 day -1 (grams CO2 per square meter per day); they are not normally distributed. Station efflux rates can vary by 7-35% during the course of the 8- to 16-h surveys. Disturbance of the upper 2 cm of ground surface causes effluxes to almost double. Semivariograms of efflux spatial covariance fit exponential or spherical models; they lack nugget effects. Efflux contour maps and total CO2 emission rates based on exponential, spherical, and linear kriging models of survey data are nearly identical; similar results are also obtained with triangulation models, suggesting that the kriging models are not seriously distorted by the lack of normal efflux distributions. In addition, model estimates of total CO2 emission rates are relatively insensitive to the measurement precision of the efflux rates and to the efflux value used to separate magmatic from forest soil sources of CO2. Surveys since 1997 indicate that, contrary to earlier speculations, a termination of elevated CO2 emissions at the HLTK is unlikely anytime soon. The HLTK CO2 efflux anomaly fluctuated greatly in size and intensity throughout the 1995-1999 surveys but maintained a N-S elongation, presumably reflecting fault control of CO2 transport from depth. Total CO2 emission rates also fluctuated greatly, ranging from 46 to 136 t day-1 (metric tons CO2 per day) and averaging 93 t day-1. The large inter-survey variations are caused primarily by external (meteorological) processes operating on time scales of hours to days. The externally caused variations can mask significant changes occurring at depth; a striking example is

Modules of co-regulated metabolites in turmeric (Curcuma longa) rhizome suggest the existence of biosynthetic modules in plant specialized metabolism.

Science.gov (United States)

Xie, Zhengzhi; Ma, Xiaoqiang; Gang, David R

2009-01-01

Turmeric is an excellent example of a plant that produces large numbers of metabolites from diverse metabolic pathways or networks. It is hypothesized that these metabolic pathways or networks contain biosynthetic modules, which lead to the formation of metabolite modules-groups of metabolites whose production is co-regulated and biosynthetically linked. To test whether such co-regulated metabolite modules do exist in this plant, metabolic profiling analysis was performed on turmeric rhizome samples that were collected from 16 different growth and development treatments, which had significant impacts on the levels of 249 volatile and non-volatile metabolites that were detected. Importantly, one of the many co-regulated metabolite modules that were indeed readily detected in this analysis contained the three major curcuminoids, whereas many other structurally related diarylheptanoids belonged to separate metabolite modules, as did groups of terpenoids. The existence of these co-regulated metabolite modules supported the hypothesis that the 3-methoxyl groups on the aromatic rings of the curcuminoids are formed before the formation of the heptanoid backbone during the biosynthesis of curcumin and also suggested the involvement of multiple polyketide synthases with different substrate selectivities in the formation of the array of diarylheptanoids detected in turmeric. Similar conclusions about terpenoid biosynthesis could also be made. Thus, discovery and analysis of metabolite modules can be a powerful predictive tool in efforts to understand metabolism in plants.

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

Science.gov (United States)

Hricová, Kristýna; Kolář, Milan

2014-12-01

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

Permeability, transport, and metabolism of solutes in Caco-2 cell monolayers: a theoretical study.

Science.gov (United States)

Sun, Huadong; Pang, K Sandy

2008-01-01

We explored the properties of a catenary model that includes the basolateral (B), apical (A), and cellular compartments via simulations under linear and nonlinear conditions to understand the asymmetric observations arising from transporters, enzymes, and permeability in Caco-2 cells. The efflux ratio (EfR; P(app,B-->A)/P(app,A-->B)), obtained from the effective permeability from the A-->B and B-->A direction under linear conditions, was unity for passively permeable drugs whose transport does not involve transporters; the value was unaffected by cellular binding or metabolism, but increased with apical efflux. Metabolism was asymmetric, showing lesser metabolite accrual for the B-->A than A-->B direction because of inherent differences in the volumes for A and B. Moreover, the net flux (total - passive permeation) due to saturable apical efflux, absorption, or metabolism showed nonconformity to simple Michaelis-Menten kinetics against C(D,0), the loading donor concentration. EfR values differed with saturable apical efflux and metabolism (>1), as well as apical absorption (EfRs transport and metabolic data in Caco-2 cells.

Long-distance transport of natrium in bean plants

International Nuclear Information System (INIS)

Marschner, H.; Ossenberg-Neuhaus, H.

1976-01-01

After Na + -application to a certain zone of the root or after application to the tip or to the base of a primary leaf or along the stem of bean plants, the long-distance transport of Na + was studied. The age of the plants was 8 d when root application took place, 10 d at the time of leaf application. After application to the root zone, the long-distance transport of Na + in the direction of the shoot was strongly prevented, and the transport in the direction of the root point could be neglected. Presence of K + in the ambient nutritive solution led to a strong increase of Na + efflux from the roots. Within 48 hrs., 30-40% of the Na + applied to a primary leaf were transported towards the roots. The Na + efflux to the ambient nutritive solution came from the basal regions; it was mostly more than 10% of the amount recepted through the leaf and was only slightly increased by the presence of K + in the external solution. In the case of Na + application through the hypokotyl, this Na + -efflux from the roots was even more than 25% within 12 hrs. Both with leaf and with stem application, only 1% of the Na + taken up was transported in the direction of the shoot point. The separation of the hypocotyl tissue in the bark and in the central cylinder showed the extremely high Na + storage capacity of the central cylinder. The transfer of Na + from the central cylinder into the bark seems to be fast in the hypocotyl, while the escape of Na + from the phloem of the bark into the central cylinder is rather limited. Long-distance transport of Na + in the phloem of the bark is highly basispetal and of high efficiency. Low Na + -contents in bean leaves are thus due to several regulation mechanisms: K + -stimulated Na + -efflux in the root, restricted long-distance transport in the xylemadue to high storage capacity of the xylemparenchyma, Na + influx pumps at the phloem in stem and leaf and strictly basipetal phloem-retransport of Na + in the root and efflux into the surrounding

Multidrug efflux pumps at the crossroad between antibiotic resistance and bacterial virulence

Directory of Open Access Journals (Sweden)

Manuel Alcalde-Rico

2016-09-01

Full Text Available Multidrug efflux pumps can be involved in bacterial resistance to antibiotics at different levels. Some efflux pumps are constitutively expressed at low levels and contribute to intrinsic resistance. In addition, their overexpression may allow higher levels of resistance. This overexpression can be transient, in the presence of an effector (phenotypic resistance, or constitutive when mutants in the regulatory elements of the expression of efflux pumps are selected (acquired resistance. Efflux pumps are present in all cells, from human to bacteria and are highly conserved, which indicates that they are ancient elements in the evolution of different organisms. Consequently, it has been suggested that, besides antibiotic resistance, bacterial multidrug efflux pumps would likely contribute to other relevant process of the microbial physiology. In the current article, we discuss some specific examples of the role that efflux pumps may have in the bacterial virulence of animals' and plants' pathogens, including the processes of intercellular communication. Based in these evidences, we propose that efflux pumps are at the crossroad between resistance and virulence of bacterial pathogens. Consequently, the comprehensive study of multidrug efflux pumps requires addressing these functions, which are of relevance for the bacterial-host interactions during infection.

Multidrug Efflux Pumps at the Crossroad between Antibiotic Resistance and Bacterial Virulence.

Science.gov (United States)

Alcalde-Rico, Manuel; Hernando-Amado, Sara; Blanco, Paula; Martínez, José L

2016-01-01

Multidrug efflux pumps can be involved in bacterial resistance to antibiotics at different levels. Some efflux pumps are constitutively expressed at low levels and contribute to intrinsic resistance. In addition, their overexpression may allow higher levels of resistance. This overexpression can be transient, in the presence of an effector (phenotypic resistance), or constitutive when mutants in the regulatory elements of the expression of efflux pumps are selected (acquired resistance). Efflux pumps are present in all cells, from human to bacteria and are highly conserved, which indicates that they are ancient elements in the evolution of different organisms. Consequently, it has been suggested that, besides antibiotic resistance, bacterial multidrug efflux pumps would likely contribute to other relevant processes of the microbial physiology. In the current article, we discuss some specific examples of the role that efflux pumps may have in the bacterial virulence of animals' and plants' pathogens, including the processes of intercellular communication. Based in these evidences, we propose that efflux pumps are at the crossroad between resistance and virulence of bacterial pathogens. Consequently, the comprehensive study of multidrug efflux pumps requires addressing these functions, which are of relevance for the bacterial-host interactions during infection.

Data-based mathematical modeling of vectorial transport across double-transfected polarized cells.

Science.gov (United States)

Bartholomé, Kilian; Rius, Maria; Letschert, Katrin; Keller, Daniela; Timmer, Jens; Keppler, Dietrich

2007-09-01

Vectorial transport of endogenous small molecules, toxins, and drugs across polarized epithelial cells contributes to their half-life in the organism and to detoxification. To study vectorial transport in a quantitative manner, an in vitro model was used that includes polarized MDCKII cells stably expressing the recombinant human uptake transporter OATP1B3 in their basolateral membrane and the recombinant ATP-driven efflux pump ABCC2 in their apical membrane. These double-transfected cells enabled mathematical modeling of the vectorial transport of the anionic prototype substance bromosulfophthalein (BSP) that has frequently been used to examine hepatobiliary transport. Time-dependent analyses of (3)H-labeled BSP in the basolateral, intracellular, and apical compartments of cells cultured on filter membranes and efflux experiments in cells preloaded with BSP were performed. A mathematical model was fitted to the experimental data. Data-based modeling was optimized by including endogenous transport processes in addition to the recombinant transport proteins. The predominant contributions to the overall vectorial transport of BSP were mediated by OATP1B3 (44%) and ABCC2 (28%). Model comparison predicted a previously unrecognized endogenous basolateral efflux process as a negative contribution to total vectorial transport, amounting to 19%, which is in line with the detection of the basolateral efflux pump Abcc4 in MDCKII cells. Rate-determining steps in the vectorial transport were identified by calculating control coefficients. Data-based mathematical modeling of vectorial transport of BSP as a model substance resulted in a quantitative description of this process and its components. The same systems biology approach may be applied to other cellular systems and to different substances.

An ace up their sleeve: a transcriptomic approach exposes the AceI efflux protein of Acinetobacter baumannii and reveals the drug efflux potential hidden in many microbial pathogens

Directory of Open Access Journals (Sweden)

Karl A Hassan

2015-04-01

Full Text Available The era of antibiotics as a cure-all for bacterial infections appears to be coming to an end. The emergence of multidrug resistance in many hospital-associated pathogens has resulted in superbugs that are effectively untreatable. Multidrug efflux pumps are well known mediators of bacterial drug resistance. Genome sequencing efforts have highlighted an abundance of putative efflux pump genes in bacteria. However, it is not clear how many of these pumps play a role in antimicrobial resistance. Several studies have demonstrated that efflux pump genes that participate in drug resistance are typically under tight regulatory control and expressed only in response to their substrates. Consequently, changes in gene expression following antimicrobial shock treatments may be used to identify efflux pumps that mediate antimicrobial resistance, informing targeted functional analyses of these proteins. Using this approach we have characterised novel efflux pumps in both Gram-negative and Gram-positive bacteria. Notably, we recently applied this strategy to characterise the AceI efflux pump from Acinetobacter. AceI is a prototype for a new family of multidrug efflux proteins that is conserved across many proteobacterial lineages. Different efflux pumps in this family have been shown to confer resistance to biocides including chlorhexidine, dequalinium, benzalkonium, proflavine and/or acriflavine. The discovery of this novel family of multidrug efflux proteins raises the possibility that additional undiscovered intrinsic resistance proteins may be encoded in the core genomes of pathogenic bacteria.

Modeling soil CO2 production and transport to investigate the intra-day variability of surface efflux and soil CO2 concentration measurements in a scots pine forest (Pinus Sylvestris, L.)

OpenAIRE

Goffin, Stéphanie; Wylock, Christophe; Haut, Benoît; Maier, Martin; Longdoz, Bernard; Aubinet, Marc

2015-01-01

Aimed:The main aim of this study is to improve the mechanistic understanding of soil CO2 efflux (Fs), especially its temporal variation at short-time scales, by investigating, through modeling, which underlying process among CO2 production and its transport up to the atmosphere is responsible for observed intra-day variation of Fs and soil CO2 concentration [CO2].Methods:In this study, a measurement campaign of Fs and vertical soil [CO2] profiles was conducted in a Scots Pine Forest soil in H...

Energetics of sodium efflux from Escherichia coli

International Nuclear Information System (INIS)

Borbolla, M.G.; Rosen, B.P.

1984-01-01

When energy-starved cells of Escherichia coli were passively loaded with 22 Na+, efflux of sodium could be initiated by addition of a source of metabolic energy. Conditions were established where the source of energy was phosphate bond energy, an electrochemical proton gradient, or both. Only an electrochemical proton gradient was required for efflux from intact cells. These results are consistent with secondary exchange of Na+ for H+ catalyzed by a sodium/proton antiporter

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

Science.gov (United States)

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.

CFTR-dependent chloride efflux in cystic fibrosis mononuclear cells is increased by ivacaftor therapy.

Science.gov (United States)

Guerra, Lorenzo; D'Oria, Susanna; Favia, Maria; Castellani, Stefano; Santostasi, Teresa; Polizzi, Angela M; Mariggiò, Maria A; Gallo, Crescenzio; Casavola, Valeria; Montemurro, Pasqualina; Leonetti, Giuseppina; Manca, Antonio; Conese, Massimo

2017-07-01

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiator ivacaftor (Kalydeco®) improves clinical outcome in G551D cystic fibrosis (CF) patients. Here, we have investigated whether ivacaftor has a clinical impact on non-G551D gating mutations and function of circulating leukocytes as well. Seven patients were treated with ivacaftor and evaluated at baseline, and at 1-3 and 6 months. Besides clinical and systemic inflammatory parameters, circulating mononuclear cells (MNC) were evaluated for CFTR-dependent chloride efflux by spectrofluorimetry, neutrophils for oxidative burst by cytofluorimetry and HVCN1 mRNA expression by real time PCR. Ivacaftor determined a significant decrease in sweat chloride concentrations at all time points during treatment. Body mass index (BMI), FEV 1 , and FVC showed an increasing trend. While C-reactive protein decreased significantly at 2 months, the opposite behavior was noticed for circulating monocytes. CFTR activity in MNC was found to increase significantly at 3 and 6 months. Neutrophil oxidative burst peaked at 2 months and then decreased to baseline. HVCN1 mRNA expression was significantly higher than baseline at 1-3 months and decreased after 6 months of treatment. The chloride efflux in MNC correlated positively with both FEV 1 and FVC. On the other hand, sweat chloride correlated positively with CRP and WBC, and negatively with both respiratory function tests. A cluster analysis confirmed that sweat chloride, FEV 1 , FVC, BMI, and MNC chloride efflux behaved as a single entity over time. In patients with non-G551D mutations, ivacaftor improved both chloride transport in sweat ducts and chloride efflux in MNC, that is, functions directly imputed to CFTR. © 2017 Wiley Periodicals, Inc.

Multidrug Efflux Pumps in Staphylococcus aureus: an Update

Science.gov (United States)

Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

2013-01-01

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions. PMID:23569469

Multidrug Efflux Pumps in Staphylococcus aureus: an Update.

Science.gov (United States)

Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

2013-01-01

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

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

Directory of Open Access Journals (Sweden)

Edward B. Neufeld

2014-12-01

Full Text Available 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.

Interplay between Mutations and Efflux in Drug Resistant Clinical Isolates of Mycobacterium tuberculosis

Directory of Open Access Journals (Sweden)

Miguel Viveiros

2017-04-01

Full Text Available Numerous studies show efflux as a universal bacterial mechanism contributing to antibiotic resistance and also that the activity of the antibiotics subject to efflux can be enhanced by the combined use of efflux inhibitors. Nevertheless, the contribution of efflux to the overall drug resistance levels of clinical isolates of Mycobacterium tuberculosis is poorly understood and still is ignored by many. Here, we evaluated the contribution of drug efflux plus target-gene mutations to the drug resistance levels in clinical isolates of M. tuberculosis. A panel of 17 M. tuberculosis clinical strains were characterized for drug resistance associated mutations and antibiotic profiles in the presence and absence of efflux inhibitors. The correlation between the effect of the efflux inhibitors and the resistance levels was assessed by quantitative drug susceptibility testing. The bacterial growth/survival vs. growth inhibition was analyzed through the comparison between the time of growth in the presence and absence of an inhibitor. For the same mutation conferring antibiotic resistance, different MICs were observed and the different resistance levels found could be reduced by efflux inhibitors. Although susceptibility was not restored, the results demonstrate the existence of a broad-spectrum synergistic interaction between antibiotics and efflux inhibitors. The existence of efflux activity was confirmed by real-time fluorometry. Moreover, the efflux pump genes mmr, mmpL7, Rv1258c, p55, and efpA were shown to be overexpressed in the presence of antibiotics, demonstrating the contribution of these efflux pumps to the overall resistance phenotype of the M. tuberculosis clinical isolates studied, independently of the genotype of the strains. These results showed that the drug resistance levels of multi- and extensively-drug resistant M. tuberculosis clinical strains are a combination between drug efflux and the presence of target-gene mutations, a reality

The Role of Macrophage Lipophagy in Reverse Cholesterol Transport

Directory of Open Access Journals (Sweden)

Se-Jin Jeong

2017-03-01

Full Text Available Macrophage cholesterol efflux is a central step in reverse cholesterol transport, which helps to maintain cholesterol homeostasis and to reduce atherosclerosis. Lipophagy has recently been identified as a new step in cholesterol ester hydrolysis that regulates cholesterol efflux, since it mobilizes cholesterol from lipid droplets of macrophages via autophagy and lysosomes. In this review, we briefly discuss recent advances regarding the mechanisms of the cholesterol efflux pathway in macrophage foam cells, and present lipophagy as a therapeutic target in the treatment of atherosclerosis.

Arsenic efflux from Microcystis aeruginosa under different phosphate regimes.

Directory of Open Access Journals (Sweden)

Changzhou Yan

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

Maintenance of asymmetric cellular localization of an auxin transport protein through interaction with the actin cytoskeleton

Science.gov (United States)

Muday, G. K.

2000-01-01

In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.

In vitro, in vivo and ex vivo characterization of ibrutinib: a potent inhibitor of the efflux function of the transporter MRP1

Science.gov (United States)

Zhang, Hui; Patel, Atish; Ma, Shao-Lin; Li, Xiao Jie; Zhang, Yun-Kai; Yang, Pei-Qi; Kathawala, Rishil J; Wang, Yi-Jun; Anreddy, Nagaraju; Fu, Li-Wu; Chen, Zhe-Sheng

2014-01-01

Background and Purpose The transporter, multidrug resistance protein 1 (MRP1, ABCC1), plays a critical role in the development of multidrug resistance (MDR). Ibrutinib is an inhibitor of Bruton's tyrosine kinase. Here we investigated the reversal effect of ibrutinib on MRP1-mediated MDR. Experimental Approach Cytotoxicity was determined by MTT assay. The expression of protein was detected by Western blot. RT-PCR and Q-PCR were performed to detect the expression of MRP1 mRNA. The intracellular accumulation and efflux of substrates for MRP1 were measured by scintillation counter and flow cytometry. HEK293/MRP1 cell xenografts in nude mice were established to study the effects of ibrutinib in vivo. Key Results Ibrutinib significantly enhanced the cytotoxicity of MRP1 substrates in HEK293/MRP1 and HL60/Adr cells overexpressing MRP1. Furthermore, ibrutinib increased the accumulation of substrates in these MRP1-overexpressing cells by inhibiting the drug efflux function of MRP1. However, mRNA and protein expression of MRP1 remained unaltered after treatment with ibrutinib in MRP1-overexpressing cells. In vivo, ibrutinib enhanced the efficacy of vincristine to inhibit the growth of HEK293/MRP1 tumour xenografts in nude mice. Importantly, ibrutinib also enhances the cytotoxicity of vincristine in primary cultures of leukaemia blasts, derived from patients. Conclusions and Implications Our results indicated that ibrutinib significantly increased the efficacy of the chemotherapeutic agents which were MRP1 substrates, in MRP1-overexpressing cells, in vitro, in vivo and ex vivo. These findings will lead to further studies on the effects of a combination of ibrutinib with chemotherapeutic agents in cancer patients overexpressing MRP1. PMID:25164592

A Novel Zn2-Cys6 Transcription Factor AtrR Plays a Key Role in an Azole Resistance Mechanism of Aspergillus fumigatus by Co-regulating cyp51A and cdr1B Expressions

Science.gov (United States)

Shimizu, Kiminori; Paul, Sanjoy; Ohba, Ayumi; Gonoi, Tohru; Watanabe, Akira; Gomi, Katsuya

2017-01-01

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B). PMID:28052140

A physico-genetic module for the polarisation of auxin efflux carriers PIN-FORMED (PIN)

Science.gov (United States)

Hernández-Hernández, Valeria; Barrio, Rafael A.; Benítez, Mariana; Nakayama, Naomi; Romero-Arias, José Roberto; Villarreal, Carlos

2018-05-01

Intracellular polarisation of auxin efflux carriers is crucial for understanding how auxin gradients form in plants. The polarisation dynamics of auxin efflux carriers PIN-FORMED (PIN) depends on both biomechanical forces as well as chemical, molecular and genetic factors. Biomechanical forces have shown to affect the localisation of PIN transporters to the plasma membrane. We propose a physico-genetic module of PIN polarisation that integrates biomechanical, molecular, and cellular processes as well as their non-linear interactions. The module was implemented as a discrete Boolean model and then approximated to a continuous dynamic system, in order to explore the relative contribution of the factors mediating PIN polarisation at the scale of single cell. Our models recovered qualitative behaviours that have been experimentally observed and enable us to predict that, in the context of PIN polarisation, the effects of the mechanical forces can predominate over the activity of molecular factors such as the GTPase ROP6 and the ROP-INTERACTIVE CRIB MOTIF-CONTAINING PROTEIN RIC1.

Arsenic and Antimony Transporters in Eukaryotes

Directory of Open Access Journals (Sweden)

Ewa Maciaszczyk-Dziubinska

2012-03-01

Full Text Available Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters.

Arsenic and Antimony Transporters in Eukaryotes

Science.gov (United States)

Maciaszczyk-Dziubinska, Ewa; Wawrzycka, Donata; Wysocki, Robert

2012-01-01

Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters. PMID:22489166

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

International Nuclear Information System (INIS)

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

1988-01-01

Expulsion of preaccumulated methyl-β-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 [ 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 [ 14 C]TMG. Since net efflux was nearly arrested when the external concentration of [ 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

Rapid efflux of Ca2+ from heart mitochondria in the presence of inorganic pyrophosphate.

Science.gov (United States)

Vercesi, A; Lehninger, A L

1984-01-13

Inorganic pyrophosphate (PPi) in the intracellular concentration range causes rapid efflux of Ca2+ from rat heart mitochondria oxidizing pyruvate + malate in a low Na+ medium. Half-maximal rates of Ca2+ efflux were given by 20 microM PPi. During and after PPi-stimulated Ca2+ efflux the mitochondria retain their structural integrity and complete respiratory control. Carboxyatractyloside inhibits PPi-stimulated Ca2+ efflux, indicating PPi must enter the matrix in order to promote Ca2+ efflux. Heart mitochondria have a much higher affinity for PPi uptake and PPi-induced Ca2+ efflux than liver mitochondria.

Efflux inhibitor suppresses Streptococcus mutans virulence properties.

Science.gov (United States)

Zeng, Huihui; Liu, Jia; Ling, Junqi

2017-04-01

It is well established that efflux pumps play important roles in bacterial pathogenicity and efflux inhibitors (EIs) have been proved to be effective in suppressing bacterial virulence properties. However, little is known regarding the EI of Streptococcus mutans, a well-known caries-inducing bacterium. In this study, we identified the EI of S. mutans through ethidium bromide efflux assay and investigated how EI affected S. mutans virulence regarding the cariogenicity and stress response. Results indicated that reserpine, the identified EI, suppressed acid tolerance, mutacin production and transformation efficiency of S. mutans, and modified biofilm architecture and extracellular polysaccharide distribution. Suppressed glycosyltransferase activity was also noted after reserpine exposure. The data from quantitative real-time-PCR demonstrated that reserpine significantly altered the expression profile of quorum-sensing and virulence-associated genes. These findings suggest that reserpine represents a promising adjunct anticariogenic agent in that it suppresses virulence properties of S. mutans. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Small intestinal efflux mediated by MRP2 and BCRP shifts sulfasalazine intestinal permeability from high to low, enabling its colonic targeting.

Science.gov (United States)

Dahan, Arik; Amidon, Gordon L

2009-08-01

Sulfasalazine is characterized by low intestinal absorption, which essentially enables its colonic targeting and therapeutic action. The mechanisms behind this low absorption have not yet been elucidated. The purpose of this study was to investigate the role of efflux transporters in the intestinal absorption of sulfasalazine as a potential mechanism for its low small-intestinal absorption and colonic targeting following oral administration. The effects of P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) inhibitors on sulfasalazine bidirectional permeability were studied across Caco-2 cell monolayers, including dose-response analysis. Sulfasalazine in vivo permeability was then investigated in the rat jejunum by single-pass perfusion, in the presence vs. absence of inhibitors. Sulfasalazine exhibited 19-fold higher basolateral-to-apical (BL-AP) than apical-to-basolateral (AP-BL) Caco-2 permeability, indicative of net mucosal secretion. MRP2 inhibitors (MK-571 and indomethacin) and BCRP inhibitors [fumitremorgin C (FTC) and pantoprazole] significantly increased AP-BL and decreased BL-AP sulfasalazine Caco-2 transport in a concentration-dependent manner. No effect was observed with the P-gp inhibitors verapamil and quinidine. The IC50 values of the specific MRP2 and BCRP inhibitors MK-571 and FTC on sulfasalazine secretion were 21.5 and 2.0 microM, respectively. Simultaneous inhibition of MRP2 and BCRP completely abolished sulfasalazine Caco-2 efflux. Without inhibitors, sulfasalazine displayed low (vs. metoprolol) in vivo intestinal permeability in the rat model. MK-571 or FTC significantly increased sulfasalazine permeability, bringing it to the low-high permeability boundary. With both MK-571 and FTC present, sulfasalazine displayed high permeability. In conclusion, efflux transport mediated by MRP2 and BCRP, but not P-gp, shifts sulfasalazine permeability from high to low, thereby enabling its

Detection of efflux pump activity among clinical isolates of ...

African Journals Online (AJOL)

Purpose: To detect efflux pump activity (EPA) and screening a suspected efflux pump inhibitor (EPI) [1- (3-(trifluoromethyl)benzyl]-piperazine (TFMBP)], which could help in reducing multi-drug resistance (MDR). Methods: Eighteen isolates, viz, 14 S. aureus, 2 S. lentus, 1 S. xylosus and 1 Micrococcus species from various ...

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

2012-05-01

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

Saikosaponin-a Attenuates Oxidized LDL Uptake and Prompts Cholesterol Efflux in THP-1 Cells.

Science.gov (United States)

He, Dan; Wang, Hongyan; Xu, Ling; Wang, Xiaoqing; Peng, Kuang; Wang, Lili; Liu, Pixu; Qu, Peng

2016-06-01

Saikosaponins-a (Ssa) is a major bioactive extract of Radix Bupleuri which is a traditional Chinese medicine. The roles of inflammatory response and lipid transportation in the process of atherosclerosis have drawn increasing attention. We explored the regulation of lipid transportation and immune-inflammatory role of Ssa in early atherosclerosis. The antiatherogenic actions and possible molecular mechanisms of Ssa were texted in THP-1 cells. We examined the effect of Ssa on oxidized low-density lipoprotein (ox-LDL)-induced lipid uptake, cholesterol efflux, immune-inflammatory response. THP-1 macrophages were treated with Ssa followed by ox-LDL for 24 hours. Results from western blot showed that Ssa obviously reduced lipoprotein uptake to block foam cell formation and the expression of Density Lipoprotein Receptor-1 and CD36. Ssa also significantly boosted cholesterol efflux and the expression of ATP binding cassettetransporter A1 and peroxisome proliferator-activated receptor γ. The results also indicated that Ssa inhibited ox-LDL-induced activation of AKT and nuclear factor-κB, assembly of NLRP3 inflammasome and production of proinflammatory cytokines. It is suggested that the ability against immune inflammatory response of Ssa is due to modulation of the PI3K/AKT/NF-κB/NLRP3 pathway. In conclusion, this study provides new insight into Ssa's molecular mechanism and its therapeutic potential in the treatment of atherosclerosis.

MCT expression and lactate influx/efflux in tanycytes involved in glia-neuron metabolic interaction.

Directory of Open Access Journals (Sweden)

Christian Cortés-Campos

Full Text Available 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.

Red blood cell sodium transport in patients with cirrhosis

DEFF Research Database (Denmark)

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

2016-01-01

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) , Psodium (r = 0·57, P......sodium efflux was higher in patients with cirrhosis (+46%, Psodium buffers showed that the F-insensitive sodium efflux was twice as high in cirrhosis as in controls (P = 0...

IP3 stimulates CA++ efflux from fusogenic carrot protoplasts

International Nuclear Information System (INIS)

Rincon, M.; Boss, W.F.

1986-01-01

Polyphosphoinositide breakdown plays an important role in signal transduction in animal cells (Berridge and Irvine, 1984, Nature, 312:315). Upon stimulation, phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol both of which act as cellular second messengers. IP 3 mobilizes Ca ++ from internal stores, hence the cytosolic free Ca ++ concentration increases and those physiological activities regulated by Ca ++ are stimulated. To test if plant cells also responded to IP 3 , Ca ++ efflux studies were done with fusogenic carrot protoplasts released in EGTA. The protoplasts were preloaded with 45 Ca ++ placed in a Ca ++ -free medium, and efflux determined as 45 Ca ++ loss from the protoplasts. IP 3 (10-20μM) caused enhanced 45 Ca ++ efflux and the response was sustained for at least 15 min. In plants, as in animals, the observed IP 3 -enhanced 45 Ca ++ efflux suggested that IP 3 released Ca ++ from internal stores, and the increased free cytosolic Ca ++ activated Ca ++ pumping mechanisms which restored the Ca ++ concentration in the cytosol to the normal level

Minireview: nuclear receptor coregulators of the p160 family: insights into inflammation and metabolism.

Science.gov (United States)

Rollins, David A; Coppo, Maddalena; Rogatsky, Inez

2015-04-01

Nuclear receptor coactivators (NCOAs) are multifunctional transcriptional coregulators for a growing number of signal-activated transcription factors. The members of the p160 family (NCOA1/2/3) are increasingly recognized as essential and nonredundant players in a number of physiological processes. In particular, accumulating evidence points to the pivotal roles that these coregulators play in inflammatory and metabolic pathways, both under homeostasis and in disease. Given that chronic inflammation of metabolic tissues ("metainflammation") is a driving force for the widespread epidemic of obesity, insulin resistance, cardiovascular disease, and associated comorbidities, deciphering the role of NCOAs in "normal" vs "pathological" inflammation and in metabolic processes is indeed a subject of extreme biomedical importance. Here, we review the evolving and, at times, contradictory, literature on the pleiotropic functions of NCOA1/2/3 in inflammation and metabolism as related to nuclear receptor actions and beyond. We then briefly discuss the potential utility of NCOAs as predictive markers for disease and/or possible therapeutic targets once a better understanding of their molecular and physiological actions is achieved.

Vertical variations in wood CO2 efflux for live emergent trees in a Bornean tropical rainforest.

Science.gov (United States)

Katayama, Ayumi; Kume, Tomonori; Komatsu, Hikaru; Ohashi, Mizue; Matsumoto, Kazuho; Ichihashi, Ryuji; Kumagai, Tomo'omi; Otsuki, Kyoichi

2014-05-01

Difficult access to 40-m-tall emergent trees in tropical rainforests has resulted in a lack of data related to vertical variations in wood CO2 efflux, even though significant variations in wood CO2 efflux are an important source of errors when estimating whole-tree total wood CO2 efflux. This study aimed to clarify vertical variations in wood CO2 efflux for emergent trees and to document the impact of the variations on the whole-tree estimates of stem and branch CO2 efflux. First, we measured wood CO2 efflux and factors related to tree morphology and environment for seven live emergent trees of two dipterocarp species at four to seven heights of up to ∼ 40 m for each tree using ladders and a crane. No systematic tendencies in vertical variations were observed for all the trees. Wood CO2 efflux was not affected by stem and air temperature, stem diameter, stem height or stem growth. The ratios of wood CO2 efflux at the treetop to that at breast height were larger in emergent trees with relatively smaller diameters at breast height. Second, we compared whole-tree stem CO2 efflux estimates using vertical measurements with those based on solely breast height measurements. We found similar whole-tree stem CO2 efflux estimates regardless of the patterns of vertical variations in CO2 efflux because the surface area in the canopy, where wood CO2 efflux often differed from that at breast height, was very small compared with that at low stem heights, resulting in little effect of the vertical variations on the estimate. Additionally, whole-tree branch CO2 efflux estimates using measured wood CO2 efflux in the canopy were considerably different from those measured using only breast height measurements. Uncertainties in wood CO2 efflux in the canopy did not cause any bias in stem CO2 efflux scaling, but affected branch CO2 efflux. © The Author 2014. Published by Oxford University Press. All rights reserved.

Estradiol receptors mediate estradiol-induced inhibition of mitochondrial Ca^{2+} efflux in rat caudate nucleus and brain stem

OpenAIRE

PETROVIC, SNJEZANA; MILOSEVIC, MAJA; RISTIC-MEDIC, DANIJELA; VELICKOVIC, NATASA; DRAKULIC, DUNJA; GRKOVIC, IVANA; HORVAT, ANICA

2015-01-01

Our earlier studies found that in vitro estradiol modulates mitochondrial Ca2+ transport in discrete brain regions. The present study examined the role of estradiol receptors (ERs) in estradiol-induced inhibition of Ca^{2+} efflux from synaptosomal mitochondria isolated from rat caudate nuclei and brain stems. Radioactively labeled CaCl_2 (0.6?0.75 µCi ^45CaCl_{2}) was used for Ca^{2+} transport monitoring. The results revealed that in the presence of ER antagonist 7\\alpha,17ß-[9[(4,4,5,5,5-...

The Glutamine Transporters and Their Role in the Glutamate/GABA-Glutamine Cycle

DEFF Research Database (Denmark)

Leke, Renata; Schousboe, Arne

2016-01-01

in this neural communication, i.e., the transporters responsible for glutamine efflux from astrocytes and influx into the neurons, such as the members of the SNAT, LAT, y(+)LAT, and ASC families of transporters. The SNAT family consists of the transporter isoforms SNAT3 and SNAT5 that are related to efflux from...... and translational mechanisms, which are induced by several determinants such as amino acid deprivation, hormones, pH, and the activity of different signaling pathways. Dysfunctional glutamine transporter activity has been associated with the pathophysiological mechanisms of certain neurologic diseases......, such as Hepatic Encephalopathy and Manganism. However, there might also be other neuropathological conditions associated with an altered GGC, in which glutamine transporters are dysfunctional. Hence, it appears to be of critical importance that the physiological and pathological aspects of glutamine transporters...

Nanoparticles as Efflux Pump and Biofilm Inhibitor to Rejuvenate Bactericidal Effect of Conventional Antibiotics

Science.gov (United States)

Gupta, Divya; Singh, Ajeet; Khan, Asad U.

2017-07-01

The universal problem of bacterial resistance to antibiotic reflects a serious threat for physicians to control infections. Evolution in bacteria results in the development of various complex resistance mechanisms to neutralize the bactericidal effect of antibiotics, like drug amelioration, target modification, membrane permeability reduction, and drug extrusion through efflux pumps. Efflux pumps acquire a wide range of substrate specificity and also the tremendous efficacy for drug molecule extrusion outside bacterial cells. Hindrance in the functioning of efflux pumps may rejuvenate the bactericidal effect of conventional antibiotics. Efflux pumps also play an important role in the exclusion or inclusion of quorum-sensing biomolecules responsible for biofilm formation in bacterial cells. This transit movement of quorum-sensing biomolecules inside or outside the bacterial cells may get interrupted by impeding the functioning of efflux pumps. Metallic nanoparticles represent a potential candidate to block efflux pumps of bacterial cells. The application of nanoparticles as efflux pump inhibitors will not only help to revive the bactericidal effect of conventional antibiotics but will also assist to reduce biofilm-forming capacity of microbes. This review focuses on a novel and fascinating application of metallic nanoparticles in synergy with conventional antibiotics for efflux pump inhibition.

Depolarization-stimulated 42K+ efflux in rat aorta is calcium- and cellular volume-dependent

International Nuclear Information System (INIS)

Magliola, L.; Jones, A.W.

1987-01-01

The purpose of this study was to investigate the factors controlling membrane permeability to potassium of smooth muscle cells from rat aorta stimulated by depolarization. The increase 42 K+ efflux (change in the rate constant) induced by depolarization (application of high concentrations of potassium chloride) was inhibited significantly by the calcium antagonists diltiazem and nisoldipine. Parallel inhibitory effects on contraction were observed. Diltiazem also inhibited potassium-stimulated 36 Cl- efflux. The addition of 25-150 mM KCl to normal physiologic solution stimulated 42 K+ efflux in a concentration-dependent manner. Diltiazem suppressed potassium-stimulated 42 K+ efflux approximately 90% at 25 mM KCl and approximately 40% at 150 mM KCl. The ability of nisoldipine to inhibit 42 K+ efflux also diminished as the potassium chloride concentration was elevated. The component of efflux that was resistant to calcium antagonists probably resulted from a decrease in the electrochemical gradient for potassium. Cellular water did not change during potassium addition. Substitution of 80 and 150 mM KCl for sodium chloride produced cellular swelling and enhanced potassium-stimulated 42 K+ efflux compared with potassium chloride addition. The addition of sucrose to prevent cellular swelling reduced efflux response to potassium substitution toward that of potassium addition. A hypoosmolar physiologic solution produced an increase in the 42 K+ efflux and a contracture that were both prevented by the addition of sucrose. We concluded that the depolarization-mediated 42 K+ efflux has three components: one is calcium dependent; a second is dependent on cellular volume; and a third is resistant to inhibition by calcium antagonists

Phagocytosis-induced /sup 45/calcium efflux in polymorphonuclear leucocytes

Energy Technology Data Exchange (ETDEWEB)

Barthelemy, A; Schell-Frederick, E [Brussels Univ. (Belgium). Institut de Recherche Interdisciplinaire; Paridaens, R [Brussels Univ. (Belgium). Faculte de Medicine

1977-10-15

The role of calcium ions in regulating the structure and function of non-muscle cells is a subject of intense study. Several lines of evidence that calcium may be essential in the function of polymorphonuclear leuocytes (PMNL) and an important control element in the process of phagocytosis. Direct studies of calcium distribution and fluxes have only recently been undertaken. To our knowledge, no report of calcium movements during normal phagocytosis has been published. In the context of an overall study of calcium dynamics in the PMNL, we report here initial studies on /sup 45/Ca efflux in prelabelled guinea pig PMNL. The results demonstrate the energy-dependence of resting calcium efflux and an increased efflux upon addition of phagocytic particles which is not dependent on particle internalization.

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

NARCIS (Netherlands)

Tena, J.J.; Alonso, M.E.; de la Calle-Mustienes, E.; Splinter, E.; de Laat, W.; Manzanares, M.; Gomez-Skarmeta, J.L.

2011-01-01

Developmental gene clusters are paradigms for the study of gene regulation; however, the mechanisms that mediate phenomena such as coregulation and enhancer sharing remain largely elusive. Here we address this issue by analysing the vertebrate Irx clusters. We first present a deep enhancer screen of

The role of efflux pumps in Bacteroides fragilis resistance to antibiotics.

Science.gov (United States)

Ghotaslou, Reza; Yekani, Mina; Memar, Mohammad Yousef

2018-05-01

The resistance of Bacteroides fragilis to the most antimicrobial agents has been reported in the world. Identification of the microbial resistance mechanisms can play an important role in controlling these resistances. Currently, B. fragilis is resistant to most antibiotics. The multi-drug efflux pumps have been shown to underlie the antimicrobial resistance in B. fragilis strains. Two types of these efflux pumps including RND and MATE can be regarded as main structures responsible for antibiotic resistance. Therefore, the strategy for suppressing of this efflux system may be useful in the treatment and control of the multidrug-resistant B. fragilis. The purpose of this study is to review the B. fragilis efflux pumps and their functions in the resistance to antibiotics. Copyright © 2018 Elsevier GmbH. All rights reserved.

Yeast Interacting Proteins Database: YNR051C, YER151C [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available that coregulates anterograde and retrograde transport between the endoplasmic reticulum and Golgi compartme...C UBP3 Ubiquitin-specific protease that interacts with Bre5p to co-regulate anterograde and retrograde...t coregulates anterograde and retrograde transport between the endoplasmic reticulum and Golgi compartments;...3 Prey description Ubiquitin-specific protease that interacts with Bre5p to co-regulate anterograde and retrograde

Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides

NARCIS (Netherlands)

Sesink, A.L.A.; Arts, I.C.W.; Boer, de V.C.J.; Breedveld, P.; Schellens, J.H.M.; Hollman, P.C.H.; Russel, F.G.M.

2005-01-01

The intestinal absorption of the flavonoid quercetin in rats is limited by the secretion of glucuronidated metabolites back into the gut lumen. The objective of this study was to determine the role of the intestinal efflux transporters breast cancer resistance protein (Bcrp1)/Abcg2 and multidrug

Breast cancer resistance protein (Bcrp1/Abcg2) limits net intestinal uptake of quercetin in rats by facilitating apical efflux of glucuronides.

NARCIS (Netherlands)

Sesink, A.L.; Arts, I.C.; Boer, V.C. de; Breedveld, P.; Schellens, J.H.; Hollman, P.C.H.; Russel, F.G.M.

2005-01-01

The intestinal absorption of the flavonoid quercetin in rats is limited by the secretion of glucuronidated metabolites back into the gut lumen. The objective of this study was to determine the role of the intestinal efflux transporters breast cancer resistance protein (Bcrp1)/Abcg2 and multidrug

Inhibitors of plant hormone transport

Czech Academy of Sciences Publication Activity Database

Klíma, Petr; Laňková, Martina; Zažímalová, Eva

2016-01-01

Roč. 253, č. 6 (2016), s. 1391-1404 ISSN 0033-183X R&D Projects: GA MŠk(CZ) LD15088 Institutional support: RVO:61389030 Keywords : polar auxin transport * acid-binding protein * gnom arf-gef * equilibrative nucleoside transporter * efflux carrier polarity * plasma-membrane-protein * cultured tobacco cells * arabidopsis-thaliana * gravitropic response * brefeldin-a * Plant hormones * Transport * Inhibitors * Auxin * Cytokinins * Strigolactones * Abscisic acid * Cell biology Subject RIV: ED - Physiology Impact factor: 2.870, year: 2016

Reduced intracellular drug accumulation in drug-resistant leukemia cells is not solely due to MDR-mediated efflux but also to decreased uptake

Directory of Open Access Journals (Sweden)

Angela Oliveira Pisco

2014-10-01

Full Text Available Expression of ABC family transporter proteins that promote drug efflux from cancer cells is a widely observed mechanism of multi-drug resistance of cancer cells. Cell adaptation in long-term culture of HL60 leukemic cells in the presence of chemotherapy leads to induction and maintenance of the ABC transporters expression, preventing further accumulation of drugs. However, we found that decreased accumulation of drugs and fluorescent dyes was also contributed by a reduced uptake by the resistant cells. Confocal time-lapse microscopy and flow cytometry revealed that fluid-phase endocytosis was diminished in drug-resistant cells compared to drug-sensitive cells. Drug uptake was increased by insulin co-treatment when cells were grown in methylcellulose and monitored under the microscope, but not when cultured in suspension. We propose that multi-drug resistance is not solely achieved by enhanced efflux capacity but also by supressed intake of the drug offering an alternative target to overcome drug resistance or potentiate chemotherapy.

Piperine analogs as potent Staphylococcus aureus NorA efflux pump inhibitors

DEFF Research Database (Denmark)

Sangwan, Payare L; Koul, Jawahir L; Koul, Surrinder

2008-01-01

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like...... reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors....

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

Science.gov (United States)

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

2015-01-01

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

The Ins and Outs of RND Efflux Pumps in Escherichia coli

Directory of Open Access Journals (Sweden)

João eAnes

2015-06-01

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

The ins and outs of RND efflux pumps in Escherichia coli

Science.gov (United States)

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

2015-01-01

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

POSSIBLE RELATED FUNCTIONS OF THE NON-HOMOLOGOUS CO-REGULATED GENE PAIR PDCD10 AND SERPINI1

Directory of Open Access Journals (Sweden)

Concetta Scimone

2017-04-01

Full Text Available Gene expression in mammalians is a very finely controlled mechanism, and bidirectional promoters can be considered one of the most compelling examples of the accuracy of genic expression coordination. As recently reported, a bidirectional promoter regulates the expression of the PDCD10(whose mutations cause familial Cerebral Cavernous Malformations (CCMs and SERPINI1 gene pair, even though they are non-homologous genes. The aim of this study was to identify any potential common roles of these two coregulated genes. An in-silico approach was used to identify functional correlations, using the BioGraph, IPA® and Cytoscape tools and the KEGG pathway database. The results obtained show that PDCD10 and SERPINI1 may co-regulate some cellular processes, particularly those related to focal adhesion maintenance. All common pathways identified for PDCD10 and SERPINI1 are closely associated with the pathogenic characteristics of CCMs; we thus hypothesize that genes involved in these networks may contribute to the development of CCMs.

Alcohol and the calcium-dependent potassium transport of human erythrocytes

International Nuclear Information System (INIS)

Harris, R.A.; Caldwell, K.K.

1985-01-01

In vitro exposure of human red blood cells to ethanol (100 and 400 mM) was found to increase the initial rate of calcium-dependent potassium efflux through the red cell membrane. This effect of ethanol was apparently not due to an elevation of the intracellular free calcium but rather to a direct action of the drug on the transport process as, (1) intracellular calcium concentrations were tightly buffered with EGTA, (2) ethanol did not alter the efflux of 45 Ca from the cells, and (3) dantrolene, which has been proposed to counteract the effect of ethanol on intracellular calcium levels in the erythrocyte, did not inhibit the stimulatory action of ethanol. The efflux of potassium from erythrocytes obtained from chronic alcoholics was not different from that of erythrocytes from non-alcoholic individuals. The relationship of these findings to neuronal potassium transport is discussed

Soil carbon dioxide (CO 2 ) efflux of two shrubs in response to plant ...

African Journals Online (AJOL)

Although plant density should affect soil carbon dioxide (CO2) efflux and carbon cycling in semi-arid regions, the effects of plant density on soil CO2 efflux are not well known. This study was performed to investigate the responses of soil CO2 efflux of two dominant shrubs (Caragana korshinkii and Salix psammophila) to ...

Greenhouse gas efflux from an impacted Malaysian tropical peat swamp (Invited)

Science.gov (United States)

Waldron, S.; Vihermaa, L. E.; Evers, S.; Garnett, M.; Newton, J.; Padfield, R.

2013-12-01

Tropical peatlands constitute ~11% of global peatland area and ~12% of the global peat C pool. Malaysia alone contains 10% of tropical peats. Due to rising global demands for food and biofuels, SE-Asia peat swamp forest ecosystems are threatened by increasing amounts of drainage, fire and conversion to plantation. These processes can change the GHG emissions and thus net ecosystem C balance. However, in comparison to temperate and boreal peatlands, there is a lack of data on terrestrial-aquatic-atmospheric carbon transfer from tropical peatlands, both those that are little disturbed and those facing anthropogenic pressures. Lateral transport of soil-respired carbon, and fluvial respiration or UV-oxidation of terrestrial DOC primes atmospheric carbon dioxide efflux. We now know that DOC lost from disturbed tropical peat swamp forests can be centuries to millennia old and originates deep within the peat column - this carbon may fuel efflux of old carbon dioxide and so anthropogenic land-use change renders the older, slower carbon cycles shorter and faster. Currently we have no knowledge of how significant ';older-slower' terrestrial-aquatic-atmospheric cycles are in disturbed tropical peatlands. Further, in some areas for commercial reasons, or by conservation bodies trying to minimise peat habitat loss, logged peats have been left to regenerate. Consequently, unpicking the legacy of multiple land uses on magnitude, age and source of GHG emissions is challenging but required to support land management decisions and projections of response to a changing climate. Here, we present the results of our first field campaign in July 2013 to the Raja Musa and Sungai Karang Peat Swamp Forest Reserves in North Selangor, Malaysia. This is one of Malaysia's largest oceanic peat swamps, and has been selectively logged and drained for 80 years, but is now subject to a 30 year logging ban to aid forest regeneration and build up wood stocks. From sites subject to different land use

Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model.

Science.gov (United States)

Lowrence, Rene Christena; Raman, Thiagarajan; Makala, Himesh V; Ulaganathan, Venkatasubramanian; Subramaniapillai, Selva Ganesan; Kuppuswamy, Ashok Ayyappa; Mani, Anisha; Chittoor Neelakantan, Sundaresan; Nagarajan, Saisubramanian

2016-11-01

Multi drug resistant (MDR) pathogens pose a serious threat to public health since they can easily render most potent drugs ineffective. Efflux pump inhibitors (EPI) can be used to counter the MDR phenotypes arising due to increased efflux. In the present study, a series of dithiazole thione derivatives were synthesized and checked for its antibacterial and efflux pump inhibitory (EPI) activity. Among 10 dithiazole thione derivatives, real-time efflux studies revealed that seven compounds were potent EPIs relative to CCCP. Zebrafish toxicity studies identified four non-toxic putative EPIs. Both DTT3 and DTT9 perturbed membrane potential and DTT6 was haemolytic. Among DTT6 and DTT10, the latter was less toxic as evidenced by histopathology studies. Since DTT10 was non-haemolytic, did not affect the membrane potential, and was least toxic, it was chosen further for in vivo study, wherein DTT10 potentiated effect of ciprofloxacin against clinical strain of MRSA and reduced bacterial burden in muscle and skin tissue of infected zebrafish by ~ 1.7 and 2.5 log fold respectively. Gene expression profiling of major efflux transport proteins by qPCR revealed that clinical isolate of MRSA, in the absence of antibiotic, upregulated NorA, NorB and MepA pump, whereas it downregulates NorC and MgrA relative to wild-type strain of Staphylococcus aureus. In vitro studies with NorA mutant strains and substrate profiling revealed that at higher concentrations DTT10 is likely to function as a competitive inhibitor of NorA efflux protein in S. aureus, whereas at lower concentrations it might inhibit ciprofloxacin efflux through NorB and MepA as implied by docking studies. A novel non-toxic, non-haemolytic dithiazole thione derivative (DTT10) was identified as a potent competitive inhibitor of NorA efflux pump in S. aureus using in silico, in vitro and in vivo studies. This study also underscores the importance of using zebrafish infection model to screen and evaluate putative EPI for

Structure and Function of Neisseria gonorrhoeae MtrF Illuminates a Class of Antimetabolite Efflux Pumps

Directory of Open Access Journals (Sweden)

Chih-Chia Su

2015-04-01

Full Text Available Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. N. gonorrhoeae MtrF is an integral membrane protein that belongs to the AbgT family of transporters for which no structural information is available. Here, we describe the crystal structure of MtrF, revealing a dimeric molecule with architecture distinct from all other families of transporters. MtrF is a bowl-shaped dimer with a solvent-filled basin extending from the cytoplasm to halfway across the membrane bilayer. Each subunit of the transporter contains nine transmembrane helices and two hairpins, posing a plausible pathway for substrate transport. A combination of the crystal structure and biochemical functional assays suggests that MtrF is an antibiotic efflux pump mediating bacterial resistance to sulfonamide antimetabolite drugs.

Impact of anatase and rutile titanium dioxide nanoparticles on uptake carriers and efflux pumps in Caco-2 gut epithelial cells

Science.gov (United States)

Dorier, M.; Brun, E.; Veronesi, G.; Barreau, F.; Pernet-Gallay, K.; Desvergne, C.; Rabilloud, T.; Carapito, C.; Herlin-Boime, N.; Carrière, M.

2015-04-01

TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken together, these data suggest that TiO2-NPs may increase the functionality of gut epithelial cells, particularly their property to form a protective barrier against exogenous toxicants and to absorb nutrients.TiO2 microparticles are widely used in food products, where they are added as a white food colouring agent. This food additive contains a significant amount of nanoscale particles; still the impact of TiO2 nanoparticles (TiO2-NPs) on gut cells is poorly documented. Our study aimed at evaluating the impact of rutile and anatase TiO2-NPs on the main functions of enterocytes, i.e. nutrient absorption driven by solute-liquid carriers (SLC transporters) and protection against other xenobiotics driven by efflux pumps from the ATP-binding cassette (ABC) family. We show that acute exposure of Caco-2 cells to both anatase (12 nm) and rutile (20 nm) TiO2-NPs induce early upregulation of a battery of efflux pumps and nutrient transporters. In addition they cause overproduction of reactive oxygen species and misbalance redox repair systems, without inducing cell mortality or DNA damage. Taken

Homology modeling, molecular dynamics, and virtual screening of NorA efflux pump inhibitors of Staphylococcus aureus.

Science.gov (United States)

Bhaskar, Baki Vijaya; Babu, Tirumalasetty Muni Chandra; Reddy, Netala Vasudeva; Rajendra, Wudayagiri

2016-01-01

Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds.

Nuclear receptor coregulator SNP discovery and impact on breast cancer risk

International Nuclear Information System (INIS)

Hartmaier, Ryan J; Ditsch, Nina; Bugert, Peter; Weber, Bernhard HF; Niederacher, Dieter; Arnold, Norbert; Varon-Mateeva, Raymonda; Wappenschmidt, Barbara; Schmutzler, Rita K; Meindl, Alfons; Bartram, Claus R; Tchatchou, Sandrine; Burwinkel, Barbara; Oesterreich, Steffi; Richter, Alexandra S; Wang, Jay; McGuire, Sean E; Skaar, Todd C; Rae, Jimmy M; Hemminki, Kari; Sutter, Christian

2009-01-01

Coregulator proteins are 'master regulators', directing transcriptional and posttranscriptional regulation of many target genes, and are critical in many normal physiological processes, but also in hormone driven diseases, such as breast cancer. Little is known on how genetic changes in these genes impact disease development and progression. Thus, we set out to identify novel single nucleotide polymorphisms (SNPs) within SRC-1 (NCoA1), SRC-3 (NCoA3, AIB1), NCoR (NCoR1), and SMRT (NCoR2), and test the most promising SNPs for associations with breast cancer risk. The identification of novel SNPs was accomplished by sequencing the coding regions of these genes in 96 apparently normal individuals (48 Caucasian Americans, 48 African Americans). To assess their association with breast cancer risk, five SNPs were genotyped in 1218 familial BRCA1/2-mutation negative breast cancer cases and 1509 controls (rs1804645, rs6094752, rs2230782, rs2076546, rs2229840). Through our resequencing effort, we identified 74 novel SNPs (30 in NCoR, 32 in SMRT, 10 in SRC-3, and 2 in SRC-1). Of these, 8 were found with minor allele frequency (MAF) >5% illustrating the large amount of genetic diversity yet to be discovered. The previously shown protective effect of rs2230782 in SRC-3 was strengthened (OR = 0.45 [0.21-0.98], p = 0.04). No significant associations were found with the other SNPs genotyped. This data illustrates the importance of coregulators, especially SRC-3, in breast cancer development and suggests that more focused studies, including functional analyses, should be conducted

Food safety regulatory systems in Europe and China：A study of how co-regulation can improve regulatory effectiveness

Institute of Scientific and Technical Information of China (English)

Kevin Chen; WANG Xin-xin; SONG Hai-ying

2015-01-01

Food safety has received a great deal of attention in both developed and developing countries in recent years. In China, the numerous food scandals and scares that have struck over the past decade have spurred signiifcant food safety regulatory reform, which has been increasingly oriented towards the public-private partnership model adopted by the Europe Union’s (EU) food safety regulatory system. This paper analyzes the development of both the EU’s and China’s food safety regu-latory systems, identiifes the current chalenges for China and additionaly considers the role of public-private partnership. The success of co-regulation in the food regulatory system would bring signiifcant beneifts and opportunities for China. Finaly, this paper recommends additional measures like training and grants to improve the private’s sector effectiveness in co-regulating China’s food safety issues.

Hsp27 promotes ABCA1 expression and cholesterol efflux through the PI3K/PKCζ/Sp1 pathway in THP-1 macrophages.

Science.gov (United States)

Kuang, Hai-Jun; Zhao, Guo-Jun; Chen, Wu-Jun; Zhang, Min; Zeng, Gao-Feng; Zheng, Xi-Long; Tang, Chao-Ke

2017-09-05

Heat shock protein 27 (Hsp27) is a putative biomarker and therapeutic target in atherosclerosis. This study was to explore the potential mechanisms underlying Hsp27 effects on ATP-binding cassette transporter A1 (ABCA1) expression and cellular cholesterol efflux. THP-1 macrophage-derived foam cells were infected with adenovirus to express wild-type Hsp27, hyper-phosphorylated Hsp27 mimic (3D Hsp27), antisense Hsp27 or hypo-phosphorylated Hsp27 mimic (3A Hsp27). Wild-type and 3D Hsp27 were found to up-regulate ABCA1 mRNA and protein expression and increase cholesterol efflux from cells. Expression of antisense or 3A Hsp27 suppressed the expression of ABCA1 and cholesterol efflux. Furthermore, over-expression of wild-type and 3D Hsp27 significantly increased the levels of phosphorylated specificity protein 1 (Sp1), protein kinase C ζ (PKCζ) and phosphatidylinositol 3-kinase (PI3K). In addition, the up-regulation of ABCA1 expression and cholesterol efflux induced by 3D Hsp27 was suppressed by inhibition of Sp1, PKCζ and PI3K with specific kinase inhibitors. Taken together, our results revealed that Hsp27 may up-regulate the expression of ABCA1 and promotes cholesterol efflux through activation of the PI3K/PKCζ/Sp1 signal pathway in THP-1 macrophage-derived foam cells. Our findings may partly explain the mechanisms underlying the anti-atherogenic effect of Hsp27. Copyright © 2017 Elsevier B.V. All rights reserved.

Interaction mediated by the putative tip regions of MdsA and MdsC in the formation of a Salmonella-specific tripartite efflux pump.

Directory of Open Access Journals (Sweden)

Saemee Song

Full Text Available To survive in the presence of a wide range of toxic compounds, gram-negative bacteria expel such compounds via tripartite efflux pumps that span both the inner and outer membranes. The Salmonella-specific MdsAB pump consists of MdsB, a resistance-nodulation-division (RND-type inner membrane transporter (IMT that requires the membrane fusion protein (MFP MdsA, and an outer membrane protein (OMP; MdsC or TolC to form a tripartite efflux complex. In this study, we investigated the role of the putative tip regions of MdsA and its OMPs, MdsC and TolC, in the formation of a functional MdsAB-mediated efflux pump. Comparative analysis indicated that although sequence homologies of MdsA and MdsC with other MFPs and OMPs, respectively, are extremely low, key residues in the putative tip regions of these proteins are well conserved. Mutagenesis studies on these conserved sites demonstrated their importance for the physical and functional interactions required to form an MdsAB-mediated pump. Our studies suggest that, despite differences in the primary amino acid sequences and functions of various OMPs and MFPs, interactions mediated by the conserved tip regions of OMP and MFP are required for the formation of functional tripartite efflux pumps in gram-negative bacteria.

High efflux pump activity and gene expression at baseline linked to ...

African Journals Online (AJOL)

Phenotypic TB drug resistance, also known as drug tolerance, has been previously attributed to slowed bacterial growth in vivo. The increased activity and expression of efflux systems can lower the intracellular concentration of many antibiotics thus reducing their efficacy. We hypothesized that efflux pump activation and ...

RND multidrug efflux pumps: what are they good for?

Science.gov (United States)

Alvarez-Ortega, Carolina; Olivares, Jorge; Martínez, José L.

2013-01-01

Multidrug efflux pumps are chromosomally encoded genetic elements capable of mediating resistance to toxic compounds in several life forms. In bacteria, these elements are involved in intrinsic and acquired resistance to antibiotics. Unlike other well-known horizontally acquired antibiotic resistance determinants, genes encoding for multidrug efflux pumps belong to the core of bacterial genomes and thus have evolved over millions of years. The selective pressure stemming from the use of antibiotics to treat bacterial infections is relatively recent in evolutionary terms. Therefore, it is unlikely that these elements have evolved in response to antibiotics. In the last years, several studies have identified numerous functions for efflux pumps that go beyond antibiotic extrusion. In this review we present some examples of these functions that range from bacterial interactions with plant or animal hosts, to the detoxification of metabolic intermediates or the maintenance of cellular homeostasis. PMID:23386844

Rapid sediment accumulation results in high methane effluxes from coastal sediments

NARCIS (Netherlands)

Egger, M.J.|info:eu-repo/dai/nl/372629199; Lenstra, W.K.|info:eu-repo/dai/nl/411295977; Jong, Dirk; Meysman, Filip; Sapart, C.J.|info:eu-repo/dai/nl/31400596X; van der Veen, C.; Röckmann, Thomas|info:eu-repo/dai/nl/304838233; Gonzalez, Santiago; Slomp, C.P.|info:eu-repo/dai/nl/159424003

2016-01-01

Globally, the methane (CH4) efflux from the ocean to the atmosphere is small, despite high rates of CH4 production in continental shelf and slope environments. This low efflux results from the biological removal of CH4 through anaerobic oxidation with sulfate in marine sediments. In some settings,

CO₂ efflux from shrimp ponds in Indonesia.

Science.gov (United States)

Sidik, Frida; Lovelock, Catherine E

2013-01-01

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

CO₂ efflux from shrimp ponds in Indonesia.

Directory of Open Access Journals (Sweden)

Frida Sidik

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

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

Science.gov (United States)

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

2016-01-01

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

Efflux Pump‑Mediated Resistance in Chemotherapy

African Journals Online (AJOL)

to elucidate their structure and mechanisms of action so as to integrate the efflux pump mechanisms in the ... resistance. c. Alteration of the penicillin binding protein (PBP) in ..... Perloff MD, von Moltke LL, Fahey JM, Daily JP, Greenblat. DJ.

Crystal structure of the Neisseria gonorrhoeae MtrD inner membrane multidrug efflux pump.

Directory of Open Access Journals (Sweden)

Jani Reddy Bolla

Full Text Available Neisseria gonorrhoeae is an obligate human pathogen and the causative agent of the sexually-transmitted disease gonorrhea. The control of this disease has been compromised by the increasing proportion of infections due to antibiotic-resistant strains, which are growing at an alarming rate. The MtrCDE tripartite multidrug efflux pump, belonging to the hydrophobic and amphiphilic efflux resistance-nodulation-cell division (HAE-RND family, spans both the inner and outer membranes of N. gonorrhoeae and confers resistance to a variety of antibiotics and toxic compounds. We here report the crystal structure of the inner membrane MtrD multidrug efflux pump, which reveals a novel structural feature that is not found in other RND efflux pumps.

Glutamate Transporters in the Blood-Brain Barrier

DEFF Research Database (Denmark)

Helms, Hans Christian Cederberg; Nielsen, Carsten Uhd; Waagepetersen, Helle S

2017-01-01

concentration of L-glutamate causes excitotoxicity. A tight control of the brain interstitial fluid L-glutamate levels is therefore imperative, in order to maintain optimal neurotransmission and to avoid such excitotoxicity. The blood-brain barrier, i.e., the endothelial lining of the brain capillaries...... cells. The mechanisms underlying transendothelial L-glutamate transport are however still not well understood. The present chapter summarizes the current knowledge on blood-brain barrier L-glutamate transporters and the suggested pathways for the brain-to-blood L-glutamate efflux......., regulates the exchange of nutrients, gases, and metabolic waste products between plasma and brain interstitial fluid. It has been suggested that brain capillary endothelial cells could play an important role in L-glutamate homeostasis by mediating brain-to-blood L-glutamate efflux. Both in vitro and in vivo...

Novel inhibitory activity of the Staphylococcus aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees.

Science.gov (United States)

Holler, Jes Gitz; Christensen, S Brøgger; Slotved, Hans-Christian; Rasmussen, Hasse B; Gúzman, Alfonso; Olsen, Carl-Erik; Petersen, Bent; Mølgaard, Per

2012-05-01

To isolate a plant-derived compound with efflux inhibitory activity towards the NorA transporter of Staphylococcus aureus. Bioassay-guided isolation was used, with inhibition of ethidium bromide efflux via NorA as a guide. Characterization of activity was carried out using MIC determination and potentiation studies of a fluoroquinolone antibiotic in combination with the isolated compound. Everted membrane vesicles of Escherichia coli cells enriched with NorA were prepared to study efflux inhibitory activity in an isolated manner. The ethanolic extract of Persea lingue was subjected to bioassay-guided fractionation and led to the isolation of the known compound kaempferol-3-O-α-L-(2,4-bis-E-p-coumaroyl)rhamnoside (compound 1). Evaluation of the dose-response relationship of compound 1 showed that ethidium bromide efflux was inhibited, with an IC(50) value of 2 μM. The positive control, reserpine, was found to have an IC(50) value of 9 μM. Compound 1 also inhibited NorA in enriched everted membrane vesicles of E. coli. Potentiation studies revealed that compound 1 at 1.56 mg/L synergistically increased the antimicrobial activity of ciprofloxacin 8-fold against a NorA overexpresser, and the synergistic activity was exerted at a fourth of the concentration necessary for reserpine. Compound 1 was not found to exert a synergistic effect on ciprofloxacin against a norA deletion mutant. The 2,3-coumaroyl isomer of compound 1 has been shown previously not to cause acute toxicity in mice at 20 mg/kg/day. Our results show that compound 1 acts through inhibition of the NorA efflux pump. Combination of compound 1 with subinhibitory concentrations of ciprofloxacin renders a wild-type more susceptible and a NorA overexpresser S. aureus susceptible.

Organic carbon efflux from a deciduous forest catchment in Korea

Directory of Open Access Journals (Sweden)

S. J. Kim

2010-04-01

Full Text Available Soil infiltration and surface discharge of precipitation are critical processes that affect the efflux of Dissolved Organic Carbon (DOC and Particulate Organic Carbon (POC in forested catchments. Concentrations of DOC and POC can be very high in the soil surface in most forest ecosystems and their efflux may not be negligible particularly under the monsoon climate. In East Asia, however, there are little data available to evaluate the role of such processes in forest carbon budget. In this paper, we address two basic questions: (1 how does stream discharge respond to storm events in a forest catchment? and (2 how much DOC and POC are exported from the catchment particularly during the summer monsoon period? To answer these questions, we collected hydrological data (e.g., precipitation, soil moisture, runoff discharge, groundwater level and conducted hydrochemical analyses (including DOC, POC, and six tracers in a deciduous forest catchment in Gwangneung National Arboretum in west-central Korea. Based on the end-member mixing analysis of the six storm events during the summer monsoon in 2005, the surface discharge was estimated as 30 to 80% of the total runoff discharge. The stream discharge responded to precipitation within 12 h during these storm events. The annual efflux of DOC and POC from the catchment was estimated as 0.04 and 0.05 t C ha⁻¹ yr⁻¹, respectively. Approximately 70% of the annual organic carbon efflux occurred during the summer monsoon period. Overall, the annual efflux of organic carbon was estimated to be about 10% of the Net Ecosystem carbon Exchange (NEE obtained by eddy covariance measurement at the same site. Considering the current trends of increasing intensity and amount of summer rainfall and the large interannual variability in NEE, ignoring the organic carbon efflux from forest catchments would result in an inaccurate estimation of the carbon sink strength of forest ecosystems in the monsoon

Applying Web-Based Co-Regulated Learning to Develop Students' Learning and Involvement in a Blended Computing Course

Science.gov (United States)

Tsai, Chia-Wen

2015-01-01

This research investigated, via quasi-experiments, the effects of web-based co-regulated learning (CRL) on developing students' computing skills. Two classes of 68 undergraduates in a one-semester course titled "Applied Information Technology: Data Processing" were chosen for this research. The first class (CRL group, n = 38) received…

Wood CO2 efflux and foliar respiration for Eucalyptus in Hawaii and Brazil

Science.gov (United States)

Michael G. Ryan; Molly A. Cavaleri; Auro C. Almeida; Ricardo Penchel; Randy S. Senock; Jose Luiz Stape

2009-01-01

We measured CO2 efflux from wood for Eucalyptus in Hawaii for 7 years and compared these measurements with those on three- and four-and-a-halfyear- old Eucalyptus in Brazil. In Hawaii, CO2 efflux from wood per unit biomass declined ~10x from age two to age five, twice as much as the decline in tree growth. The CO2 efflux from wood in Brazil was 8-10· lower than that...

Sodium Is Not Required for Chloride Efflux via Chloride/Bicarbonate Exchanger from Rat Thymic Lymphocytes

Directory of Open Access Journals (Sweden)

Donatas Stakišaitis

2014-01-01

Full Text Available Sodium-dependent Cl−/HCO3- exchanger acts as a chloride (Cl− efflux in lymphocytes. Its functional characterization had been described when Cl− efflux was measured upon substituting extracellular sodium (Na+ by N-methyl-D-glucamine (NMDG. For Na+ and Cl− substitution, we have used D-mannitol or NMDG. Thymocytes of male Wistar rats aged 7–9 weeks were used and intracellular Cl− was measured by spectrofluorimetry using MQAE dye in bicarbonate buffers. Chloride efflux was measured in a Cl−-free buffer (Cl− substituted with isethionate acid and in Na+ and Cl−-free buffer with D-mannitol or with NMDG. The data have shown that Cl− efflux is mediated in the absence of Na+ in a solution containing D-mannitol and is inhibited by H2DIDS. Mathematical modelling has shown that Cl− efflux mathematical model parameters (relative membrane permeability, relative rate of exchanger transition, and exchanger efficacy were the same in control and in the medium in which Na+ had been substituted by D-mannitol. The net Cl− efflux was completely blocked in the NMDG buffer. The same blockage of Cl− efflux was caused by H2DIDS. The study results allow concluding that Na+ is not required for Cl− efflux via Cl−/HCO3- exchanger. NMDG in buffers cannot be used for substituting Na+ because NMDG inhibits the exchanger.

Assessing Biobehavioural Self-Regulation and Coregulation in Early Childhood: The Parent-Child Challenge Task.

Science.gov (United States)

Lunkenheimer, Erika; Kemp, Christine J; Lucas-Thompson, Rachel G; Cole, Pamela M; Albrecht, Erin C

2017-01-01

Researchers have argued for more dynamic and contextually relevant measures of regulatory processes in interpersonal interactions. In response, we introduce and examine the effectiveness of a new task, the Parent-Child Challenge Task, designed to assess the self-regulation and coregulation of affect, goal-directed behavior, and physiology in parents and their preschoolers in response to an experimental perturbation. Concurrent and predictive validity was examined via relations with children's externalizing behaviors. Mothers used only their words to guide their 3-year-old children to complete increasingly difficult puzzles in order to win a prize ( N = 96). A challenge condition was initiated mid-way through the task with a newly introduced time limit. The challenge produced decreases in parental teaching and dyadic behavioral variability and increases in child negative affect and dyadic affective variability, measured by dynamic systems-based methods. Children rated lower on externalizing showed respiratory sinus arrhythmia (RSA) suppression in response to challenge, whereas those rated higher on externalizing showed RSA augmentation. Additionally, select task changes in affect, behavior, and physiology predicted teacher-rated externalizing behaviors four months later. Findings indicate the Parent-Child Challenge Task was effective in producing regulatory changes and suggest its utility in assessing biobehavioral self-regulation and coregulation in parents and their preschoolers.

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

OpenAIRE

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

2014-01-01

Many ant species construct subterranean nests. The presence of their nests may explain soil respiration “hot spots”, an important factor in the high CO2 efflux from tropical forests. However, no studies have directly measured CO2 efflux from ant nests. We established 61 experimental plots containing 13 subterranean ant species to evaluate the CO2 efflux from subterranean ant nests in a tropical seasonal forest, Thailand. We examined differences in nest CO2 efflux among ant species. We determi...

ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium

DEFF Research Database (Denmark)

Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H.

2016-01-01

Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC ...

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

Science.gov (United States)

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

2015-08-01

Recently, the cationic polymer thiolated chitosan has been reported to modulate drug absorption by inhibition of intestinal efflux pumps. The objective of this study was to evaluate in vitro and in vivo whether thiolated anionic biopolymers also show an efflux pump inhibitory effect in order to improve intestinal transcellular drug uptake. Therefore, three thiomers have been synthesized due covalent attachment of cysteine to various polymer backbones: pectin-cysteine (pect-cys), carboxymethylcellulose-cysteine (CMC-cys) and alginate-cysteine (alg-cys). In vitro, the permeation enhancing properties of these thiomers and their corresponding unmodified polymers have been evaluated on rat small intestine in Ussing-type chambers, using sulforhodamine 101 (SR-101) as MRP2 model substrate. In comparison to buffer only, SR-101 transport in presence of pect-cys, CMC-cys and alg-cys was improved 1.5-fold, 1.8-fold and 3.0-fold, respectively. Due to the comparatively best in vitro performance of thiolated alginate, it has been chosen for in vivo studies: a SR-101 solution containing 4% (w/v) alg-cys led to an AUC0 ≥ 12 of SR-101 of 109 ng ml(-1)h in rats representing a 3.8-fold improvement in comparison to a SR-101 buffer solution. Unmodified alginate improved the AUC0 ≥ 12 of SR-101 by a factor of 1.9. These findings suggest thiolated alginate as promising auxiliary agent for drugs being anionic efflux pump substrates, since the oral bioavailability of a MRP2 substrate could be significantly improved. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibition of the NLRP3 inflammasome attenuates foam cell formation of THP-1 macrophages by suppressing ox-LDL uptake and promoting cholesterol efflux.

Science.gov (United States)

Chen, Liang; Yao, Qiying; Xu, Siwei; Wang, Hongyan; Qu, Peng

2018-01-01

The NOD-like receptor family, pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the development of atherosclerosis. The activated NLRP3 inflammasome has been reported to promote macrophage foam cell formation, but not all studies have obtained the same result, and how NLRP3 inflammasome is involved in the formation of foam cells remains elusive. We used selective NLRP3 inflammasome inhibitors and NLRP3-deficient THP-1 cells to assess the effect of NLRP3 inflammasome inhibition on macrophage foam cell formation, oxidized low-density lipoprotein (ox-LDL) uptake, esterification, and cholesterol efflux, as well as the expression of associated proteins. Inhibition of the NLRP3 inflammasome attenuated foam cell formation, diminished ox-LDL uptake, and promoted cholesterol efflux from THP-1 macrophages. Moreover, it downregulated CD36, acyl coenzyme A: cholesterol acyltransferase-1 and neutral cholesterol ester hydrolase expression; upregulated ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression; but had no effect on the expression of scavenger receptor class A and ATP-binding cassette transporter G1. Collectively, our findings show that inhibition of the NLRP3 inflammasome decreases foam cell formation of THP-1 macrophages via suppression of ox-LDL uptake and enhancement of cholesterol efflux, which may be due to downregulation of CD36 expression and upregulation of ABCA1 and SR-BI expression, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

A Transcriptomic Approach to Identify Novel Drug Efflux Pumps in Bacteria.

Science.gov (United States)

Li, Liping; Tetu, Sasha G; Paulsen, Ian T; Hassan, Karl A

2018-01-01

The core genomes of most bacterial species include a large number of genes encoding putative efflux pumps. The functional roles of most of these pumps are unknown, however, they are often under tight regulatory control and expressed in response to their substrates. Therefore, one way to identify pumps that function in antimicrobial resistance is to examine the transcriptional responses of efflux pump genes to antimicrobial shock. By conducting complete transcriptomic experiments following antimicrobial shock treatments, it may be possible to identify novel drug efflux pumps encoded in bacterial genomes. In this chapter we describe a complete workflow for conducting transcriptomic analyses by RNA sequencing, to determine transcriptional changes in bacteria responding to antimicrobials.

Computational model for speed of efflux in liquids | Ikata | Journal of ...

African Journals Online (AJOL)

We have looked at the efflux of a viscous liquid from an orifice. Assuming the steady flow of a Newtonian fluid, a model for the energy loss due to viscous shearing stress is derived, and a first-order non-linear ordinary differential equation of second degree is obtained for the speed of efflux. Numerically, the equation is ...

The importance of protoporphyrin IX efflux for ALA-PDT dosimetry

International Nuclear Information System (INIS)

Milanetto, M C; Imasato, H; Perussi, J R

2009-01-01

One of the major advances in PDT is the use of 5-aminolevulinic acid (ALA) to induce the production of an endogenous photosensitizer inside the cells using intracellular enzymatic pathways. ALA is the first intermediate in heme biosynthesis and a precursor of the protoporphyrin IX (PpIX). When activated by light, this efficient photosensitizer accumulated in the target cells can produce cytotoxicity. The aim of this study was to find the best conditions for cell killing using ALA to temporarily increase the concentration of PpIX in two cell lines. It was shown that a considerable efflux of synthesized PpIX occurs. Since this efflux is time-dependent, it is essential to know the optimum time for irradiation after ALA administration. So, the efflux of PpIX from the cells is an important parameter to be considered for ALA-PDT dosimetry

Interaction of dipeptide prodrugs of saquinavir with multidrug resistance protein-2 (MRP-2): evasion of MRP-2 mediated efflux.

Science.gov (United States)

Jain, Ritesh; Agarwal, Sheetal; Mandava, Nanda Kishore; Sheng, Ye; Mitra, Ashim K

2008-10-01

Saquinavir (SQV), the first protease inhibitor approved by FDA to treat HIV-1 infection. This drug is a well-known substrate for multidrug resistance protein-2 (MRP-2). The objective of this study was to investigate whether derivatization of SQV to dipeptide prodrugs, valine-valine-saquinavir (Val-Val-SQV) and glycine-valine-saquinavir (Gly-Val-SQV), targeting peptide transporter can circumvent MRP-2 mediated efflux. Uptake and transport studies were carried out across MDCKII-MRP2 cell monolayers to investigate the interaction of SQV and its prodrugs with MRP-2. In situ single pass intestinal perfusion experiments in rat jejunum were performed to calculate intestinal absorption rate constants and permeabilities of SQV, Val-Val-SQV and Gly-Val-SQV. Uptake studies demonstrated that the prodrugs have significantly lower interaction with MRP-2 relative to SQV. Transepithelial transport of Val-Val-SQV and Gly-Val-SQV across MDCKII-MRP2 cells exhibited an enhanced absorptive flux and reduced secretory flux as compared to SQV. Intestinal perfusion studies revealed that synthesized prodrugs have higher intestinal permeabilities relative to SQV. Enhanced absorption of Val-Val-SQV and Gly-Val-SQV relative to SQV can be attributed to their translocation by the peptide transporter in the jejunum. In the presence of MK-571, a MRP family inhibitor, there was a significant increase in the permeabilities of SQV and Gly-Val-SQV indicating that these compounds are probably substrates for MRP-2. However, there was no change in the permeability of Val-Val-SQV with MK-571 indicating lack of any interaction of Val-Val-SQV with MRP-2. In conclusion, peptide transporter targeted prodrug modification of MRP-2 substrates may lead to shielding of these drug molecules from MRP-2 efflux pumps.

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

Science.gov (United States)

Querejeta, José Ignacio; Allen, Michael F

2008-01-01

Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar 15N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought. PMID:19704776

The role of active efflux in antibiotic - resistance of clinical isolates of Helicobacter pylori

Directory of Open Access Journals (Sweden)

Falsafi T

2009-01-01

Full Text Available Purpose: In gram-negative bacteria, active efflux pumps that excrete drugs can confer resistance to antibiotics however, in Helicobacter pylori this role is not well established. The purpose of this study is to evaluate the role of active efflux in resistance of H. pylori isolates to antibiotics. Materials and Methods: Twelve multiple antibiotic resistant (MAR isolates resistant to at least four antibiotics, including β-lactams, metronidazole, tetracycline, erythromycin, and ciprofloxacin; three resistant to only β-lactams, and two hyper-susceptible isolates, were obtained from screening of 96 clinical isolates of H. pylori . Their minimal inhibitory concentrations (MICs for antibiotics and ethidium-bromide (EtBr were compared in the presence- and absence of a proton-conductor, carbonyl cyanide-m chlorophenyl-hydrazone (CCCP using agar-dilution and disc diffusion. Drug accumulation studies for EtBr and antibiotics were assessed in the presence and absence of CCCP using spectrofluorometry. Results: MIC of EtBr for eight MAR-isolates was decreased two- to four-folds in the presence of CCCP, of which five showed reduced MICs for β-lactam, metronidazole, tetracycline, and ciprofloxacin with CCCP. Accumulation of EtBr by the MAR-isolates was rapid and not dependant on the pattern of multiple resistance. Antibiotic accumulation assay confirmed the presence of energy-dependant efflux of β-lactam, metronidazole, tetracycline, and ciprofloxacin, but no erythromycin in five MAR isolates. Energy-dependant efflux of EtBr or antibiotics was not observed for four MAR-isolates, and three isolates were resistant only to β-lactams. Conclusion: Energy-dependant efflux plays a role in the resistance of H. pylori clinical isolates to structurally unrelated antibiotics in a broadly specific multidrug efflux manner. Difference in the efflux potential of MAR isolates may be related to the presence or absence of functional efflux-pumps in diverse H. pylori

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

LENUS (Irish Health Repository)

Costa, Sofia SANTOS

2011-10-27

Abstract 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

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

Science.gov (United States)

Acosta, M.; Pavelka, M.

2012-04-01

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

Integrated co-regulation of bacterial arsenic and phosphorus metabolisms.

Science.gov (United States)

Kang, Yoon-Suk; Heinemann, Joshua; Bothner, Brian; Rensing, Christopher; McDermott, Timothy R

2012-12-01

Arsenic ranks first on the US Environmental Protection Agency Superfund List of Hazardous Substances. Its mobility and toxicity depend upon chemical speciation, which is significantly driven by microbial redox transformations. Genome sequence-enabled surveys reveal that in many microorganisms genes essential to arsenite (AsIII) oxidation are located immediately adjacent to genes coding for functions associated with phosphorus (Pi) acquisition, implying some type of functional importance to the metabolism of As, Pi or both. We extensively document how expression of genes key to AsIII oxidation and the Pi stress response are intricately co-regulated in the soil bacterium Agrobacterium tumefaciens. These observations significantly expand our understanding of how environmental factors influence microbial AsIII metabolism and contribute to the current discussion of As and P metabolism in the microbial cell. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Relationship between insulin release and 65zinc efflux from rat pancreatic islets maintained in tissue culture

International Nuclear Information System (INIS)

Formby, B.; Schmid-Formby, F.; Grodsky, G.M.

1984-01-01

In short-term batch-incubation or perfusion experiments, we studied insulin release and associated 65 Zn efflux from rat pancreatic islets loaded with 65 Zn by 24-h tissue culture in low-glucose medium. The fractional basal insulin release and 65 Zn efflux were 0.4% and 3% of total content/h/islet, respectively. Thus, basal 65 Zn efflux was much greater than that to be accounted for if zinc was released proportionally with insulin release only; extragranular zinc flux was suggested. Two millimolar glucose, with or without 1 mM 3-isobutyl-1-methylxanthine (IBMX), affected neither insulin release nor associated 65 Zn efflux. Twenty-five millimolar glucose produced a significant threefold increase in insulin release above baseline, but somewhat decreased 65 Zn efflux at marginal significance. Glucose (25 mM) plus 1 mM IBMX provoked a high increase in insulin release and an associated 30% increase in fractional 65 Zn efflux over basal. Calculations based on previous estimations of 65 Zn distribution and equilibrium with islet zinc indicated that molar zinc efflux was more than sufficient to account for a 2-zinc-insulin hexamer. L-Leucine (2 or 20 mM) plus 1 mM IBMX caused far greater 65 Zn efflux for the amount of insulin released, indicating additional 65 Zn mobilization not directly related to insulin secretion. To evaluate 65 Zn efflux during inhibited insulin secretion, batch incubations were performed in 100% D 2 O or at 27 degrees C, conditions that inhibited insulin release stimulated by high glucose plus IBMX. These agents decreased the 65 Zn efflux far below the basal value (35% and 50%, respectively) and greater than could be accounted for by the attendent inhibition of insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Directory of Open Access Journals (Sweden)

Tatiane eCoelho

2015-04-01

Full Text Available 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.

Regulation of taurine transport at the blood-placental barrier by calcium ion, PKC activator and oxidative stress conditions

Directory of Open Access Journals (Sweden)

Lee Na-Young

2010-08-01

Full Text Available Abstract Background In the present study, we investigated the changes of uptake and efflux transport of taurine under various stress conditions using rat conditionally immortalized syncytiotrophoblast cell line (TR-TBT cells, as in vitro blood-placental barrier (BPB model. Methods The transport of taurine in TR-TBT cells were characterized by cellular uptake study using radiolabeled taurine. The efflux of taurine was measured from the amount of radiolabeled taurine remaining in the cells after the uptake of radiolabeled taurine for 60 min. Results Taurine uptake was significantly decreased by phosphorylation of protein kinase C (PKC activator in TR-TBT cells. Also, calcium ion (Ca2+ was involved in taurine transport in TR-TBT cells. Taurine uptake was inhibited and efflux was enhanced under calcium free conditions in the cells. In addition, oxidative stress induced the change of taurine transport in TR-TBT cells, but the changes were different depending on the types of oxidative stress inducing agents. Tumor necrosis factor-α (TNF-α, lipopolysaccharide (LPS and diethyl maleate (DEM significantly increased taurine uptake, but H2O2 and nitric oxide (NO donor decreased taurine uptake in the cells. Taurine efflux was down-regulated by TNF-α in TR-TBT cells. Conclusion Taurine transport in TR-TBT cells were regulated diversely at extracellular Ca2+ level, PKC activator and oxidative stress conditions. It suggested that variable stresses affected the taurine supplies from maternal blood to fetus and taurine level of fetus.

Calibration and analysis of soil carbon efflux estimates with closed chambers at Forsmark and Laxemar

International Nuclear Information System (INIS)

Tagesson, Torbern

2006-08-01

The Forsmark and the Laxemar investigation areas are examined by the Swedish Nuclear Fuel and Waste Management Co. for a possible construction of a deep repository for nuclear waste. In the case of a future leakage of waste, the radioactive isotopes could end up in the ecosystems above the repository. The fate of the radionuclides and their possible radiological impacts are then highly determined by ecosystem carbon cycling. An important part of the carbon cycling is the soil carbon effluxes, and in the investigation areas soil carbon effluxes have been examined with the closed chamber technique. This paper is divided into two parts. Firstly, there were problems with the equipment measuring the soil carbon dioxide efflux, and the first part is a description of the problem, how it was corrected and its possible causes. The second part is a manual in how to analyse data and calculate annual estimates of soil carbon efflux. The field measurement by the EGM-4 is just an occasional estimate of the soil carbon efflux at a certain spot and at a certain point in time. To make an interpretation of the measurements, it is essential to analyse the data and to temporally extrapolate them. It is necessary to prepare the raw data for the analysis. The problems with the EGM-4 doing the measurements at the Forsmark and the Laxemar investigation area makes it necessary to correct the data taken up by this EGM-4. The data should also be separated into soil respiration and gross primary production (GPP). Soil carbon dioxide effluxes should be changed to soil carbon effluxes. Soil carbon effluxes are strongly controlled by abiotic factors; temperature is the main factor to influence soil respiration and photosynthetically active radiation (PAR) and air temperature are the main factors to influence GPP. Regression with soil respiration against temperature and with GPP against PAR or temperature can therefore be done. These equations can then be used on datasets with temperature and PAR

Calibration and analysis of soil carbon efflux estimates with closed chambers at Forsmark and Laxemar

Energy Technology Data Exchange (ETDEWEB)

Tagesson, Torbern (Dept. of Physical Geography and Ecosystem Analysis, Lund Univ., Lund (SE))

2006-08-15

The Forsmark and the Laxemar investigation areas are examined by the Swedish Nuclear Fuel and Waste Management Co. for a possible construction of a deep repository for nuclear waste. In the case of a future leakage of waste, the radioactive isotopes could end up in the ecosystems above the repository. The fate of the radionuclides and their possible radiological impacts are then highly determined by ecosystem carbon cycling. An important part of the carbon cycling is the soil carbon effluxes, and in the investigation areas soil carbon effluxes have been examined with the closed chamber technique. This paper is divided into two parts. Firstly, there were problems with the equipment measuring the soil carbon dioxide efflux, and the first part is a description of the problem, how it was corrected and its possible causes. The second part is a manual in how to analyse data and calculate annual estimates of soil carbon efflux. The field measurement by the EGM-4 is just an occasional estimate of the soil carbon efflux at a certain spot and at a certain point in time. To make an interpretation of the measurements, it is essential to analyse the data and to temporally extrapolate them. It is necessary to prepare the raw data for the analysis. The problems with the EGM-4 doing the measurements at the Forsmark and the Laxemar investigation area makes it necessary to correct the data taken up by this EGM-4. The data should also be separated into soil respiration and gross primary production (GPP). Soil carbon dioxide effluxes should be changed to soil carbon effluxes. Soil carbon effluxes are strongly controlled by abiotic factors; temperature is the main factor to influence soil respiration and photosynthetically active radiation (PAR) and air temperature are the main factors to influence GPP. Regression with soil respiration against temperature and with GPP against PAR or temperature can therefore be done. These equations can then be used on datasets with temperature and PAR

The barley MATE gene, HvAACT1, increases citrate efflux and Al3+ tolerance when expressed in wheat and barley

Science.gov (United States)

Zhou, Gaofeng; Delhaize, Emmanuel; Zhou, Meixue; Ryan, Peter R.

2013-01-01

Background and Aims Aluminium is toxic in acid soils because the soluble Al3+ inhibits root growth. A mechanism of Al3+ tolerance discovered in many plant species involves the release of organic anions from root apices. The Al3+-activated release of citrate from the root apices of Al3+-tolerant genotypes of barley is controlled by a MATE gene named HvAACT1 that encodes a citrate transport protein located on the plasma membrane. The aim of this study was to investigate whether expressing HvAACT1 with a constitutive promoter in barley and wheat can increase citrate efflux and Al3+ tolerance of these important cereal species. Methods HvAACT1 was over-expressed in wheat (Triticum aestivum) and barley (Hordeum vulgare) using the maize ubiquitin promoter. Root apices of transgenic and control lines were analysed for HvAACT1 expression and organic acid efflux. The Al3+ tolerance of transgenic and control lines was assessed in both hydroponic solution and acid soil. Key Results and Conclusions Increased HvAACT1 expression in both cereal species was associated with increased citrate efflux from root apices and enhanced Al3+ tolerance, thus demonstrating that biotechnology can complement traditional breeding practices to increase the Al3+ tolerance of important crop plants. PMID:23798600

Dual effects of the PI3K inhibitor ZSTK474 on multidrug efflux pumps in resistant cancer cells.

Science.gov (United States)

Muthiah, Divya; Callaghan, Richard

2017-11-15

ZSTK474 is a potent phosphoinositide 3-kinase (PI3K) inhibitor that reduces cell proliferation via G 1 -arrest. However, there is little information on the susceptibility of this anticancer drug to resistance conferred by the multidrug pumps P-glycoprotein (ABCB1) and ABCG2. We have demonstrated that ZSTK474 generated cytotoxicity in cells over-expressing either pump with potency similar to that in drug sensitive cells. In addition, the co-administration of ZSTK474 with the cytotoxic anti-cancer drugs vinblastine and mitoxantrone caused a potentiated cytotoxic effect in both drug sensitive and efflux pump expressing cells. These observations suggest that ZSTK474 is unaffected by the presence of multidrug efflux pumps and may circumvent their activities. Indeed, ZSTK474 increased the cellular accumulation of calcein-AM and mitoxantrone in cells expressing ABCB1 and ABCG2, respectively. ZSTK474 treatment also resulted in reduced expression of both efflux pumps in multidrug resistant cancer cells. Measurement of ABCB1 or ABCG2 mRNA levels demonstrated that the reduction was not due to altered transcription. Similarly, inhibitor studies showed that the proteasomal degradation pathway for ABCB1 and the lysosomal route for ABCG2 degradation were unaffected by ZSTK474. Thus the mechanism underlying reduced ABCB1 and ABCG2 levels caused by ZSTK474 was due to a reduction in overall protein synthesis; a process influenced by the PI3K pathway. In summary, ZSTK474 is not susceptible to efflux by the resistance mediators ABCB1 and ABCG2. Moreover, it inhibits the drug transport function of the pumps and leads to a reduction in their cellular expression levels. Our observations demonstrate that ZSTK474 is a powerful anticancer drug. Copyright © 2017 Elsevier B.V. All rights reserved.

ATP-binding cassette transporters in reproduction: a new frontier

Science.gov (United States)

Bloise, E.; Ortiga-Carvalho, T.M.; Reis, F.M.; Lye, S.J.; Gibb, W.; Matthews, S.G.

2016-01-01

BACKGROUND The transmembrane ATP-binding cassette (ABC) transporters actively efflux an array of clinically relevant compounds across biological barriers, and modulate biodistribution of many physiological and pharmacological factors. To date, over 48 ABC transporters have been identified and shown to be directly and indirectly involved in peri-implantation events and fetal/placental development. They efflux cholesterol, steroid hormones, vitamins, cytokines, chemokines, prostaglandins, diverse xenobiotics and environmental toxins, playing a critical role in regulating drug disposition, immunological responses and lipid trafficking, as well as preventing fetal accumulation of drugs and environmental toxins. METHODS This review examines ABC transporters as important mediators of placental barrier functions and key reproductive processes. Expression, localization and function of all identified ABC transporters were systematically reviewed using PubMed and Google Scholar websites to identify relevant studies examining ABC transporters in reproductive tissues in physiological and pathophysiological states. Only reports written in English were incorporated with no restriction on year of publication. While a major focus has been placed on the human, extensive evidence from animal studies is utilized to describe current understanding of the regulation and function of ABC transporters relevant to human reproduction. RESULTS ABC transporters are modulators of steroidogenesis, fertilization, implantation, nutrient transport and immunological responses, and function as ‘gatekeepers’ at various barrier sites (i.e. blood-testes barrier and placenta) against potentially harmful xenobiotic factors, including drugs and environmental toxins. These roles appear to be species dependent and change as a function of gestation and development. The best-described ABC transporters in reproductive tissues (primarily in the placenta) are the multidrug transporters p-glycoprotein and

Cholesterol efflux is differentially regulated in neurons and astrocytes: implications for brain cholesterol homeostasis

Science.gov (United States)

Chen, Jing; Zhang, Xiaolu; Kusumo, Handojo; Costa, Lucio G.; Guizzetti, Marina

2012-01-01

Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood-brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS. PMID:23010475

Gly[14]-humanin inhibits ox-LDL uptake and stimulates cholesterol efflux in macrophage-derived foam cells.

Science.gov (United States)

Zhu, Wa-Wa; Wang, Shu-Rong; Liu, Zhi-Hua; Cao, Yong-Jun; Wang, Fen; Wang, Jing; Liu, Chun-Feng; Xie, Ying; Xie, Ying; Zhang, Yan-Lin

2017-01-01

Foam cell formation, which is caused by imbalanced cholesterol influx and efflux by macrophages, plays a vital role in the occurrence and development of atherosclerosis. Humanin (HN), a mitochondria-derived peptide, can prevent the production of reactive oxygen species and death of human aortic endothelial cells exposed to oxidized low-density lipoprotein (ox-LDL) and has a protective effect on patients with in early atherosclerosis. However, the effects of HN on the regulation of cholesterol metabolism in RAW 264.7 macrophages are still unknown. This study was designed to investigate the role of [Gly14]-humanin (HNG) in lipid uptake and cholesterol efflux in RAW 264.7 macrophages. Flow cytometry and live cell imaging results showed that HNG reduced Dil-ox-LDL accumulation in the RAW 264.7 macrophages. A similar result was obtained for lipid accumulation by measuring cellular cholesterol content. Western blot analysis showed that ox-LDL treatment upregulated not only the protein expression of CD36 and LOX-1, which mediate ox-LDL endocytosis, but also ATP-binding cassette (ABC) transporter A1 and ABCG1, which mediate ox-LDL exflux. HNG pretreatment inhibited the upregulation of CD36 and LOX-1 levels, prompting the upregulation of ABCA1 and ABCG1 levels induced by ox-LDL. Therefore we concluded that HNG could inhibit ox-LDL-induced macrophage-derived foam cell formation, which occurs because of a decrease in lipid uptake and an increase in cholesterol efflux from macrophage cells. Copyright © 2016 Elsevier Inc. All rights reserved.

AtNPF2.5 Modulates Chloride (Cl−) Efflux from Roots of Arabidopsis thaliana

KAUST Repository

Li, Bo

2017-01-05

The accumulation of high concentrations of chloride (Cl) in leaves can adversely affect plant growth. When comparing different varieties of the same Cl sensitive plant species those that exclude relatively more Cl from their shoots tend to perform better under saline conditions; however, the molecular mechanisms involved in maintaining low shoot Cl remain largely undefined. Recently, it was shown that the NRT1/PTR Family 2.4 protein (NPF2.4) loads Cl into the root xylem, which affects the accumulation of Cl in Arabidopsis shoots. Here we characterize NPF2.5, which is the closest homolog to NPF2.4 sharing 83.2% identity at the amino acid level. NPF2.5 is predominantly expressed in root cortical cells and its transcription is induced by salt. Functional characterisation of NPF2.5 via its heterologous expression in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes indicated that NPF2.5 is likely to encode a Cl permeable transporter. Arabidopsis npf2.5 T-DNA knockout mutant plants exhibited a significantly lower Cl efflux from roots, and a greater Cl accumulation in shoots compared to salt-treated Col-0 wild-type plants. At the same time, NO- content in 3 the shoot remained unaffected. Accumulation of Cl in the shoot increased following (1) amiRNA-induced knockdown of NPF2.5 transcript abundance in the root, and (2) constitutive over-expression of NPF2.5. We suggest that both these findings are consistent with a role for NPF2.5 in modulating Cl transport. Based on these results, we propose that NPF2.5 functions as a pathway for Cl efflux from the root, contributing to exclusion of Cl from the shoot of Arabidopsis.

Soil CO2 efflux of a larch forest in northern Japan

Directory of Open Access Journals (Sweden)

Y. Fujinuma

2010-11-01

Full Text Available We had continuously measured soil CO2 efflux (Rs in a larch forest in northern Japan at hourly intervals for the snow-free period in 2003 with an automated chamber system and partitioned Rs into heterotrophic respiration (Rh and autotrophic respiration (Rr by using the trench method. In addition, we applied the soil CO2 concentration gradients method to continuously measure soil CO2 profiles under snowpack in the snowy period and to partition Rs into topsoil (Oa and A horizons CO2 efflux (Ft with a depth of 0.13 m and sub-soil (C horizon CO2 efflux (Fc. We found that soil CO2 effluxes were strongly affected by the seasonal variation of soil temperature but weakly correlated with soil moisture, probably because the volumetric soil moisture (30–40% at 95% confidence interval was within a plateau region for root and microbial activities. The soil CO2 effluxes changed seasonally in parallel with soil temperature in topsoil with the peak in late summer. On the other hand, the contribution of Rr to Rs was the largest at about 50% in early summer, when canopy photosynthesis and plant growth were more active. The temperature sensitivity (Q10 of Rr peaked in June. Under snowpack, Rs was stable until mid-March and then gradually increased with snow melting. Rs summed up to 79 gC m−2 during the snowy season for 4 months. The annual Rs was determined at 934 gC m−2 y−1 in 2003, which accounted for 63% of ecosystem respiration. The annual contributions of Rh and Rs to Rs were 57% and 43%, respectively. Based on the gradient approach, Rs was partitioned vertically into litter (Oi and Oe horizons with a depth of 0.01–0.02 m, topsoil and sub-soil respirations with proportions of 6, 72 and 22%, respectively, on an annual basis. The vertical distribution of CO2 efflux was consistent with those of soil carbon and root biomass.

The Glutamine Transporters and Their Role in the Glutamate/GABA-Glutamine Cycle

DEFF Research Database (Denmark)

Leke, Renata; Schousboe, Arne

2016-01-01

in this neural communication, i.e., the transporters responsible for glutamine efflux from astrocytes and influx into the neurons, such as the members of the SNAT, LAT, y(+)LAT, and ASC families of transporters. The SNAT family consists of the transporter isoforms SNAT3 and SNAT5 that are related to efflux from......Glutamine is a key amino acid in the CNS, playing an important role in the glutamate/GABA-glutamine cycle (GGC). In the GGC, glutamine is transferred from astrocytes to neurons, where it will replenish the inhibitory and excitatory neurotransmitter pools. Different transporters participate...... the astrocytic compartment, and SNAT1 and SNAT2 that are associated with glutamine uptake into the neuronal compartment. The isoforms SNAT7 and SNAT8 do not have their role completely understood, but they likely also participate in the GGC. The isoforms LAT2 and y(+)LAT2 facilitate the exchange of neutral amino...

Evolutionarily Conserved Roles for Blood-Brain Barrier Xenobiotic Transporters in Endogenous Steroid Partitioning and Behavior.

Science.gov (United States)

Hindle, Samantha J; Munji, Roeben N; Dolghih, Elena; Gaskins, Garrett; Orng, Souvinh; Ishimoto, Hiroshi; Soung, Allison; DeSalvo, Michael; Kitamoto, Toshihiro; Keiser, Michael J; Jacobson, Matthew P; Daneman, Richard; Bainton, Roland J

2017-10-31

Central nervous system (CNS) chemical protection depends upon discrete control of small-molecule access by the blood-brain barrier (BBB). Curiously, some drugs cause CNS side-effects despite negligible transit past the BBB. To investigate this phenomenon, we asked whether the highly BBB-enriched drug efflux transporter MDR1 has dual functions in controlling drug and endogenous molecule CNS homeostasis. If this is true, then brain-impermeable drugs could induce behavioral changes by affecting brain levels of endogenous molecules. Using computational, genetic, and pharmacologic approaches across diverse organisms, we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice). Furthermore, we show that MDR1-interacting drugs are associated with anxiety-related behaviors in humans. We propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates and implicates the BBB in behavioral regulation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Hydrodynamic Controls on Carbon Dioxide Efflux from Inland Waters

Science.gov (United States)

Long, H. E.; Waldron, S.; Hoey, T.; Newton, J.; Quemin, S.

2013-12-01

Intensive research has been undertaken on carbon dioxide efflux from lakes, estuaries and oceans, but much less attention has been given to rivers and streams, especially lower order streams. River systems are often over-saturated with carbon dioxide and so tend to act as sources of carbon dioxide to the atmosphere. It has been thought that rivers act as pipes carrying this terrestrial carbon to the oceans. However, recent studies have shown that a significant amount of the carbon is reprocessed within the system in a series of transformations and losses. Fluvial evasion of carbon dioxide is now recognised to be a significant component of carbon cycles, however the factors controlling carbon dioxide efflux and its magnitude remain poorly understood and quantified. This research aims to quantify, and better understand the controls on, freshwater carbon dioxide evasion. Data are presented here from field measurements that commenced in Sept 2013 in two contrasting Scottish rivers: the River Kelvin which has a large (335 km.sq) part-urban catchment with predominantly non-peat soils and Drumtee Water, a small (9.6 km.sq) rural catchment of peat soils and agricultural land. Using a floating chamber with the headspace connected to an infrared gas analyser to measure changes in carbon dioxide concentration, efflux rates from 0.22 - 47.4 μmol CO2/m.sq/sec were measured, these close to the middle of the range of previously reported values. At one site on the River Kelvin in May 2013 an influx of -0.61 - -3.53 μmol CO2/m.sq/sec was recorded. Whereas previous research finds carbon dioxide efflux to increase with decreasing river size and a more organic-rich soil catchment, here the controls on carbon dioxide evasion are similar across the contrasting catchments. Carbon dioxide evasion shows seasonality, with maximum fluxes in the summer months being up to twice as high as the winter maxima. Linear regression demonstrates that evasion increases with increased flow velocity

Efflux pump genes of the resistance-nodulation-division family in Burkholderia cenocepacia genome

Directory of Open Access Journals (Sweden)

Manina Giulia

2006-07-01

Full Text Available Abstract Background Burkholderia cenocepacia is recognized as opportunistic pathogen that can cause lung infections in cystic fibrosis patients. A hallmark of B. cenocepacia infections is the inability to eradicate the organism because of multiple intrinsic antibiotic resistance. As Resistance-Nodulation-Division (RND efflux systems are responsible for much of the intrinsic multidrug resistance in Gram-negative bacteria, this study aims to identify RND genes in the B. cenocepacia genome and start to investigate their involvement into antimicrobial resistance. Results Genome analysis and homology searches revealed 14 open reading frames encoding putative drug efflux pumps belonging to RND family in B. cenocepacia J2315 strain. By reverse transcription (RT-PCR analysis, it was found that orf3, orf9, orf11, and orf13 were expressed at detectable levels, while orf10 appeared to be weakly expressed in B. cenocepacia. Futhermore, orf3 was strongly induced by chloramphenicol. The orf2 conferred resistance to fluoroquinolones, tetraphenylphosphonium, streptomycin, and ethidium bromide when cloned and expressed in Escherichia coli KAM3, a strain lacking the multidrug efflux pump AcrAB. The orf2-overexpressing E. coli also accumulate low concentrations of ethidium bromide, which was restored to wild type level in the presence of CCCP, an energy uncoupler altering the energy of the drug efflux pump. Conclusion The 14 RND pumps gene we have identified in the genome of B. cenocepacia suggest that active efflux could be a major mechanism underlying antimicrobial resistance in this microorganism. We have characterized the ORF2 pump, one of these 14 potential RND efflux systems. Its overexpression in E. coli conferred resistance to several antibiotics and to ethidium bromide but it remains to be determined if this pump play a significant role in the antimicrobial intrinsic resistance of B. cenocepacia. The characterization of antibiotic efflux pumps in B

McMYB12 Transcription Factors Co-regulate Proanthocyanidin and Anthocyanin Biosynthesis in Malus Crabapple

OpenAIRE

Tian, Ji; Zhang, Jie; Han, Zhen-yun; Song, Ting-ting; Li, Jin-yan; Wang, Ya-ru; Yao, Yun-cong

2017-01-01

The flavonoid compounds, proanthocyanidins (PAs), protect plants from biotic stresses, contribute to the taste of many fruits, and are beneficial to human health in the form of dietary antioxidants. In this study, we functionally characterized two Malus crabapple R2R3-MYB transcription factors, McMYB12a and McMYB12b, which co-regulate PAs and anthocyanin biosynthesis. McMYB12a was shown to be mainly responsible for upregulating the expression of anthocyanin biosynthetic genes by binding to th...

Stress Introduction Rate Alters the Benefit of AcrAB-TolC Efflux Pumps.

Science.gov (United States)

Langevin, Ariel M; Dunlop, Mary J

2018-01-01

Stress tolerance studies are typically conducted in an all-or-none fashion. However, in realistic settings-such as in clinical or metabolic engineering applications-cells may encounter stresses at different rates. Therefore, how cells tolerate stress may depend on its rate of appearance. To address this, we studied how the rate of stress introduction affects bacterial stress tolerance by focusing on a key stress response mechanism. Efflux pumps, such as AcrAB-TolC of Escherichia coli , are membrane transporters well known for the ability to export a wide variety of substrates, including antibiotics, signaling molecules, and biofuels. Although efflux pumps improve stress tolerance, pump overexpression can result in a substantial fitness cost to the cells. We hypothesized that the ideal pump expression level would involve a rate-dependent trade-off between the benefit of pumps and the cost of their expression. To test this, we evaluated the benefit of the AcrAB-TolC pump under different rates of stress introduction, including a step, a fast ramp, and a gradual ramp. Using two chemically diverse stresses, the antibiotic chloramphenicol and the jet biofuel precursor pinene, we assessed the benefit provided by the pumps. A mathematical model describing these effects predicted the benefit as a function of the rate of stress introduction. Our findings demonstrate that as the rate of introduction is lowered, stress response mechanisms provide a disproportionate benefit to pump-containing strains, allowing cells to survive beyond the original inhibitory concentrations. IMPORTANCE Efflux pumps are ubiquitous in nature and provide stress tolerance in the cells of species ranging from bacteria to mammals. Understanding how pumps provide tolerance has far-reaching implications for diverse fields, from medicine to biotechnology. Here, we investigated how the rate of stressor appearance impacts tolerance. We focused on two distinct substrates of AcrAB-TolC efflux pumps, the

ABC transporters affect the elimination and toxicity of CdTe quantum dots in liver and kidney cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Mingli; Yin, Huancai; Bai, Pengli [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); Miao, Peng [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Deng, Xudong [Department of Chemical Engineering, McMaster University, Hamilton, Ontario, L8S 4L7 (Canada); Xu, Yingxue [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Hu, Jun [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); Yin, Jian, E-mail: yinj@sibet.ac.cn [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China)

2016-07-15

This paper aimed to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters on the efflux and the toxicity of nanoparticles in liver and kidney cells. In this study, we synthesized CdTe quantum dots (QDs) that were monodispersed and emitted green fluorescence (maximum peak at 530 nm). Such QDs tended to accumulate in human hepatocellular carcinoma cells (HepG2), human kidney cells 2 (HK-2), and Madin-Darby canine kidney (MDCK) cells, and cause significant toxicity in all the three cell lines. Using specific inhibitors and inducers of P-glycoprotein (Pgp) and multidrug resistance associated proteins (Mrps), the cellular accumulation and subsequent toxicity of QDs in HepG2 and HK-2 cells were significantly affected, while only slight changes appeared in MDCK cells, corresponding well with the functional expressions of ABC transporters in cells. Moreover, treatment of QDs caused concentration- and time- dependent induction of ABC transporters in HepG2 and HK-2 cells, but such phenomenon was barely found in MDCK cells. Furthermore, the effects of CdTe QDs on ABC transporters were found to be greater than those of CdCl{sub 2} at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd{sup 2+} and specific properties of QDs. Overall, these results indicated a strong dependence between the functional expressions of ABC transporters and the efflux of QDs, which could be an important reason for the modulation of QDs toxicity by ABC transporters. - Highlights: • ABC transporters contributed actively to the cellular efflux of CdTe quantum dots. • ABC transporters affected the cellular toxicity of CdTe quantum dots. • Treatment of CdTe quantum dots induced the gene expression of ABC transporters. • Free Cd{sup 2+} should be partially involved in the effects of QDs on ABC transporters. • Cellular efflux of quantum dots could be an important modulator for its toxicity.

Distribution and physiology of ABC-Type transporters contributing to multidrug resistance in bacteria

NARCIS (Netherlands)

Lubelski, Jacek; Konings, Wil N.; Driessen, Arnold J. M.

Membrane proteins responsible for the active efflux of structurally and functionally unrelated drugs were first characterized in higher eukalyotes. To date, a vast number of transporters contributing to multidrug resistance (MDR transporters) have been reported for a large variety of organisms.

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

International Nuclear Information System (INIS)

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

1989-01-01

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

Pharmacophore-Based Repositioning of Approved Drugs as Novel Staphylococcus aureus NorA Efflux Pump Inhibitors.

Science.gov (United States)

Astolfi, Andrea; Felicetti, Tommaso; Iraci, Nunzio; Manfroni, Giuseppe; Massari, Serena; Pietrella, Donatella; Tabarrini, Oriana; Kaatz, Glenn W; Barreca, Maria L; Sabatini, Stefano; Cecchetti, Violetta

2017-02-23

An intriguing opportunity to address antimicrobial resistance is represented by the inhibition of efflux pumps. Focusing on NorA, the most important efflux pump of Staphylococcus aureus, an efflux pump inhibitors (EPIs) library was used for ligand-based pharmacophore modeling studies. By exploitation of the obtained models, an in silico drug repositioning approach allowed for the identification of novel and potent NorA EPIs.

A multichannel automated chamber system for continuous measurement of forest soil CO2 efflux

International Nuclear Information System (INIS)

Liang, N.; Inoue, G.; Fujinuma, Y.

2003-01-01

Development of a fast-response multi-chamber system for measuring soil-surface carbon dioxide efflux is described. The sixteen-chamber automated system continuously monitors surface carbon dioxide efflux at different locations within a forest ecosystem using a single infrared gas analyzer that successively measures gas samples from each of the sixteen chambers. The chambers have lids that open and close automatically, and are connected in parallel to the single carbon dioxide analyzer which is equipped with a sixteen-channel gas sampler. Air is withdrawn continuously from the inlets and outlets of each chamber and fed sequentially to the gas analyzer. Using this instrument, surface carbon dioxide efflux was measured in a 40-year old pine forest during a three-month period (February to May) in 2001. Results showed a steady increase in mean carbon dioxide efflux during the period. A statistically significant correlation between soil-surface carbon dioxide efflux and surface temperature was also established. Spatial variation of carbon dioxide efflux was found to be higher in the non-growing season than in the growing season. It was concluded that the multi-channel automated chamber system can provide large amounts of high quality data on soil carbon dioxide efflux over a large surface area and simultaneously evaluate both spatial and temporal variation. The system uses a relatively small amount of power (70 W maximum) which can be further reduced (to 15 W) by minimizing the pressure difference between inside and outside the chamber. The system requires no maintenance other than the calibration of the gas analyzer and measurement of the flow rate through the chambers. 34 refs., 8 figs

Large-scale analysis of Arabidopsis transcription reveals a basal co-regulation network

Directory of Open Access Journals (Sweden)

Chamovitz Daniel A

2009-09-01

Full Text Available Abstract Background Analyses of gene expression data from microarray experiments has become a central tool for identifying co-regulated, functional gene modules. A crucial aspect of such analysis is the integration of data from different experiments and different laboratories. How to weigh the contribution of different experiments is an important point influencing the final outcomes. We have developed a novel method for this integration, and applied it to genome-wide data from multiple Arabidopsis microarray experiments performed under a variety of experimental conditions. The goal of this study is to identify functional globally co-regulated gene modules in the Arabidopsis genome. Results Following the analysis of 21,000 Arabidopsis genes in 43 datasets and about 2 × 108 gene pairs, we identified a globally co-expressed gene network. We found clusters of globally co-expressed Arabidopsis genes that are enriched for known Gene Ontology annotations. Two types of modules were identified in the regulatory network that differed in their sensitivity to the node-scoring parameter; we further showed these two pertain to general and specialized modules. Some of these modules were further investigated using the Genevestigator compendium of microarray experiments. Analyses of smaller subsets of data lead to the identification of condition-specific modules. Conclusion Our method for identification of gene clusters allows the integration of diverse microarray experiments from many sources. The analysis reveals that part of the Arabidopsis transcriptome is globally co-expressed, and can be further divided into known as well as novel functional gene modules. Our methodology is general enough to apply to any set of microarray experiments, using any scoring function.

Measurements of low energy neutral hydrogen efflux during ICRF heating

International Nuclear Information System (INIS)

Cohen, S.A.; Ruzic, D.; Voss, D.E.

1984-09-01

Using the Low Energy Neutral Atom Spectrometer, measurements were made of the H 0 and D 0 efflux from PLT during ion cyclotron heating experiments. The application of rf power at frequencies appropriate to fundamental and 2nd-harmonic heating results in a rapid, toroidally uniform rise in the charge-exchange efflux at a rate of about 10 15 cm -2 s -1 MW -1 . This flux increase is larger at lower plasma currents. The cause of this flux and its impact on plasma behavior are discussed

Sequential induction of auxin efflux and influx carriers regulates lateral root emergence.

Science.gov (United States)

Péret, Benjamin; Middleton, Alistair M; French, Andrew P; Larrieu, Antoine; Bishopp, Anthony; Njo, Maria; Wells, Darren M; Porco, Silvana; Mellor, Nathan; Band, Leah R; Casimiro, Ilda; Kleine-Vehn, Jürgen; Vanneste, Steffen; Sairanen, Ilkka; Mallet, Romain; Sandberg, Göran; Ljung, Karin; Beeckman, Tom; Benkova, Eva; Friml, Jiří; Kramer, Eric; King, John R; De Smet, Ive; Pridmore, Tony; Owen, Markus; Bennett, Malcolm J

2013-10-22

In Arabidopsis, lateral roots originate from pericycle cells deep within the primary root. New lateral root primordia (LRP) have to emerge through several overlaying tissues. Here, we report that auxin produced in new LRP is transported towards the outer tissues where it triggers cell separation by inducing both the auxin influx carrier LAX3 and cell-wall enzymes. LAX3 is expressed in just two cell files overlaying new LRP. To understand how this striking pattern of LAX3 expression is regulated, we developed a mathematical model that captures the network regulating its expression and auxin transport within realistic three-dimensional cell and tissue geometries. Our model revealed that, for the LAX3 spatial expression to be robust to natural variations in root tissue geometry, an efflux carrier is required--later identified to be PIN3. To prevent LAX3 from being transiently expressed in multiple cell files, PIN3 and LAX3 must be induced consecutively, which we later demonstrated to be the case. Our study exemplifies how mathematical models can be used to direct experiments to elucidate complex developmental processes.

Volume-dependent K+ transport in rabbit red blood cells comparison with oxygenated human SS cells

Energy Technology Data Exchange (ETDEWEB)

Al-Rohil, N.; Jennings, M.L.

1989-07-01

In this study the volume-dependent or N-ethylmaleimide (NEM)-stimulated, ouabain-insensitive K+ influx and efflux were measured with the tracer 86Rb+ in rabbit red blood cells. The purpose of the work was to examine the rabbit as a potential model for cell volume regulation in human SS red blood cells and also to investigate the relationship between the NEM-reactive sulfhydryl group(s) and the signal by which cell swelling activates the transport. Ouabain-resistant K+ efflux and influx increase nearly threefold in cells swollen hypotonically by 15%. Pretreatment with 2 mM NEM stimulates efflux 5-fold and influx 10-fold (each measured in an isotonic medium). The ouabain-resistant K+ efflux was dependent on the major anion in the medium. The anion dependence of K+ efflux in swollen or NEM-stimulated cells was as follows: Br- greater than Cl- much greater than NO3- = acetate. The magnitudes of both the swelling- and the NEM-stimulated fluxes are much higher in young cells (density separated but excluding reticulocytes) than in older cells. Swelling- or NEM-stimulated K+ efflux in rabbit red blood cells was inhibited 50% by 1 mM furosemide, and the inhibitory potency of furosemide was enhanced by extracellular K+, as is known to be true for human AA and low-K+ sheep red blood cells. The swelling-stimulated flux in both rabbit and human SS cells has a pH optimum at approximately 7.4. We conclude that rabbit red blood cells are a good model for swelling-stimulated K+ transport in human SS cells.

Molecular characterization of ABC transporters in marine ciliate, Euplotes crassus: Identification and response to cadmium and benzo[a]pyrene.

Science.gov (United States)

Kim, Hokyun; Yim, Bora; Kim, Jisoo; Kim, Haeyeon; Lee, Young-Mi

2017-11-30

ATP-binding cassette (ABC) transporters participate in transporting various substances, including xenobiotics, in or out of cells. However, their genetic information and function in ciliates remain still unclear. In this study, we sequenced and characterized two ABC transporter genes (EcABCB and EcABCC), and investigated the effect of cadmium (Cd) and benzo[a]pyrene (B[a]P) on their function and gene expression, using efflux assay and real-time reverse transcription-polymerase chain reaction (qRT-PCR), respectively, in the marine ciliate, Euplotes crassus. Sequencing analysis and efflux assay showed that EcABCB and EcABCC are typical ABC transporters, possessing conserved function. Exposure to Cd (≥5mg/L) and B[a]P (≥50.5μg/L) enhanced accumulation of a substrate. A significant increase in the expression of EcABCB and EcABC mRNA was observed at lower concentration in response to Cd and B[a]P. Our findings indicate that Cd and B[a]P could inhibit the efflux function of ABC transporters, leading to cellular toxicity in the ciliate. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2011-10-01

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

Ligand Promiscuity between the Efflux Pumps Human P-Glycoprotein and S. aureus NorA.

Science.gov (United States)

Brincat, Jean Pierre; Broccatelli, Fabio; Sabatini, Stefano; Frosini, Maria; Neri, Annalisa; Kaatz, Glenn W; Cruciani, Gabriele; Carosati, Emanuele

2012-03-08

Thirty-two diverse compounds were evaluated for their ability to inhibit both Pgp-mediated efflux in mouse T-lymphoma L5178 MDR1 and NorA-mediated efflux in S. aureus SA-1199B. Only four compounds were strong inhibitors of both efflux pumps. Three compounds were found to inhibit Pgp exclusively and strongly, while seven compounds inhibited only NorA. These results demonstrate that Pgp and NorA inhibitors do not necessarily overlap, opening the way to safer therapeutic use of effective NorA inhibitors.

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.

Directory of Open Access Journals (Sweden)

Wei-Ming Chen

Full Text Available 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.

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.

Science.gov (United States)

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.

The actin cytoskeleton may control the polar distribution of an auxin transport protein

Science.gov (United States)

Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

2000-01-01

The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

Efflux pump, the masked side of beta-lactam resistance in Klebsiella pneumoniae clinical isolates.

Directory of Open Access Journals (Sweden)

Jean-Marie Pages

Full Text Available BACKGROUND: Beta-lactamase production and porin decrease are the well-recognized mechanisms of acquired beta-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX and susceptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this beta-lactam phenotype was the aim of this study. METHODOLOGY/FINDINGS: MICs of 9 beta-lactams, including cloxacillin (CLX, and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI, then with both CLX (subinhibitory concentrations and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other beta-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also 16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other beta-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of beta-lactams. CONCLUSION: This is the first study demonstrating that efflux mechanism plays a key role in the beta-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in beta-lactam resistance is specially underestimated in clinical isolates.

Peptide mediators of cholesterol efflux

Energy Technology Data Exchange (ETDEWEB)

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.

How cholesterol interacts with proteins and lipids during its intracellular transport

DEFF Research Database (Denmark)

Wüstner, Daniel; Solanko, Katarzyna

2015-01-01

as well as by non-vesicular sterol exchange between organelles. In this article, we will review recent progress in elucidating sterol-lipid and sterol-protein interactions contributing to proper sterol transport in living cells. We outline recent biophysical models of cholesterol distribution and dynamics...... for characterization of sterol-protein interactions and for monitoring intracellular sterol transport. Finally, we review recent work on the molecular mechanisms underlying lipoprotein-mediated cholesterol import into mammalian cells and describe the process of cellular cholesterol efflux. Overall, we emphasize how......Sterols, as cholesterol in mammalian cells and ergosterol in fungi, are indispensable molecules for proper functioning and nanoscale organization of the plasma membrane. Synthesis, uptake and efflux of cholesterol are regulated by a variety of protein-lipid and protein-protein interactions...

Calcium efflux systems in stress signalling and adaptation in plants

Directory of Open Access Journals (Sweden)

Jayakumar eBose

2011-12-01

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

Divalent phosphate is a counterion for carboxyatractyloside-insensitive adenine nucleotide transport in rat liver mitochondria

International Nuclear Information System (INIS)

Nosek, M.T.; Aprille, J.R.

1986-01-01

Unidirectional, carboxyatractyloside(CAT)-insensitive adenine nucleotide (AdN) fluxes have been studied in isolated rat liver mitochondria (mito). Previous work has shown that ATP x Mg transport in one direction is coupled to ATP x Mg or P/sub i/ transport in the opposite direction. The purpose of this study was to determine whether divalent HPO 4 2- or monovalent H 2 PO 4 - is the transported phosphate species. The authors used the monofluorophosphate (PO 3 F 2- ) and difluorophosphate (PO 2 F 2 - ) analogues as potential counterions forAdN efflux. After a preincubation on ice with 14 C-ADP to label the matrix AdN, efflux was measured at 30 0 C, pH 7.4, in 225mM sucrose, 10mM KCl, 5mM MgCl 2 , 5mM glutamate, 5mM malate, 10mM Tris, 0.5mM P/sub i/, 1mM ATP, and 5μM CAT. With no other additions efflux was -0.62 +/- 0.20 nmole/minute/mg protein. The data supports the hypothesis that divalent but not monovalent phosphate can act as a counterion for ATPx Mg transport over this CAT-insensitive carrier

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

Science.gov (United States)

Branco, Patrícia; Albergaria, Helena; Arneborg, Nils; Prista, Catarina

2018-05-01

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

The role of nucleoside/nucleotide transport and metabolism in the uptake and retention of 3'-fluoro-3'-deoxythymidine in human B-lymphoblast cells

International Nuclear Information System (INIS)

Plotnik, David A.; McLaughlin, Lena J.; Chan, Jenny; Redmayne-Titley, Joshua N.; Schwartz, Jeffrey L.

2011-01-01

Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3'-fluoro-3'-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention. Methods: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished. Results: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer. Conclusions: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides.

Layers of Self- and Co-Regulation: Teachers Working Collaboratively to Support Adolescents' Self-Regulated Learning through Reading

Directory of Open Access Journals (Sweden)

Deborah L. Butler

2013-01-01

Full Text Available This paper reports findings from a longitudinal project in which secondary teachers were working collaboratively to support adolescents' self-regulated learning through reading (LTR in subject-area classrooms. We build from prior research to “connect the dots” between teachers' engagement in self- and co-regulated inquiry, associated shifts in classroom practice, and student self-regulation. More specifically, we investigated whether and how teachers working within a community of inquiry were mobilizing research to shape classroom practice and advance student learning. Drawing on evidence from 18 teachers and their respective classrooms, we describe findings related to the following research questions: (1 While engaged in self- and co-regulated inquiry, what types of practices did teachers enact to support LTR in their subject-area classrooms? (2 How did teachers draw on research-based resources to inform practice development? (3 What kinds of practices could be associated with gains in students' self-regulated LTR? In our discussion, we highlight contributions to understanding how teachers can be supported to situate research in authentic classroom environments and about qualities of practices supportive of students' self-regulated LTR. We also identify limitations of this work and important future directions.

Comparative effects of auxin and abscisic acid on growth, hydrogen ion efflux and gravitropism in primary roots of maize

Science.gov (United States)

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

1984-01-01

In order to test the idea that auxin action on root growth may be mediated by H(+) movement, the correlation of auxin action on growth and H(+) movement in roots was examined along with changes in H(+) efflux patterns associated with the asymmetric growth which occurs during gravitropism. The effects of indoleacetic acid (IAA) and abscisic acid (AbA) on growth, H(+) secretion, and gravitropism in roots were compared. Results show a close correlation existent between H(+) efflux and growth in maize roots. In intact roots there is strong H(+) efflux from the elongation zone. Growth-promoting concentrations of IAA stimulate H(+) efflux. During gravitropism the H(+) efflux from the elongation zone becomes asymmetric; the evidence indicates that auxin redistribution contributes to the development of acid efflux asymmetry. That AbA stimulates root growth is reflected in its ability to stimulate H(+) efflux from apical root segments.

Efflux pump-mediated benzalkonium chloride resistance in Listeria monocytogenes isolated from retail food.

Science.gov (United States)

Jiang, Xiaobing; Yu, Tao; Liang, Yu; Ji, Shengdong; Guo, Xiaowei; Ma, Jianmin; Zhou, Lijun

2016-01-18

In this study, efflux pump-mediated benzalkonium chloride (BC) resistance, including plasmid-encoded (Qac protein family and BcrABC) and chromosome-borne efflux pumps, was investigated in Listeria monocytogenes from retail food in China. Among the 59 L. monocytogenes strains, 13 (22.0%) strains were resistant to BC. The PCR results showed that bcrABC was harbored by 2 of 13 BC resistant strains. However, none of the qac genes were detected among the 59 strains. The bcrABC was absent in both of the plasmid cured strains, indicating that this BC resistance determinant was plasmid-encoded in the two bcrABC-positive strains. In the presence of reserpine, most of the bcrABC-negative strains had decreases in the MICs of BC, suggesting the existence of other efflux pumps and their role in BC resistance. After exposed to reserpine, the reduction in BC MICs was observed in the two cured strains, indicating that efflux pumps located on chromosome was also involved in BC resistance. Our findings suggest that food products may act as reservoirs for BC resistant isolates of L. monocytogenes and plasmid- and chromosome-encoded efflux pumps could mediate the BC resistance of L. monocytogenes, which is especially relevant to the adaption of this organism in food-related environments with frequent BC use. Copyright © 2015 Elsevier B.V. All rights reserved.

A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics

DEFF Research Database (Denmark)

Saaby, Lasse; Brodin, Birger

2017-01-01

Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties...... and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may...... mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate...

Evaluation of the tannic acid inhibitory effect against the NorA efflux pump of Staphylococcus aureus.

Science.gov (United States)

Tintino, Saulo R; Oliveira-Tintino, Cícera D M; Campina, Fábia F; Silva, Raimundo L P; Costa, Maria do S; Menezes, Irwin R A; Calixto-Júnior, João T; Siqueira-Junior, José P; Coutinho, Henrique D M; Leal-Balbino, Tereza C; Balbino, Valdir Q

2016-08-01

During the early periods of antibiotic usage, bacterial infections were considered tamed. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains. Active efflux is a mechanism for bacterial resistance to inhibitory substances, known simply as drug efflux pumps. The bacterium Staphylococcus aureus is an important pathogenic bacterium responsible for an array of infections. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus. The inhibition of the efflux pump was assayed using a sub-inhibitory concentration of standard efflux pump inhibitors and tannic acid (MIC/8), where its capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due to the possible inhibitory effect of these substances was observed. The MICs of EtBr and antibiotics were significantly reduced in the presence of tannic acid, indicating the inhibitory effect of this agent against the efflux pumps of both strains causing a three-fold reduction of the MIC when compared with the control. These results indicate the possible usage of tannic acid as an adjuvant in antibiotic therapy against multidrug resistant bacteria (MDR). Copyright © 2016 Elsevier Ltd. All rights reserved.

New Roads Leading to Old Destinations: Efflux Pumps as Targets to Reverse Multidrug Resistance in Bacteria

Directory of Open Access Journals (Sweden)

Gabriella Spengler

2017-03-01

Full Text Available Multidrug resistance (MDR has appeared in response to selective pressures resulting from the incorrect use of antibiotics and other antimicrobials. This inappropriate application and mismanagement of antibiotics have led to serious problems in the therapy of infectious diseases. Bacteria can develop resistance by various mechanisms and one of the most important factors resulting in MDR is efflux pump-mediated resistance. Because of the importance of the efflux-related multidrug resistance the development of new therapeutic approaches aiming to inhibit bacterial efflux pumps is a promising way to combat bacteria having over-expressed MDR efflux systems. The definition of an efflux pump inhibitor (EPI includes the ability to render the bacterium increasingly more sensitive to a given antibiotic or even reverse the multidrug resistant phenotype. In the recent years numerous EPIs have been developed, although so far their clinical application has not yet been achieved due to their in vivo toxicity and side effects. In this review, we aim to give a short overview of efflux mediated resistance in bacteria, EPI compounds of plant and synthetic origin, and the possible methods to investigate and screen EPI compounds in bacterial systems.

Sandwich-Cultured Hepatocytes for Mechanistic Understanding of Hepatic Disposition of Parent Drugs and Metabolites by Transporter-Enzyme Interplay.

Science.gov (United States)

Matsunaga, Norikazu; Fukuchi, Yukina; Imawaka, Haruo; Tamai, Ikumi

2018-05-01

Functional interplay between transporters and drug-metabolizing enzymes is currently one of the hottest topics in the field of drug metabolism and pharmacokinetics. Uptake transporter-enzyme interplay is important to determine intrinsic hepatic clearance based on the extended clearance concept. Enzyme and efflux transporter interplay, which includes both sinusoidal (basolateral) and canalicular efflux transporters, determines the fate of metabolites formed in the liver. As sandwich-cultured hepatocytes (SCHs) maintain metabolic activities and form a canalicular network, the whole interplay between uptake and efflux transporters and drug-metabolizing enzymes can be investigated simultaneously. In this article, we review the utility and applicability of SCHs for mechanistic understanding of hepatic disposition of both parent drugs and metabolites. In addition, the utility of SCHs for mimicking species-specific disposition of parent drugs and metabolites in vivo is described. We also review application of SCHs for clinically relevant prediction of drug-drug interactions caused by drugs and metabolites. The usefulness of mathematical modeling of hepatic disposition of parent drugs and metabolites in SCHs is described to allow a quantitative understanding of an event in vitro and to develop a more advanced model to predict in vivo disposition. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

The Impact of Diesel Oil Pollution on the Hydrophobicity and CO2 Efflux of Forest Soils.

Science.gov (United States)

Hewelke, Edyta; Szatyłowicz, Jan; Hewelke, Piotr; Gnatowski, Tomasz; Aghalarov, Rufat

2018-01-01

The contamination of soil with petroleum products is a major environmental problem. Petroleum products are common soil contaminants as a result of human activities, and they are causing substantial changes in the biological (particularly microbiological) processes, chemical composition, structure and physical properties of soil. The main objective of this study was to assess the impact of soil moisture on CO 2 efflux from diesel-contaminated albic podzol soils. Two contamination treatments (3000 and 9000 mg of diesel oil per kg of soil) were prepared for four horizons from two forest study sites with different initial levels of soil water repellency. CO 2 emissions were measured using a portable infrared gas analyser (LCpro+, ADC BioScientific, UK) while the soil samples were drying under laboratory conditions (from saturation to air-dry). The assessment of soil water repellency was performed using the water drop penetration time test. An analysis of variance (ANVOA) was conducted for the CO 2 efflux data. The obtained results show that CO 2 efflux from diesel-contaminated soils is higher than efflux from uncontaminated soils. The initially water-repellent soils were found to have a bigger CO 2 efflux. The non-linear relationship between soil moisture content and CO 2 efflux only existed for the upper soil horizons, while for deeper soil horizons, the efflux is practically independent of soil moisture content. The contamination of soil by diesel leads to increased soil water repellency.

Soil CO2 efflux among four coniferous forest types of Kashmir Himalaya, India.

Science.gov (United States)

Dar, Javid Ahmad; Ganie, Khursheed Ahmad; Sundarapandian, Somaiah

2015-11-01

Soil CO2 efflux was measured in four different coniferous forest types (Cedrus deodara (CD), Pinus wallichiana (PW), mixed coniferous (MC), and Abies pindrow (AP)) for a period of 2 years (April 2012 to December 2013). The monthly soil CO2 efflux ranged from 0.8 to 4.1 μmoles CO2 m(-2) s(-1) in 2012 and 1.01 to 5.48 μmoles CO2 m(-2) s(-1) in 2013. The soil CO2 efflux rate was highest in PW forest type in both the years, while it was lowest in MC and CD forest types during 2012 and 2013, respectively. Soil temperature (TS) at a depth of 10 cm ranged from 3.8 to 19.4 °C in 2012 and 3.5 to 19.1 °C in 2013 in all the four forest types. Soil moisture (MS) ranged from 19.8 to 58.6% in 2012 and 18.5 to 58.6% in 2013. Soil CO2 efflux rate was found to be significantly higher in summer than the other seasons and least during winter. Soil CO2 efflux showed a significant positive relationship with TS (R2=0.52 to 0.74), SOC% (R2=0.67), pH (R2=0.68), and shrub biomass (R2=0.51), whereas, only a weak positive relationship was found with soil moisture (R2=0.16 to 0.41), tree density (R2=0.25), tree basal area (R2=0.01), tree biomass (R2=0.07), herb biomass (R2=0.01), and forest floor litter (R2=0.02). Thus, the study indicates that soil CO2 efflux in high mountainous areas is greatly influenced by seasons, soil temperature, and other environmental factors.

Intestinal Transport Characteristics and Metabolism of C-Glucosyl Dihydrochalcone, Aspalathin

Directory of Open Access Journals (Sweden)

Sandra Bowles

2017-03-01

Full Text Available Insight into the mechanisms of intestinal transport and metabolism of aspalathin will provide important information for dose optimisation, in particular for studies using mouse models. Aspalathin transportation across the intestinal barrier (Caco-2 monolayer tested at 1–150 µM had an apparent rate of permeability (Papp typical of poorly absorbed compounds (1.73 × 10−6 cm/s. Major glucose transporters, sodium glucose linked transporter 1 (SGLT1 and glucose transporter 2 (GLUT2, and efflux protein (P-glycoprotein, PgP (1.84 × 10−6 cm/s; efflux ratio: 1.1 were excluded as primary transporters, since the Papp of aspalathin was not affected by the presence of specific inhibitors. The Papp of aspalathin was also not affected by constituents of aspalathin-enriched rooibos extracts, but was affected by high glucose concentration (20.5 mM, which decreased the Papp value to 2.9 × 10−7 cm/s. Aspalathin metabolites (sulphated, glucuronidated and methylated were found in mouse urine, but not in blood, following an oral dose of 50 mg/kg body weight of the pure compound. Sulphates were the predominant metabolites. These findings suggest that aspalathin is absorbed and metabolised in mice to mostly sulphate conjugates detected in urine. Mechanistically, we showed that aspalathin is not actively transported by the glucose transporters, but presumably passes the monolayer paracellularly.

Intestinal Transport Characteristics and Metabolism of C-Glucosyl Dihydrochalcone, Aspalathin.

Science.gov (United States)

Bowles, Sandra; Joubert, Elizabeth; de Beer, Dalene; Louw, Johan; Brunschwig, Christel; Njoroge, Mathew; Lawrence, Nina; Wiesner, Lubbe; Chibale, Kelly; Muller, Christo

2017-03-30

Insight into the mechanisms of intestinal transport and metabolism of aspalathin will provide important information for dose optimisation, in particular for studies using mouse models. Aspalathin transportation across the intestinal barrier (Caco-2 monolayer) tested at 1-150 µM had an apparent rate of permeability (P app ) typical of poorly absorbed compounds (1.73 × 10 -6 cm/s). Major glucose transporters, sodium glucose linked transporter 1 (SGLT1) and glucose transporter 2 (GLUT2), and efflux protein (P-glycoprotein, PgP) (1.84 × 10 -6 cm/s; efflux ratio: 1.1) were excluded as primary transporters, since the P app of aspalathin was not affected by the presence of specific inhibitors. The P app of aspalathin was also not affected by constituents of aspalathin-enriched rooibos extracts, but was affected by high glucose concentration (20.5 mM), which decreased the P app value to 2.9 × 10 -7 cm/s. Aspalathin metabolites (sulphated, glucuronidated and methylated) were found in mouse urine, but not in blood, following an oral dose of 50 mg/kg body weight of the pure compound. Sulphates were the predominant metabolites. These findings suggest that aspalathin is absorbed and metabolised in mice to mostly sulphate conjugates detected in urine. Mechanistically, we showed that aspalathin is not actively transported by the glucose transporters, but presumably passes the monolayer paracellularly.

Cerebellar modulation of frontal cortex dopamine efflux in mice: relevance to autism and schizophrenia.

Science.gov (United States)

Mittleman, Guy; Goldowitz, Daniel; Heck, Detlef H; Blaha, Charles D

2008-07-01

Cerebellar and frontal cortical pathologies have been commonly reported in schizophrenia, autism, and other developmental disorders. Whether there is a relationship between prefrontal and cerebellar pathologies is unknown. Using fixed potential amperometry, dopamine (DA) efflux evoked by cerebellar or, dentate nucleus electrical stimulation (50 Hz, 200 muA) was recorded in prefrontal cortex of urethane anesthetized lurcher (Lc/+) mice with 100% loss of cerebellar Purkinje cells and wildtype (+/+) control mice. Cerebellar stimulation with 25 and 100 pulses evoked prefrontal cortex DA efflux in +/+ mice that persisted for 12 and 25 s poststimulation, respectively. In contrast, 25 pulse cerebellar stimulation failed to evoke prefrontal cortex DA efflux in Lc/+ mice indicating a dependency on cerebellar Purkinje cell outputs. Dentate nucleus stimulation (25 pulses) evoked a comparable but briefer (baseline recovery within 7 s) increase in prefrontal cortex DA efflux compared to similar cerebellar stimulation in +/+ mice. However, in Lc/+ mice 25 pulse dentate nucleus evoked prefrontal cortex DA efflux was attenuated by 60% with baseline recovery within 4 s suggesting that dentate nucleus outputs to prefrontal cortex remain partially functional. DA reuptake blockade enhanced 100 pulse stimulation evoked prefrontal cortex responses, while serotonin or norepinephrine reuptake blockade were without effect indicating the specificity of the amperometric recordings to DA. Results provide neurochemical evidence that the cerebellum can modulate DA efflux in the prefrontal cortex. Together, these findings may explain why cerebellar and frontal cortical pathologies co-occur, and may provide a mechanism that accounts for the diversity of symptoms common to multiple developmental disorders.

Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

Science.gov (United States)

Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

2011-01-25

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11

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

International Nuclear Information System (INIS)

Quast, U.; Cook, N.S.

1988-01-01

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

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

International Nuclear Information System (INIS)

Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.; Alarcon, Jose C.; Fariña, Macarena A.; Amigo, Romina F.; Muñoz-Godoy, Natalia A.; Pinilla, Mabel G.; Peña, Eduardo A.; Gonzalez-Chavarria, Ivan; Toledo, Jorge R.; Rivas, Coralia I.; Vera, Juan C.; McNerney, Eileen M.; Onate, Sergio A.

2015-01-01

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co

Bone stroma-derived cells change coregulators recruitment to androgen receptor and decrease cell proliferation in androgen-sensitive and castration-resistant prostate cancer cells

Energy Technology Data Exchange (ETDEWEB)

Villagran, Marcelo A.; Gutierrez-Castro, Francisco A.; Pantoja, Diego F.; Alarcon, Jose C.; Fariña, Macarena A.; Amigo, Romina F.; Muñoz-Godoy, Natalia A. [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Pinilla, Mabel G. [Department of Medical Specialties, School of Medicine, University of Concepcion, Concepcion (Chile); Peña, Eduardo A.; Gonzalez-Chavarria, Ivan; Toledo, Jorge R.; Rivas, Coralia I.; Vera, Juan C. [Department of Physiopathology, School of Biological Sciences, University of Concepcion, Concepcion (Chile); McNerney, Eileen M. [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Onate, Sergio A., E-mail: sergio.onate@udec.cl [Molecular Endocrinology and Oncology Laboratory, University of Concepcion, Concepcion (Chile); Department of Medical Specialties, School of Medicine, University of Concepcion, Concepcion (Chile); Department of Urology, State University of New York at Buffalo, NY (United States)

2015-11-27

Prostate cancer (CaP) bone metastasis is an early event that remains inactive until later-stage progression. Reduced levels of circulating androgens, due to andropause or androgen deprivation therapies, alter androgen receptor (AR) coactivator expression. Coactivators shift the balance towards enhanced AR-mediated gene transcription that promotes progression to androgen-resistance. Disruptions in coregulators may represent a molecular switch that reactivates latent bone metastasis. Changes in AR-mediated transcription in androgen-sensitive LNCaP and androgen-resistant C4-2 cells were analyzed for AR coregulator recruitment in co-culture with Saos-2 and THP-1. The Saos-2 cell line derived from human osteosarcoma and THP-1 cell line representing human monocytes were used to display osteoblast and osteoclast activity. Increased AR activity in androgen-resistant C4-2 was due to increased AR expression and SRC1/TIF2 recruitment and decreased SMRT/NCoR expression. AR activity in both cell types was decreased over 90% when co-cultured with Saos-2 or THP-1 due to dissociation of AR from the SRC1/TIF2 and SMRT/NCoR coregulators complex, in a ligand-dependent and cell-type specific manner. In the absence of androgens, Saos-2 decreased while THP-1 increased proliferation of LNCaP cells. In contrast, both Saos-2 and THP-1 decreased proliferation of C4-2 in absence and presence of androgens. Global changes in gene expression from both CaP cell lines identified potential cell cycle and androgen regulated genes as mechanisms for changes in cell proliferation and AR-mediated transactivation in the context of bone marrow stroma cells. - Highlights: • Decreased corepressor expression change AR in androgen-resistance prostate cancer. • Bone stroma-derived cells change AR coregulator recruitment in prostate cancer. • Bone stroma cells change cell proliferation in androgen-resistant cancer cells. • Global gene expression in CaP cells is modified by bone stroma cells in co

[The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii].

Science.gov (United States)

Abramycheva, N Iu; Govorun, V M

2000-01-01

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.

The role of nucleoside/nucleotide transport and metabolism in the uptake and retention of 3'-fluoro-3'-deoxythymidine in human B-lymphoblast cells

Energy Technology Data Exchange (ETDEWEB)

Plotnik, David A.; McLaughlin, Lena J.; Chan, Jenny; Redmayne-Titley, Joshua N.; Schwartz, Jeffrey L., E-mail: jschwart@uw.edu

2011-10-15

Introduction: Recent studies in the human adenocarcinoma cell line A549 have identified cell growth-dependent equilibrative nucleoside transporter-1 (hENT1) as a modifier of 3'-fluoro-3'-deoxythymidine (FLT) uptake and retention. In the present study, we used the ability to isolate human lymphoblastoid clones deficient in thymidine kinase 1 (TK1) to study how metabolism and nucleoside transport influence FLT uptake and retention. Methods: Transport and metabolism of FLT were measured in the human lymphoblastoid cell line TK6 and in eight clones isolated from TK6. Four clones were TK1-proficient, while four were TK1-deficient. Both influx and efflux of FLT were measured under conditions where concentrative and equilibrative transport could be distinguished. Results: Sodium-dependent concentrative FLT transport dominated over equilibrative transport mechanisms and while inhibition of hENT1 reduced FLT uptake, there were no correlations between clonal variations in hENT1 levels and FLT uptake. There was an absolute requirement of TK1 for concentration of FLT in TK6 cells. FLT uptake reached a peak after 60 min of incubation with FLT after which intracellular levels of FLT and FLT metabolites declined. Efflux was rapid and was associated with reductions in FLT and each of its metabolites. Both FLT and FLT-monophosphate were found in the efflux buffer. Conclusions: Initial rates of FLT uptake were a function of both concentrative and equilibrative transporters. TK1 activity was an absolute requirement for the accumulation of FLT. Retention was dependent on nucleoside/nucleotide efflux and retrograde metabolism of FLT nucleotides.

Gadoxetate-enhanced MR imaging and compartmental modelling to assess hepatocyte bidirectional transport function in rats with advanced liver fibrosis

Energy Technology Data Exchange (ETDEWEB)

Giraudeau, Celine; Leporq, Benjamin; Doblas, Sabrina [University Paris Diderot, Sorbonne Paris Cite, Hopital Beaujon, Laboratory of Imaging Biomarkers, UMR1149 Inserm, Clichy (France); Lagadec, Matthieu; Daire, Jean-Luc; Van Beers, Bernard E. [University Paris Diderot, Sorbonne Paris Cite, Hopital Beaujon, Laboratory of Imaging Biomarkers, UMR1149 Inserm, Clichy (France); Beaujon University Hospital Paris Nord, Department of Radiology, Clichy (France); Pastor, Catherine M. [University Paris Diderot, Sorbonne Paris Cite, Hopital Beaujon, Laboratory of Imaging Biomarkers, UMR1149 Inserm, Clichy (France); Hopitaux Universitaires de Geneve, Departement d' Imagerie et des Sciences de l' Information Medicale, Geneva (Switzerland)

2017-05-15

Changes in the expression of hepatocyte membrane transporters in advanced fibrosis decrease the hepatic transport function of organic anions. The aim of our study was to assess if these changes can be evaluated with pharmacokinetic analysis of the hepatobiliary transport of the MR contrast agent gadoxetate. Dynamic gadoxetate-enhanced MRI was performed in 17 rats with advanced fibrosis and 8 normal rats. After deconvolution, hepatocyte three-compartmental analysis was performed to calculate the hepatocyte influx, biliary efflux and sinusoidal backflux rates. The expression of Oatp1a1, Mrp2 and Mrp3 organic anion membrane transporters was assessed with reverse transcription polymerase chain reaction. In the rats with advanced fibrosis, the influx and efflux rates of gadoxetate decreased and the backflux rate increased significantly (p = 0.003, 0.041 and 0.010, respectively). Significant correlations were found between influx and Oatp1a1 expression (r = 0.78, p < 0.001), biliary efflux and Mrp2 (r = 0.50, p = 0.016) and sinusoidal backflux and Mrp3 (r = 0.61, p = 0.002). These results show that changes in the bidirectional organic anion hepatocyte transport function in rats with advanced liver fibrosis can be assessed with compartmental analysis of gadoxetate-enhanced MRI. (orig.)

Gadoxetate-enhanced MR imaging and compartmental modelling to assess hepatocyte bidirectional transport function in rats with advanced liver fibrosis

International Nuclear Information System (INIS)

Giraudeau, Celine; Leporq, Benjamin; Doblas, Sabrina; Lagadec, Matthieu; Daire, Jean-Luc; Van Beers, Bernard E.; Pastor, Catherine M.

2017-01-01

Changes in the expression of hepatocyte membrane transporters in advanced fibrosis decrease the hepatic transport function of organic anions. The aim of our study was to assess if these changes can be evaluated with pharmacokinetic analysis of the hepatobiliary transport of the MR contrast agent gadoxetate. Dynamic gadoxetate-enhanced MRI was performed in 17 rats with advanced fibrosis and 8 normal rats. After deconvolution, hepatocyte three-compartmental analysis was performed to calculate the hepatocyte influx, biliary efflux and sinusoidal backflux rates. The expression of Oatp1a1, Mrp2 and Mrp3 organic anion membrane transporters was assessed with reverse transcription polymerase chain reaction. In the rats with advanced fibrosis, the influx and efflux rates of gadoxetate decreased and the backflux rate increased significantly (p = 0.003, 0.041 and 0.010, respectively). Significant correlations were found between influx and Oatp1a1 expression (r = 0.78, p < 0.001), biliary efflux and Mrp2 (r = 0.50, p = 0.016) and sinusoidal backflux and Mrp3 (r = 0.61, p = 0.002). These results show that changes in the bidirectional organic anion hepatocyte transport function in rats with advanced liver fibrosis can be assessed with compartmental analysis of gadoxetate-enhanced MRI. (orig.)

Stem compression reversibly reduces phloem transport in Pinus sylvestris trees.

Science.gov (United States)

Henriksson, Nils; Tarvainen, Lasse; Lim, Hyungwoo; Tor-Ngern, Pantana; Palmroth, Sari; Oren, Ram; Marshall, John; Näsholm, Torgny

2015-10-01

Manipulating tree belowground carbon (C) transport enables investigation of the ecological and physiological roles of tree roots and their associated mycorrhizal fungi, as well as a range of other soil organisms and processes. Girdling remains the most reliable method for manipulating this flux and it has been used in numerous studies. However, girdling is destructive and irreversible. Belowground C transport is mediated by phloem tissue, pressurized through the high osmotic potential resulting from its high content of soluble sugars. We speculated that phloem transport may be reversibly blocked through the application of an external pressure on tree stems. Thus, we here introduce a technique based on compression of the phloem, which interrupts belowground flow of assimilates, but allows trees to recover when the external pressure is removed. Metal clamps were wrapped around the stems and tightened to achieve a pressure theoretically sufficient to collapse the phloem tissue, thereby aiming to block transport. The compression's performance was tested in two field experiments: a (13)C canopy labelling study conducted on small Scots pine (Pinus sylvestris L.) trees [2-3 m tall, 3-7 cm diameter at breast height (DBH)] and a larger study involving mature pines (∼15 m tall, 15-25 cm DBH) where stem respiration, phloem and root carbohydrate contents, and soil CO2 efflux were measured. The compression's effectiveness was demonstrated by the successful blockage of (13)C transport. Stem compression doubled stem respiration above treatment, reduced soil CO2 efflux by 34% and reduced phloem sucrose content by 50% compared with control trees. Stem respiration and soil CO2 efflux returned to normal within 3 weeks after pressure release, and (13)C labelling revealed recovery of phloem function the following year. Thus, we show that belowground phloem C transport can be reduced by compression, and we also demonstrate that trees recover after treatment, resuming C

Macrophage cholesterol efflux correlates with lipoprotein subclass distribution and risk of obstructive coronary artery disease in patients undergoing coronary angiography

Directory of Open Access Journals (Sweden)

Kremer Werner

2009-04-01

Full Text Available Abstract Background Studies in patients with low HDL have suggested that impaired cellular cholesterol efflux is a heritable phenotype increasing atherosclerosis risk. Less is known about the association of macrophage cholesterol efflux with lipid profiles and CAD risk in normolipidemic subjects. We have therefore measured macrophage cholesterol efflux in142 normolipidemic subjects undergoing coronary angiography. Methods Monocytes isolated from blood samples of patients scheduled for cardiac catheterization were differentiated into macrophages over seven days. Isotopic cholesterol efflux to exogenously added apolipoprotein A-I and HDL2 was measured. Quantitative cholesterol efflux from macrophages was correlated with lipoprotein subclass distribution in plasma from the same individuals measured by NMR-spectroscopy of lipids and with the extent of coronary artery disease seen on coronary angiography. Results Macrophage cholesterol efflux was positively correlated with particle concentration of smaller HDL and LDL particles but not with total plasma concentrations of HDL or LDL-cholesterol. We observed an inverse relationship between macrophage cholesterol efflux and the concntration of larger and triglyceride rich particles (VLDL, chylomicrons. Subjects with significant stenosis on coronary angiography had lower cholesterol efflux from macrophages compared to individuals without significant stenosis (adjusted p = 0.02. Conclusion Macrophage cholesterol efflux is inversely correlated with lipoprotein particle size and risk of CAD.

Dopamine inhibits maitotoxin-stimulated pituitary 45Ca2+ efflux and prolactin release

International Nuclear Information System (INIS)

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

1986-01-01

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

Effect of shear stress on 86Rb+ efflux and cytosolic Ca2+ of calf pulmonary artery endothelial cells (CPAEs)

International Nuclear Information System (INIS)

Alevriadou, B.R.; Mo, M.; Rickman, D.S.; Eskin, S.G.; McIntire, L.V.; Schilling, W.P.

1991-01-01

The effect of flow-induced shear stress (SS) on membrane K + permeability and cytosolic free Ca 2+ , [Ca 2+ ] i , was investigated by measuring 86 Rb + efflux and fura-2 fluorescence in CPAEs using a parallel plate flow chamber. Increasing SS from 1 to 2.4, 4.8 or 10 dyn/cm 2 produced a graded, transient increase in 86 Rb + efflux which peaked within 1 min and subsequently declined rapidly towards pre-stimulus levels. Mathematical modeling confirmed that the transient increase in 86 Rb + efflux did not reflect a washout phenomenon. Upon returning SS to 1 dyn/cm 2 , 86 Rb + efflux initially decreased, but returned slowly to basal values. In contrast, application of bradykinin (BK) at a constant SS of either 0.33 or 1 dyn/cm 2 produced a transient increase in 86 Rb + efflux that was followed by a sustained elevated phase during which time efflux gradually returned to pre-stimulus levels. To determine the mechanism by which shear stress increases K + permeability, the effect of tetrabutylammonium ion (TBA), a selective inhibitor of Ca 2+ -dependent K + channels (K Ca ), on both the BK- and SS-induced increases in 86 Rb + efflux, was examined. TBA inhibited the BK-stimulated increase in 86 Rb + efflux >90% under both stationary and flow conditions and significantly reduced SS-dependent 86 Rb + efflux 38.3%. These results suggest that increased 86 Rb + efflux from CPAEs with SS occurs, at least in part, via K Ca and suggests that SS increases cytosolic Ca 2+ . However, when measured using fura-2-loaded CPAEs, SS was without significant effect on [Ca 2+ ] i

Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release

DEFF Research Database (Denmark)

Rickhag, Karl Mattias; Owens, WA; Winkler, Marie-Therese

2013-01-01

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate......-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects....

MDR1 P-glycoprotein transports endogenous opioid peptides

NARCIS (Netherlands)

Oude Elferink, R. P.; Zadina, J.

2001-01-01

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore

Evolutionarily Conserved Roles for Blood-Brain Barrier Xenobiotic Transporters in Endogenous Steroid Partitioning and Behavior

Directory of Open Access Journals (Sweden)

Samantha J. Hindle

2017-10-01

Full Text Available Summary: Central nervous system (CNS chemical protection depends upon discrete control of small-molecule access by the blood-brain barrier (BBB. Curiously, some drugs cause CNS side-effects despite negligible transit past the BBB. To investigate this phenomenon, we asked whether the highly BBB-enriched drug efflux transporter MDR1 has dual functions in controlling drug and endogenous molecule CNS homeostasis. If this is true, then brain-impermeable drugs could induce behavioral changes by affecting brain levels of endogenous molecules. Using computational, genetic, and pharmacologic approaches across diverse organisms, we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice. Furthermore, we show that MDR1-interacting drugs are associated with anxiety-related behaviors in humans. We propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates and implicates the BBB in behavioral regulation. : Hindle et al. shed light on the curious finding that some drugs cause behavioral side-effects despite negligible access into the brain. These authors propose a unifying hypothesis that links blood-brain barrier drug transporter function and brain access of circulating steroids to common CNS adverse drug responses. Keywords: drug side effect mechanisms, central nervous system, blood brain barrier, behavior, toxicology, drug transporters, endobiotics, steroid hormones

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.

Directory of Open Access Journals (Sweden)

Qian Liu

Full Text Available 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.

Novel structural analogues of piperine as inhibitors of the NorA efflux pump of Staphylococcus aureus

DEFF Research Database (Denmark)

Kumar, Ashwani; Khan, Inshad Ali; Koul, Surrinder

2008-01-01

OBJECTIVES: Evaluation of novel synthetic analogues of piperine as inhibitors of multidrug efflux pump NorA of Staphylococcus aureus. METHODS: A library of piperine-derived compounds was evaluated for their potential to inhibit ethidium bromide efflux in NorA-overexpressing S. aureus SA 1199B...... inhibitors of the NorA efflux pump. These inhibitors acted in a synergistic manner with ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. These analogues were 2- to 4-fold more potent than piperine at a significantly lower minimal...... of ciprofloxacin through the inhibition of the NorA efflux pump. These molecules may prove useful in augmenting the antibacterial activities of fluoroquinolones in a clinical setting....

Response to Bile Salts in Clinical Strains of Acinetobacter baumannii Lacking the AdeABC Efflux Pump: Virulence Associated with Quorum Sensing

Directory of Open Access Journals (Sweden)

Maria López

2017-05-01

Full Text Available Introduction:Acinetobacter baumannii is an opportunistic nosocomial pathogen associated with multiple infections. This pathogen usually colonizes (first stage of microbial infection host tissues that are in contact with the external environment. As one of the sites of entry in human hosts is the gastrointestinal tract, the pathogen must be capable of tolerating bile salts. However, studies analyzing the molecular characteristics involved in the response to bile salts in clinical strains of A. baumannii are scarce.Material and Methods: Microbiological and transcriptional studies (arrays and RT-PCR in the response to bile salts were carried out in isogenic (A. baumanni ΔadeB ATCC 17978 and A. baumannii ΔadeL ATCC 17978 and clinical strains from clone ST79/PFGE-HUI-1 which is characterized by lacking the AdeABC efflux pump and by overexpression the AdeFGH efflux pump.Results and Discussion: In presence of bile salts, in addition to the glutamate/aspartate transporter were found overexpressed in A. baumannii ΔadeB ATCC 17978, the virulence factors (surface motility, biofilm, and Type VI Secretion System which are associated with activation of the Quorum Sensing system. Overexpression of these factors was confirmed in clinical strains of clone ST79/PFGE-HUI-1.Conclusions: This the first study about the adaptive response to bile salts investigating the molecular and microbiological characteristics in response to bile salts of an isogenic model of A. baumannii ATCC 17978 and clinical isolates of A. baumannii (clinical strains of ST79/PFGE-HUI-1 lacking the main RND efflux pump (AdeABC. Clinical isolates of A. baumannii lacking the AdeABC efflux pump (clone ST79/PFGE-HUI-1 displayed a new clinical profile (increased invasiveness possibly associated with the response to stress conditions (such as the presence of bile salts.

Response to Bile Salts in Clinical Strains of Acinetobacter baumannii Lacking the AdeABC Efflux Pump: Virulence Associated with Quorum Sensing.

Science.gov (United States)

López, Maria; Blasco, Lucia; Gato, Eva; Perez, Astrid; Fernández-Garcia, Laura; Martínez-Martinez, Luis; Fernández-Cuenca, Felipe; Rodríguez-Baño, Jesús; Pascual, Alvaro; Bou, German; Tomás, Maria

2017-01-01

Introduction: Acinetobacter baumannii is an opportunistic nosocomial pathogen associated with multiple infections. This pathogen usually colonizes (first stage of microbial infection) host tissues that are in contact with the external environment. As one of the sites of entry in human hosts is the gastrointestinal tract, the pathogen must be capable of tolerating bile salts. However, studies analyzing the molecular characteristics involved in the response to bile salts in clinical strains of A. baumannii are scarce. Material and Methods: Microbiological and transcriptional studies (arrays and RT-PCR) in the response to bile salts were carried out in isogenic ( A. baumanni Δ adeB ATCC 17978 and A. baumannii Δ adeL ATCC 17978) and clinical strains from clone ST79/PFGE-HUI-1 which is characterized by lacking the AdeABC efflux pump and by overexpression the AdeFGH efflux pump. Results and Discussion: In presence of bile salts, in addition to the glutamate/aspartate transporter were found overexpressed in A. baumannii Δ adeB ATCC 17978, the virulence factors (surface motility, biofilm, and Type VI Secretion System) which are associated with activation of the Quorum Sensing system. Overexpression of these factors was confirmed in clinical strains of clone ST79/PFGE-HUI-1. Conclusions: This the first study about the adaptive response to bile salts investigating the molecular and microbiological characteristics in response to bile salts of an isogenic model of A. baumannii ATCC 17978 and clinical isolates of A. baumannii (clinical strains of ST79/PFGE-HUI-1) lacking the main RND efflux pump (AdeABC). Clinical isolates of A. baumannii lacking the AdeABC efflux pump (clone ST79/PFGE-HUI-1) displayed a new clinical profile (increased invasiveness) possibly associated with the response to stress conditions (such as the presence of bile salts).

Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters.

Science.gov (United States)

Lebedeva, Irina V; Pande, Praveen; Patton, Wayne F

2011-01-01

An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC(2)(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments.

Chloride transport in human fibroblasts is activated by hypotonic shock

Energy Technology Data Exchange (ETDEWEB)

Rugolo, M.; Mastocola, T.; Flamigni, A.; Lenaz, G. (Universita' di Bologna (Italy))

1989-05-15

Incubation of human skin fibroblasts in hypotonic media induced the activation of {sup 36}Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of {sup 36}Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also {sup 36}Cl- influx was enhanced by hypotonic medium.

Development of novel, 384-well high-throughput assay panels for human drug transporters: drug interaction and safety assessment in support of discovery research.

Science.gov (United States)

Tang, Huaping; Shen, Ding Ren; Han, Yong-Hae; Kong, Yan; Balimane, Praveen; Marino, Anthony; Gao, Mian; Wu, Sophie; Xie, Dianlin; Soars, Matthew G; O'Connell, Jonathan C; Rodrigues, A David; Zhang, Litao; Cvijic, Mary Ellen

2013-10-01

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.

Old carbon efflux from tropical peat swamp drainage waters

Science.gov (United States)

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

2014-05-01

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

The lactococcal secondary multidrug transporter LmrP confers resistance to lincosamides, macrolides, streptogramins and tetracyclines

NARCIS (Netherlands)

Putman, M; van Veen, HW; Degener, JE; Konings, WN

2001-01-01

The active efflux of toxic compounds by (multi)drug transporters is one of the mechanisms that bacteria have developed to resist cytotoxic drugs. The authors describe the role of the lactococcal secondary multidrug transporter LmrP in the resistance to a broad range of clinically important

Interactions between organic anions on multiple transporters in Caco-2 cells

DEFF Research Database (Denmark)

Grandvuinet, Anne Sophie; Steffansen, Bente

2011-01-01

Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E1S) were used as model substrates. Possible inhibitors studied were TCA, E1S, taurolithocholic acid, fluvastatin, and glipizide......-dependent bile acid transporter and the organic solute transporter α/β, and to less extent by the organic anion transporting polypeptide 2B1. However, interactions on efflux transporters were not detected, although they were expected from the literature on the investigated compounds. Biosimulation methods may...

Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi

DEFF Research Database (Denmark)

Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar

2015-01-01

Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim...... of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug......-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874...

Effects of L-arabinose efflux on λ Red recombination-mediated gene knockout in multiple-antimicrobial-resistant Salmonella enterica serovar Choleraesuis.

Science.gov (United States)

Liao, Shi-Wei; Lee, Jen-Jie; Ptak, Christopher P; Wu, Ying-Chen; Hsuan, Shih-Ling; Kuo, Chih-Jung; Chen, Ter-Hsin

2018-03-01

In this study, six swine-derived multiple-antimicrobial-resistant (MAR) strains of Salmonella Choleraesuis (S. Choleraesuis) were demonstrated to possess higher efflux pump activity than the wild-type (WT). L-Arabinose, a common inducer for gene expression, modulated S. Choleraesuis efflux pump activity in a dose-dependent manner. At low L-arabinose concentrations, increasing L-arabinose led to a corresponding increase in fluorophore efflux, while at higher L-arabinose concentrations, increasing L-arabinose decreased fluorophore efflux activity. The WT S. Choleraesuis that lacks TolC (ΔtolC), an efflux protein associated with bacterial antibiotic resistance and virulence, was demonstrated to possess a significantly reduced ability to extrude L-arabinose. Further, due to the rapid export of L-arabinose, an efficient method for recombination-mediated gene knockout, the L-arabinose-inducible bacteriophage λ Red recombinase system, has a reduced recombination frequency (~ 12.5%) in clinically isolated MAR Salmonella strains. An increased recombination frequency (up to 60%) can be achieved using a higher concentration of L-arabinose (fivefold) for genetic manipulation and functional analysis for MAR Salmonella using the λ Red system. The study suggests that L-arabinose serves not only as an inducer of the TolC-dependent efflux system but also acts as a competitive substrate of the efflux system. In addition, understanding the TolC-dependent efflux of L-arabinose should facilitate the optimization of L-arabinose induction in strains with high efflux activity.

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

Directory of Open Access Journals (Sweden)

Momen Askoura

2011-05-01

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

Total HDL cholesterol efflux capacity in healthy children - Associations with adiposity and dietary intakes of mother and child.

Science.gov (United States)

Khalil, H; Murrin, C; O'Reilly, M; Viljoen, K; Segurado, R; O'Brien, J; Somerville, R; McGillicuddy, F; Kelleher, C C

2017-01-01

High-density lipoprotein (HDL) cholesterol efflux capacity in adults may be a measure of the atheroprotective property of HDL. Little however, is known about HDL cholesterol efflux capacity in childhood. We aimed to investigate the relationship between HDL cholesterol efflux capacity and childhood anthropometrics in a longitudinal study. Seventy-five children (mean age = 9.4 ± 0.4 years) were followed from birth until the age of 9 years. HDL cholesterol efflux capacity was determined at age 9 by incubating serum-derived HDL-supernatants with 3 H-cholesterol labeled J774 macrophages and percentage efflux determined. Mothers provided dietary information by completing food frequency questionnaires in early pregnancy and then 5 years later on behalf of themselves and their children. Pearson's correlations and multiple regression analyses were conducted to confirm independent associations with HDL efflux. There was a negative correlation between HDL cholesterol efflux capacity and waist circumference at age 5 (r = -0.3, p = 0.01) and age 9 (r = -0.24, p = 0.04) and BMI at age 5 (r = -0.45, p = 0.01) and age 9 (r = -0.19, p = 0.1). Multiple regression analysis showed that BMI at age 5 remained significantly associated with reduced HDL cholesterol efflux capacity (r = -0.45, p < 0.001). HDL-C was negatively correlated with energy-adjusted fat intake (r = -0.24, p = 0.04) and positively correlated with energy-adjusted protein (r = 0.24, p = 0.04) and starch (r = 0.29, p = 0.01) intakes during pregnancy. HDL-C was not significantly correlated with children dietary intake at age 5. There were no significant correlations between maternal or children dietary intake and HDL cholesterol efflux capacity. This novel analysis shows that efflux capacity is negatively associated with adiposity in early childhood independent of HDL-C. Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the

CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation.

Science.gov (United States)

Tang, Tiantian; Lang, Xueting; Xu, Congfei; Wang, Xiaqiong; Gong, Tao; Yang, Yanqing; Cui, Jun; Bai, Li; Wang, Jun; Jiang, Wei; Zhou, Rongbin

2017-08-04

The NLRP3 inflammasome can sense different pathogens or danger signals, and has been reported to be involved in the development of many human diseases. Potassium efflux and mitochondrial damage are both reported to mediate NLRP3 inflammasome activation, but the underlying, orchestrating signaling events are still unclear. Here we show that chloride intracellular channels (CLIC) act downstream of the potassium efflux-mitochondrial reactive oxygen species (ROS) axis to promote NLRP3 inflammasome activation. NLRP3 agonists induce potassium efflux, which causes mitochondrial damage and ROS production. Mitochondrial ROS then induces the translocation of CLICs to the plasma membrane for the induction of chloride efflux to promote NEK7-NLRP3 interaction, inflammasome assembly, caspase-1 activation, and IL-1β secretion. Thus, our results identify CLICs-dependent chloride efflux as an essential and proximal upstream event for NLRP3 activation.The NLRP3 inflammasome is key to the regulation of innate immunity against pathogens or stress, but the underlying signaling regulation is still unclear. Here the authors show that chloride intracellular channels (CLIC) interface between mitochondria stress and inflammasome activation to modulate inflammatory responses.

Virulence and the Environment: a Novel Role for Vibrio cholerae Toxin-Coregulated Pili in Biofilm Formation on Chitin

Science.gov (United States)

Reguera, Gemma; Kolter, Roberto

2005-01-01

The toxin-coregulated pilus (TCP) of Vibrio cholerae is required for intestinal colonization and cholera toxin acquisition. Here we report that TCP mediates bacterial interactions required for biofilm differentiation on chitinaceous surfaces. We also show that undifferentiated TCP− biofilms have reduced ecological fitness and, thus, that chitin colonization may represent an ecological setting outside the host in which selection for a host colonization factor may take place. PMID:15866944

Switch loop flexibility affects substrate transport of the AcrB efflux pump

International Nuclear Information System (INIS)

Muller, Reinke T.; Travers, Timothy; Cha, Hi-jea; Phillips, Joshua L.

2017-01-01

The functionally important switch-loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11 amino acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1 proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway towards the deep binding pocket. Here, two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.

Studies on alterations of the 86-rubidium efflux from rat pancreatic islets caused by thiol and thiol oxidants

International Nuclear Information System (INIS)

Wahl, M.A.

1983-01-01

The following findings were revealed by this study: 1) Oxidation-reduction (redox) of the intracellular system of glutathione influences the potassium efflux by way of an increase in the 86-rubidium efflux brought about by the oxidation of intracellular thiols. 2) The 86-rubidium efflux is not subject to change by oxidation of extracellular thiols located in the membrane, nor can it in any way be influenced by reduced glutathione of exogenous origin. 3) The potassium efflux from rat pancreatic islets, being generally known to trigger the electric activities of the beta-cell, is controlled by the oxidation-reduction of intracellular thiols rather than by that of extracellular thiols. (TRV) [de

Bicarbonate-dependent transport of acetate and butyrate across the basolateral membrane of sheep rumen epithelium.

Science.gov (United States)

Dengler, F; Rackwitz, R; Benesch, F; Pfannkuche, H; Gäbel, G

2014-02-01

This study aimed to assess the role of HCO₃⁻ in the transport of acetate and butyrate across the basolateral membrane of rumen epithelium and to identify transport proteins involved. The effects of basolateral variation in HCO₃⁻ concentrations on acetate and butyrate efflux out of the epithelium and the transepithelial flux of these short-chain fatty acids were tested in Ussing chamber experiments using (14)C-labelled substrates. HCO₃⁻-dependent transport mechanisms were characterized by adding specific inhibitors of candidate proteins to the serosal side. Effluxes of acetate and butyrate out of the epithelium were higher to the serosal side than to the mucosal side. Acetate and butyrate effluxes to both sides of rumen epithelium consisted of HCO₃⁻-independent and -dependent parts. HCO₃⁻-dependent transport across the basolateral membrane was confirmed in studies of transepithelial fluxes. Mucosal to serosal fluxes of acetate and butyrate decreased with lowering serosal HCO₃⁻ concentrations. In the presence of 25 mm HCO₃⁻, transepithelial flux of acetate was inhibited effectively by p-hydroxymercuribenzoic acid or α-cyano-4-hydroxycinnamic acid, while butyrate flux was unaffected by the blockers. Fluxes of both acetate and butyrate from the serosal to the mucosal side were diminished largely by the addition of NO₃⁻ to the serosal side, with this effect being more pronounced for acetate. Our results indicate the existence of a basolateral short-chain fatty acid/HCO₃⁻ exchanger, with monocarboxylate transporter 1 as a primary candidate for acetate transfer. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Small Molecule Membrane Transporters in the Mammalian Podocyte: A Pathogenic and Therapeutic Target

Directory of Open Access Journals (Sweden)

Cristina Zennaro

2014-11-01

Full Text Available The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development.

Biases of chamber methods for measuring soil CO2 efflux demonstrated with a laboratory apparatus.

Science.gov (United States)

S. Mark Nay; Kim G. Mattson; Bernard T. Bormann

1994-01-01

Investigators have historically measured soil CO2 efflux as an indicator of soil microbial and root activity and more recently in calculations of carbon budgets. The most common methods estimate CO2 efflux by placing a chamber over the soil surface and quantifying the amount of CO2 entering the...

Detection of inhibitors of Candida albicans Cdr transporters using a diS-C3(3 fluorescence

Directory of Open Access Journals (Sweden)

Joanna eSzczepaniak

2015-03-01

Full Text Available Candida albicans is a major cause of opportunistic and life-threatening, systemic fungal infections. Hence new antifungal agents, as well as new methods to treat fungal infections, are still needed. The application of inhibitors of drug-efflux pumps may increase the susceptibility of C. albicans to drugs. We developed a new fluorescence method that allows the in vivo activity evaluation of compounds inhibiting of C. albicans transporters. We show that the potentiometric dye 3,3′-dipropylthiacarbocyanine iodide diS-C3(3 is pumped out by both Cdr1 and Cdr2 transporters. The fluorescence labeling with diS-C3(3 enables a real-time observation of the activity of C. albicans Cdr1 and Cdr2 transporters. We demonstrate that enniatin A and beauvericin show different specificities toward these transporters. Enniatin A inhibits diS-C3(3 efflux by Cdr1 while beauvericin inhibits both Cdr1p and Cdr2p.

Study of the role of efflux pump in ciprofloxacin resistance in Salmonella enterica serotype Typhi

Directory of Open Access Journals (Sweden)

V Sharma

2013-01-01

Full Text Available Purpose: There are increasing reports on failure of clinical response to ciprofloxacin in typhoid fever despite the strain being sensitive to drug in in-vitro using standard guidelines and showing mutations in DNA gyrase. But this increased MIC and clinical failures with ciprofloxacin are not always co-related with mutations presently identified in gyrA and parC genes. This shows that there may be other mechanisms such as an active drug efflux pump responsible as has been shown in other Enterobacteriaceae. This study was carried out to determine the role of efflux pump in Salmonella Typhi isolates. Materials and Methods : Total 25 already characterized nalidixic acid sensitive and nalidixic acid resistant S. Typhi strains with different range of ciprofloxacin MIC were included to study the role of efflux pump in the presence of CCCP (efflux pump inhibitor. For genotypic characterization, the entire acrR gene was sequenced to confirm the presence of any mutation in the gene. Results: The MIC of ciprofloxacin remained same in the presence and absence of CCCP in the studied strains and no significant mutations were found in the acrR gene in any of the isolates studied. Conclusions: No role of efflux pump in ciprofloxacin resistance was found in strains studied. There is a need to explore further mechanism of ciprofloxacin resistance in Salmonella Typhi.

Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux.

Directory of Open Access Journals (Sweden)

Soazig Le Lay

Full Text Available Caveolin-1 (Cav1, a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs derived from wild type (WT or Cav1-deficient (Cav1(-/- animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/- cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/- MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/- cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.

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

Science.gov (United States)

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

2014-12-01

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Manganese Transport and Toxicity in Polarized WIF-B Hepatocytes.

Science.gov (United States)

Thompson, Khristy J; Hein, Jennifer; Baez, Andrew; Sosa, Jose Carlo; Wessling-Resnick, Marianne

2018-05-24

Mn toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains while ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to > 250 M MnCl2. However, the hepatocytes were resistant to 4 h exposures of up to 500 M MnCl2 despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor brefeldin A did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile.

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

Directory of Open Access Journals (Sweden)

Sylvie eBaucheron

2014-01-01

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

Effect of perfusion and irrigation flow rate variations on NaCl efflux from the isolated, perfused head of the marine teleost, Myoxocephalus octodecimspinosus

Energy Technology Data Exchange (ETDEWEB)

Claiborne, J.B. (Miami Univ., Coral Gables, FL (USA)); Evans, D.H. (Mt. Desert Island Biological Laboratory, Salsbury Cove, ME, USA)

1981-06-01

In vivo branchial blood pressure and unidirectional efflux values for NaCl were determined in the marine teleost, Myoxocephalus octodecimspinosus. Utilizing an isolated, perfused head preparation, perfused at in vivo pressure levels, NaCl efflux was measured and compared to in vivo values. The effect of variations in perfusion or irrigation rates on the ion efflux across the gills of the isolated head was also studied. The efflux of /sup 22/Na from the isolated, perfused head was found to be similar to in vivo values and dependent on perfusion flow and pressure. In vitro /sup 36/Cl efflux was lower than the efflux from intact animals and was determined to be flow/pressure independent. Irrigation rate changes at all rates tested did not affect the unidirectional efflux of either ion.

L-lactate transport in Ehrlich ascites-tumour cells.

Science.gov (United States)

Spencer, T L; Lehninger, A L

1976-01-01

Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids. PMID:7237

Punigratane, a novel pyrrolidine alkaloid from Punica granatum rind with putative efflux inhibition activity.

Science.gov (United States)

Rafiq, Zumaana; Narasimhan, Sreevidya; Vennila, Rosy; Vaidyanathan, Rama

2016-02-25

A new pyrrolidine alkaloid named Punigratane was isolated from the rind of Punica granatum. This is the first report of a pyrrolidine-like structure from the rind. The activity of this compound was tested in a representative MDR Klebsiella pneumoniae strain which exhibited high efflux pump activity. At a concentration of 6 mg, this compound Punigratane was found to have efflux inhibition activity.

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

Science.gov (United States)

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

2015-04-01

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

Co-regulation analysis of closely linked genes identifies a highly recurrent gain on chromosome 17q25.3 in prostate cancer

International Nuclear Information System (INIS)

Bermudo, Raquel; Martínez-A, Carlos; Ortiz, Ángel R; Fernández, Pedro L; Thomson, Timothy M; Abia, David; Ferrer, Berta; Nayach, Iracema; Benguria, Alberto; Zaballos, Ángel; Rey, Javier del; Miró, Rosa; Campo, Elías

2008-01-01

Transcriptional profiling of prostate cancer (PC) has unveiled new markers of neoplasia and allowed insights into mechanisms underlying this disease. Genomewide analyses have also identified new chromosomal abnormalities associated with PC. The combination of both classes of data for the same sample cohort might provide better criteria for identifying relevant factors involved in neoplasia. Here we describe transcriptional signatures identifying distinct normal and tumoral prostate tissue compartments, and the inference and demonstration of a new, highly recurrent copy number gain on chromosome 17q25.3. We have applied transcriptional profiling to tumoral and non-tumoral prostate samples with relatively homogeneous epithelial representations as well as pure stromal tissue from peripheral prostate and cultured cell lines, followed by quantitative RT-PCR validations and immunohistochemical analysis. In addition, we have performed in silico colocalization analysis of co-regulated genes and validation by fluorescent in situ hybridization (FISH). The transcriptomic analysis has allowed us to identify signatures corresponding to non-tumoral luminal and tumoral epithelium, basal epithelial cells, and prostate stromal tissue. In addition, in silico analysis of co-regulated expression of physically linked genes has allowed us to predict the occurrence of a copy number gain at chromosomal region 17q25.3. This computational inference was validated by fluorescent in situ hybridization, which showed gains in this region in over 65% of primary and metastatic tumoral samples. Our approach permits to directly link gene copy number variations with transcript co-regulation in association with neoplastic states. Therefore, transcriptomic studies of carefully selected samples can unveil new diagnostic markers and transcriptional signatures highly specific of PC, and lead to the discovery of novel genomic abnormalities that may provide additional insights into the causes and mechanisms

Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

Energy Technology Data Exchange (ETDEWEB)

Bhattacharjee, Sourav, E-mail: sourav.bhattacharjee@wur.nl [Wageningen University, Laboratory of Organic Chemistry (Netherlands); Opstal, Edward J. van; Alink, Gerrit M. [Wageningen University, Division of Toxicology (Netherlands); Marcelis, Antonius T. M.; Zuilhof, Han [Wageningen University, Laboratory of Organic Chemistry (Netherlands); Rietjens, Ivonne M. C. M. [Wageningen University, Division of Toxicology (Netherlands)

2013-06-15

The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size {approx}45 nm) and polystyrene nanoparticles (PSNPs/size {approx}50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

Science.gov (United States)

Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

2013-06-01

The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size 45 nm) and polystyrene nanoparticles (PSNPs/size 50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

International Nuclear Information System (INIS)

Bhattacharjee, Sourav; Opstal, Edward J. van; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

2013-01-01

The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size ∼45 nm) and polystyrene nanoparticles (PSNPs/size ∼50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

Enhancing isoprenoid production through systematically assembling and modulating efflux pumps in Escherichia coli.

Science.gov (United States)

Wang, Jian-Feng; Xiong, Zhi-Qiang; Li, Shi-Yuan; Wang, Yong

2013-09-01

Enhancement of the cellular exportation of heterologous compounds is an important aspect to improve the product yield in microbial cell factory. Efflux pumps can expel various intra- or extra-cellular substances out of microbial hosts and increase the cellular tolerance. Thus in this study, by using the hydrophobic sesquiterpene (amorphadiene) and diterpene (kaurene) as two model compounds, we attempted to improve isoprenoid production through systematically engineering the efflux pumps in Escherichia coli BL21(DE3). The pleiotropic resistant pumps, AcrAB-TolC, MdtEF-TolC from E. coli and heterologous MexAB-OprM pump from Pseudomonas aeruginosa, were overexpressed, assembled, and finely modulated. We found that overexpression of AcrB and TolC components can effectively enhance the specific yield of amorphadiene and kaurene, e.g., 31 and 37 % improvement for amorphadiene compared with control, respectively. The heterologous MexB component can enhance kaurene production with 70 % improvement which is more effective than TolC and AcrB. The results suggest that the three components of tripartite efflux pumps play varied effect to enhance isoprenoid production. Considering the highly organized structure of efflux pumps and importance of components interaction, various component combinations were constructed and the copy number of key components AcrB and TolC was finely modulated as well. The results exhibit that the combination TolC and TolC and AcrB improved the specific yield of amorphadiene with 118 %, and AcrA and TolC and AcrB improved that of kaurene with 104 %. This study indicates that assembling and finely modulating efflux pumps is an effective strategy to improve the production of heterologous compounds in E. coli.

Novel structural analogues of piperine as inhibitors of the NorA efflux pump of Staphylococcus aureus.

Science.gov (United States)

Kumar, Ashwani; Khan, Inshad Ali; Koul, Surrinder; Koul, Jawahir Lal; Taneja, Subhash Chandra; Ali, Intzar; Ali, Furqan; Sharma, Sandeep; Mirza, Zahid Mehmood; Kumar, Manoj; Sangwan, Pyare Lal; Gupta, Pankaj; Thota, Niranjan; Qazi, Ghulam Nabi

2008-06-01

Evaluation of novel synthetic analogues of piperine as inhibitors of multidrug efflux pump NorA of Staphylococcus aureus. A library of piperine-derived compounds was evaluated for their potential to inhibit ethidium bromide efflux in NorA-overexpressing S. aureus SA 1199B. The active compounds were then individually combined with ciprofloxacin to study the potentiation of ciprofloxacin's activity. Based on the efflux inhibition assay, a library of 200 compounds was screened. Three piperine analogues, namely SK-20, SK-56 and SK-29, were found to be the most potent inhibitors of the NorA efflux pump. These inhibitors acted in a synergistic manner with ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. These analogues were 2- to 4-fold more potent than piperine at a significantly lower minimal effective concentration. Furthermore, these inhibitors also significantly suppressed the in vitro emergence of ciprofloxacin-resistant S. aureus. A newly identified class of compounds derived from a natural amide, piperine, is more potent than the parent molecule in potentiating the activity of ciprofloxacin through the inhibition of the NorA efflux pump. These molecules may prove useful in augmenting the antibacterial activities of fluoroquinolones in a clinical setting.

Increased maternal and fetal cholesterol efflux capacity and placental CYP27A1 expression in preeclampsia.

Science.gov (United States)

Mistry, Hiten D; Kurlak, Lesia O; Mansour, Yosef T; Zurkinden, Line; Mohaupt, Markus G; Escher, Geneviève

2017-06-01

Preeclampsia is a pregnancy-specific condition that leads to increased cardiovascular risk in later life. A decrease in cholesterol efflux capacity is linked to CVD. We hypothesized that in preeclampsia there would be a disruption of maternal/fetal plasma to efflux cholesterol, as well as differences in the concentrations of both placental sterol 27-hydroxylase (CYP27A1) and apoA1 binding protein (AIBP). Total, HDL-, and ABCA1-mediated cholesterol effluxes were performed with maternal and fetal plasma from women with preeclampsia and normotensive controls (both n = 17). apoA1 and apoE were quantified by chemiluminescence, and 27-hydroxycholesterol (27-OHC) by GC-MS. Immunohistochemistry was used to determine placental expression/localization of CYP27A1, AIBP, apoA1, apoE, and SRB1. Maternal and fetal total and HDL-mediated cholesterol efflux capacities were increased in preeclampsia (by 10-20%), but ABCA1-mediated efflux was decreased (by 20-35%; P preeclampsia. Fetal plasma 27-OHC levels were decreased in preeclamptic samples ( P preeclampsia ( P = 0.04). Placental 27-OHC concentrations were also raised in preeclampsia ( P preeclampsia, to remove cholesterol from cells to limit lipid peroxidation and increase placental angiogenesis. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Transport characteristics of guanidino compounds at the blood-brain barrier and blood-cerebrospinal fluid barrier: relevance to neural disorders

Directory of Open Access Journals (Sweden)

Tachikawa Masanori

2011-02-01

Full Text Available Abstract Guanidino compounds (GCs, such as creatine, phosphocreatine, guanidinoacetic acid, creatinine, methylguanidine, guanidinosuccinic acid, γ-guanidinobutyric acid, β-guanidinopropionic acid, guanidinoethane sulfonic acid and α-guanidinoglutaric acid, are present in the mammalian brain. Although creatine and phosphocreatine play important roles in energy homeostasis in the brain, accumulation of GCs may induce epileptic discharges and convulsions. This review focuses on how physiologically important and/or neurotoxic GCs are distributed in the brain under physiological and pathological conditions. Transporters for GCs at the blood-brain barrier (BBB and the blood-cerebrospinal fluid (CSF barrier (BCSFB have emerged as substantial contributors to GCs distribution in the brain. Creatine transporter (CRT/solute carrier (SLC 6A8 expressed at the BBB regulates creatine concentration in the brain, and represents a major pathway for supply of creatine from the circulating blood to the brain. CRT may be a key factor facilitating blood-to-brain guanidinoacetate transport in patients deficient in S-adenosylmethionine:guanidinoacetate N-methyltransferase, the creatine biosynthetic enzyme, resulting in cerebral accumulation of guanidinoacetate. CRT, taurine transporter (TauT/SLC6A6 and organic cation transporter (OCT3/SLC22A3 expressed at the BCSFB are involved in guanidinoacetic acid or creatinine efflux transport from CSF. Interestingly, BBB efflux transport of GCs, including guanidinoacetate and creatinine, is negligible, though the BBB has a variety of efflux transport systems for synthetic precursors of GCs, such as amino acids and neurotransmitters. Instead, the BCSFB functions as a major cerebral clearance system for GCs. In conclusion, transport of GCs at the BBB and BCSFB appears to be the key determinant of the cerebral levels of GCs, and changes in the transport characteristics may cause the abnormal distribution of GCs in the brain seen

Expression of Genes for Drug Transporters in the Human Female Genital Tract and Modulatory Effect of Antiretroviral Drugs.

Directory of Open Access Journals (Sweden)

Karolin Hijazi

Full Text Available Anti-retroviral (ARV -based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on

Expression of Genes for Drug Transporters in the Human Female Genital Tract and Modulatory Effect of Antiretroviral Drugs.

Science.gov (United States)

Hijazi, Karolin; Cuppone, Anna M; Smith, Kieron; Stincarelli, Maria A; Ekeruche-Makinde, Julia; De Falco, Giulia; Hold, Georgina L; Shattock, Robin; Kelly, Charles G; Pozzi, Gianni; Iannelli, Francesco

2015-01-01

Anti-retroviral (ARV) -based microbicides are one of the strategies pursued to prevent HIV-1 transmission. Delivery of ARV drugs to subepithelial CD4+ T cells at concentrations for protection is likely determined by drug transporters expressed in the cervicovaginal epithelium. To define the role of drug transporters in mucosal disposition of topically applied ARV-based microbicides, these must be tested in epithelial cell line-based biopharmaceutical assays factoring the effect of relevant drug transporters. We have characterised gene expression of influx and efflux drug transporters in a panel of cervicovaginal cell lines and compared this to expression in cervicovaginal tissue. We also investigated the effect of dapivirine, darunavir and tenofovir, currently at advanced stages of microbicides development, on expression of drug transporters in cell lines. Expression of efflux ABC transporters in cervical tissue was best represented in HeLa, Ect1/E6E7 and End1/E6E7 cell lines. Expression of influx OCT and ENT transporters in ectocervix matched expression in Hela while expression of influx SLCO transporters in vagina was best reflected in VK2/E6E7 cell line. Stimulation with darunavir and dapivirine upregulated MRP transporters, including MRP5 involved in transport of tenofovir. Dapivirine also significantly downregulated tenofovir substrate MRP4 in cervical cell lines. Treatment with darunavir and dapivirine showed no significant effect on expression of BCRP, MRP2 and P-glycoprotein implicated in efflux of different ARV drugs. Darunavir strongly induced expression in most cell lines of CNT3 involved in cell uptake of nucleotide/nucleoside analogue reverse transcriptase inhibitors and SLCO drug transporters involved in cell uptake of protease inhibitors. This study provides insight into the suitability of cervicovaginal cell lines for assessment of ARV drugs in transport kinetics studies. The modulatory effect of darunavir and dapivirine on expression of drug

Variability in soil CO2 production and surface CO2 efflux across riparian-hillslope transitions

Science.gov (United States)

Vincent Jerald. Pacific

2007-01-01

The spatial and temporal controls on soil CO2 production and surface CO2 efflux have been identified as an outstanding gap in our understanding of carbon cycling. I investigated both the spatial and temporal variability of soil CO2 concentrations and surface CO2 efflux across eight topographically distinct riparian-hillslope transitions in the ~300 ha subalpine upper-...

Alterations of monocarboxylate transporter densities during hypoxia in brain and breast tumour cells

DEFF Research Database (Denmark)

Cheng, Chang; Edin, Nina F Jeppesen; Lauritzen, Knut H

2012-01-01

Tumour cells are characterized by aerobic glycolysis, which provides biomass for tumour proliferation and leads to extracellular acidification through efflux of lactate via monocarboxylate transporters (MCTs). Deficient and spasm-prone tumour vasculature causes variable hypoxia, which favours...

In-silico identification and phylogenetic analysis of auxin efflux ...

African Journals Online (AJOL)

ufuoma

2014-01-08

Jan 8, 2014 ... PIN proteins of Arabidopsis viz., PIN1,PIN4 and PIN7 show plasma membrane .... The central hydrophilic loop is dynamic in nature and differs from each other in terms ... research of this plant at the molecular level. Auxin efflux.

Enhanced Boron Tolerance in Plants Mediated by Bidirectional Transport Through Plasma Membrane Intrinsic Proteins.

Science.gov (United States)

Mosa, Kareem A; Kumar, Kundan; Chhikara, Sudesh; Musante, Craig; White, Jason C; Dhankher, Om Parkash

2016-02-23

High boron (B) concentration is toxic to plants that limit plant productivity. Recent studies have shown the involvement of the members of major intrinsic protein (MIP) family in controlling B transport. Here, we have provided experimental evidences showing the bidirectional transport activity of rice OsPIP1;3 and OsPIP2;6. Boron transport ability of OsPIP1;3 and OsPIP2;6 were displayed in yeast HD9 mutant strain (∆fps1∆acr3∆ycf1) as a result of increased B sensitivity, influx and accumulation by OsPIP1;3, and rapid efflux activity by OsPIP2;6. RT-PCR analysis showed strong upregulation of OsPIP1;3 and OsPIP2;6 transcripts in roots by B toxicity. Transgenic Arabidopsis lines overexpressing OsPIP1;3 and OsPIP2;6 exhibited enhanced tolerance to B toxicity. Furthermore, B concentration was significantly increased after 2 and 3 hours of tracer boron ((10)B) treatment. Interestingly, a rapid efflux of (10)B from the roots of the transgenic plants was observed within 1 h of (10)B treatment. Boron tolerance in OsPIP1;3 and OsPIP2;6 lines was inhibited by aquaporin inhibitors, silver nitrate and sodium azide. Our data proved that OsPIP1;3 and OsPIP2;6 are indeed involved in both influx and efflux of boron transport. Manipulation of these PIPs could be highly useful in improving B tolerance in crops grown in high B containing soils.

Partitioning of soil CO2 efflux in un-manipulated and experimentally flooded plots of a temperate fen

Science.gov (United States)

Wunderlich, S.; Borken, W.

2012-08-01

Peatlands store large amounts of organic carbon, but the carbon stock is sensitive to changes in precipitation or water table manipulations. Restoration of drained peatlands by drain blocking and flooding is a common measure to conserve and augment the carbon stock of peatland soils. Here, we report to what extent flooding affected the contribution of heterotrophic and rhizosphere respiration to soil CO2 efflux in a grass-dominated mountain fen in Germany. Soil CO2 efflux was measured in three un-manipulated control plots and three flooded plots in two consecutive years. Flooding was achieved by permanent irrigation during the growing seasons. Radiocarbon signatures of CO2 from different sources including soil CO2 efflux, incubated peat cores and live grass roots were repeatedly analyzed for partitioning of soil CO2 efflux. Additionally, heterotrophic respiration and its radiocarbon signature were determined by eliminating rhizosphere respiration in trenched subplots (only control). In the control plots, rhizosphere respiration determined by 14C signatures contributed between 47 and 61% during the growing season, but was small (4 ± 8%) immediately before budding. Trenching revealed a smaller rhizosphere contribution of 33 ± 8% (2009) and 22 ± 9% (2010) during growing seasons. Flooding reduced annual soil CO2 efflux of the fen by 42% in 2009 and by 30% in 2010. The reduction was smaller in 2010 mainly through naturally elevated water level in the control plots. A one-week interruption of irrigation caused a strong short-lived increase in soil CO2 efflux, demonstrating the sensitivity of the fen to water table drawdown near the peat surface. The reduction in soil CO2 efflux in the flooded plots diminished the relative proportion of rhizosphere respiration from 56 to 46%, suggesting that rhizosphere respiration was slightly more sensitive to flooding than heterotrophic respiration.

Genes co-regulated with LBD16 in nematode feeding sites inferred from in silico analysis show similarities to regulatory circuits mediated by the auxin/cytokinin balance in Arabidopsis.

Science.gov (United States)

Cabrera, Javier; Fenoll, Carmen; Escobar, Carolina

2015-01-01

Plant endoparasitic nematodes, root-knot and cyst nematodes (RKNs and CNs) induce within the root vascular cylinder transfer cells used for nourishing, termed giant cells (GCs) and syncytia. Understanding the molecular mechanisms behind this process is essential to develop tools for nematode control. Based on the crucial role in gall development of LBD16, also a key component of the auxin pathway leading to the divisions in the xylem pole pericycle during lateral root (LR) formation, we investigated genes co-regulated with LBD16 in different transcriptomes and analyzed their similarities and differences with those of RKNs and CNs feeding sites (FS). This analysis confirmed LBD16 and its co-regulated genes, integrated in signaling cascades mediated by auxins during LR and callus formation, as a particular feature of RKN-FS distinct to CNs. However, LBD16, and its positively co-regulated genes, were repressed in syncytia, suggesting a selective down- regulation of the LBD16 auxin mediated pathways in CNs-FS. Interestingly, cytokinin-induced genes are enriched in syncytia and we encountered similarities between the transcriptome of shoot regeneration from callus, modulated by cytokinins, and that of syncytia. These findings establish differences in the regulatory networks leading to both FS formation, probably modulated by the auxin/cytokinin balance.

The effect of an exceptionally wet summer on methane effluxes from a 15-year re-wetted fen in north-east Germany

Directory of Open Access Journals (Sweden)

V. Huth

2013-10-01

Full Text Available Re-wetting minerotrophic fens has become an important strategy to mitigate climate change in Germany. However, recent studies report raised methane (CH4 effluxes during the first years after flooding. A minerotrophic fen in north-east Germany that was re-wetted 15 years ago was exposed to exceptionally heavy rainfall and freshwater flooding in August 2011. We measured CH4 effluxes from wetland vegetation stands dominated by Phragmites australis (Cav. Trin. ex Steud., Typha latifolia L. and Carex acutiformis Ehrh., using the closed-chamber method, fortnightly from March 2011 to March 2012 with extra sampling during the flooding. The respective annual effluxes of CH4 (mean ± 1 standard error from the three vegetation types were 18.5 ± 1.3, 21.1 ± 1.2 and 47.5 ± 5.0 g m-2 a-1, with the August effluxes contributing 40 %, 50 % and 10 % of the annual effluxes. Despite the freshwater flooding in August, annual CH4 effluxes from the 15-year re-wetted fen are similar to those reported from pristine fens. These results are promising because they indicate that, although CH4 effluxes are elevated after re-wetting, they may return to values typical for pristine fens after 15 years. Hence, re-wetting can achieve the purpose of reducing greenhouse gas effluxes from drained minerotrophic fens.

YnfA , a SMR family efflux pump is abundant in Escherichia coli isolates from urinary infection

Directory of Open Access Journals (Sweden)

S K Sarkar

2015-01-01

Full Text Available A quantitative study was undertaken to determine the expression level of different efflux pumps in multi-drug-resistant (MDR Escherichia coli isolates from urinary infection. We have determined the presence of different efflux pumps and measured the expression level of tolC, mdfA, norE and ynfA genes among 48 isolates by quantitative real-time PCR. The expression level of tolC and ynfA was constantly high and observed among 75-80% of isolates, whereas mdfA and norE were expressed occasionally. Our findings suggest that ynfA, a new SMR efflux pump gene family member increases the antibiotics′ resistance in E. coli.

The effect of perfusion and irrigation flow rate variations on NaCl efflux from the isolated, perfused head of the marine teleost, Myoxocephalus octodecimspinosus

International Nuclear Information System (INIS)

Claiborne, J.B.; Evans, D.H.

1981-01-01

In vivo branchial blood pressure and unidirectional efflux values for NaCl were determined in the marine teleost, Myoxocephalus octodecimspinosus. Utilizing an isolated, perfused head preparation, perfused at in vivo pressure levels, NaCl efflux was measured and compared to in vivo values. The effect of variations in perfusion or irrigation rates on the ion efflux across the gills of the isolated head was also studied. The efflux of 22 Na from the isolated, perfused head was found to be similar to in vivo values and dependent on perfusion flow and pressure. In vitro 36 Cl efflux was lower than the efflux from intact animals and was determined to be flow/pressure independent. Irrigation rate changes at all rates tested did not affect the unidirectional efflux of either ion. (Auth.)

NorA efflux pump inhibitory activity of coumarins from Mesua ferrea.

Science.gov (United States)

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

2013-10-01

The purpose of this investigation was to study the modulator and efflux pump inhibitor activity of coumarins isolated from Mesua ferrea against clinical strains as well as NorA-over expressed strain of Staphylococcus aureus 1199B. Seven coumarins were tested for modulator activity using ethidium bromide (EtBr) as a substrate. Compounds 1, 4-7 modulated the MIC of EtBr by ≥ 2 fold against wild type clinical strains of S. aureus 1199 and S. aureus 1199B, whereas compounds 4-7 modulated the MIC of EtBr by ≥ 16 fold against MRSA 831. Compounds 1, 4-7 also reduced the MIC of norfloxacin by ≥ 8 fold against S. aureus 1199B, and 4-6 reduced the MIC of norfloxacin by ≥ 8 fold against MRSA 831 at half of their MICs. Inhibition of EtBr efflux by NorA-overproducing S. aureus 1199B and MRSA 831 confirmed the role of compounds 4-6 as NorA efflux pump inhibitors (EPI). Dose-dependent activity at sub-inhibitory concentration (6.25 μg/mL) suggested that compounds 4 and 5 are promising EPI compared to verapamil against 1199B and MRSA 831 strains. © 2013.

A Conserved Circular Network of Coregulated Lipids Modulates Innate Immune Responses.

Science.gov (United States)

Köberlin, Marielle S; Snijder, Berend; Heinz, Leonhard X; Baumann, Christoph L; Fauster, Astrid; Vladimer, Gregory I; Gavin, Anne-Claude; Superti-Furga, Giulio

2015-07-02

Lipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Functional characterization of Citrus macrophylla BOR1 as a boron transporter.

Science.gov (United States)

Cañon, Paola; Aquea, Felipe; Rodríguez-Hoces de la Guardia, Amparo; Arce-Johnson, Patricio

2013-11-01

Plants have evolved to develop an efficient system of boron uptake and transport using a range of efflux carriers named BOR proteins. In this work we isolated and characterized a boron transporter of citrus (Citrus macrophylla), which was named CmBOR1 for its high homology to AtBOR1. CmBOR1 has 4403 bp and 12 exons. Its coding region has 2145 bp and encodes for a protein of 714 amino acids. CmBOR1 possesses the molecular features of BORs such as an anion exchanger domain and the presence of 10 transmembrane domains. Functional analysis in yeast indicated that CmBOR1 has an efflux boron transporter activity, and transformants have increased tolerance to excess boron. CmBOR1 is expressed in leaves, stem and flowers and shows the greatest accumulation in roots. The transcript accumulation was significantly increased under boron deficiency conditions in shoots. In contrast, the accumulation of the transcript did not change in boron toxicity conditions. Finally, we observed that constitutive expression of CmBOR1 was able to increase tolerance to boron deficiency conditions in Arabidopsis thaliana, suggesting that CmBOR1 is a xylem loading boron transporter. Based on these results, it was determined that CmBOR1 encodes a boric acid/borate transporter involved in tolerance to boron deficiency in plants. © 2013 Scandinavian Plant Physiology Society.

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective

Directory of Open Access Journals (Sweden)

Nicholas P. Greene

2018-05-01

Full Text Available The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.

Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective

Science.gov (United States)

Greene, Nicholas P.; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

2018-01-01

The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking. PMID:29892271

[Effects of plastic film mulching on soil CO2 efflux and CO2 concentration in an oasis cotton field].

Science.gov (United States)

Yu, Yong-xiang; Zhao, Cheng-yi; Jia, Hong-tao; Yu, Bo; Zhou, Tian-he; Yang, Yu-guang; Zhao, Hua

2015-01-01

A field study was conducted to compare soil CO2 efflux and CO2 concentration between mulched and non-mulched cotton fields by using closed chamber method and diffusion chamber technique. Soil CO2 efflux and CO2 concentration exhibited a similar seasonal pattern, decreasing from July to October. Mulched field had a lower soil CO2 efflux but a higher CO2 concentration, compared to those of non-mulched fields. Over the measurement period, cumulative CO2 efflux was 1871.95 kg C . hm-2 for mulched field and 2032.81 kg C . hm-2 for non-mulched field. Soil CO2 concentration was higher in mulched field (ranging from 5137 to 25945 µL . L-1) than in non- mulched field (ranging from 2165 to 23986 µL . L-1). The correlation coefficients between soil CO2 concentrations at different depths and soil CO2 effluxes were 0.60 to 0.73 and 0.57 to 0.75 for the mulched and non-mulched fields, indicating that soil CO2 concentration played a crucial role in soil CO2 emission. The Q10 values were 2.77 and 2.48 for the mulched and non-mulched fields, respectively, suggesting that CO2 efflux in mulched field was more sensitive to the temperature.

Antibacterial and efflux pump inhibitors of thymol and carvacrol against food-borne pathogens.

Science.gov (United States)

Miladi, Hanene; Zmantar, Tarek; Chaabouni, Yassine; Fedhila, Kais; Bakhrouf, Amina; Mahdouani, Kacem; Chaieb, Kamel

2016-10-01

In this study thymol (THY) and carvacrol (CAR), two monoterpenic phenol produced by various aromatic plants, was tested for their antibacterial and efflux pump inhibitors potencies against a panel of clinical and foodborne pathogenes. Our results demonstrated a substantial susceptibility of the tested bacteria toward THY and CAR. Especially, THY displayed a strong inhibitory activity (MIC's values ranged from 32 to 64 μg/mL) against the majority of the tested strains compared to CAR. Moreover, a significant reduction in MIC's of TET and benzalkonium chloride (QAC) were noticed when tested in combinations with THY and CAR. Their synergic effect was more significant in the case of THY which resulted a reduction of MIC's values of TET (2-8 fold) and QAC (2-8 fold). We noted also that THY and CAR inhibited the ethidium bromide (EtBr) cell efflux in a concentration-dependent manner. The rate of EtBr accumulation in food-borne pathogen was enhanced with THY and CAR (0, 250 and 500 μg/mL). The lowest concentration causing 50% of EtBr efflux inhibition (IC 50) was noticed in Salmonella enteritidis (1129) at 150 μg/mL of THY and 190 μg/mL of CAR respectively. These findings indicate that THY and CAR may serve as potential sources of efflux pump inhibitor in food-borne pathogens. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of Na+-dependent hexose transport in cultured renal epithelial cells (LLC-PK1)

International Nuclear Information System (INIS)

Weiss, E.R.; Amsler, K.; Dawson, W.D.; Cook, J.S.

1984-01-01

A number of factors were explored to analyze how they interact to yield the increasing transport capacity in differentiating cell populations. These factors include the number of functional transporters in the population, the distribution of these transporters among the individual cells, the Na + chemical gradient, the transmembrane potential, the pathways and activities of these pathways for efflux of glucoside, and cell-cell coupling between accumulating and non-accumulating cells. 35 references, 9 figures, 2 tables

Is P-glycoprotein (ABCB1) a phase 0 or a phase 3 colchicine transporter depending on colchicine exposure conditions?

International Nuclear Information System (INIS)

Decleves, Xavier.; Niel, Elisabeth; Debray, Marcel; Scherrmann, Jean-Michel

2006-01-01

This study investigates the P-glycoprotein (Pgp)-mediated transport of its substrates in accumulation or efflux modes under steady-state conditions. The kinetics of colchicine uptake and efflux, a substrate of both Pgp and intracellular tubulin, were studied in HL60 and HL60/DNR cells; HL60/DNR cells contain 25 times more Pgp than do HL60 cells. HL60/DNR cells in a medium containing 6.25 nM colchicine, which mimics therapeutic conditions, reached steady-state twice as rapidly as did HL60 cells, and accumulated 24-times less colchicine than did HL60 cells. The Pgp inhibitor GF120918, increased colchicine uptake by HL60 cells 1.2-fold and that of HL60/DNR cells 17-fold, while it had no effect on colchicine efflux from either cell line that had been incubated with colchicine for 24 h. Colchicine kinetics fitted well a two closed-compartment model, showing that the low intracellular accumulation of colchicine in HL60/DNR cells resulted from a 11-fold decrease in colchicine uptake and a 2.3-fold increase in colchicine efflux, that could be attributed to Pgp-mediated efflux activity in HL60/DNR cells. Intracellular colchicine was mainly and similarly distributed in the cytosol in both cell lines. These data demonstrate that the kinetics of the intracellular colchicine accumulation depend on the density of Pgp and that Pgp is more a phase 0 (preventing cellular uptake) than a phase 3 (effluxing intracellular substrate) transporter under steady-state conditions, although the situation is reversed after a short incubation time (30 min), when intracellular free colchicine concentration is probably high enough for it to be removed from the cell by Pgp

Clathrin-Mediated Auxin Efflux and Maxima Regulate Hypocotyl Hook Formation and Light-Stimulated Hook Opening in Arabidopsis.

Science.gov (United States)

Yu, Qinqin; Zhang, Ying; Wang, Juan; Yan, Xu; Wang, Chao; Xu, Jian; Pan, Jianwei

2016-01-04

The establishment of auxin maxima by PIN-FORMED 3 (PIN3)- and AUXIN RESISTANT 1/LIKE AUX1 (LAX) 3 (AUX1/LAX3)-mediated auxin transport is essential for hook formation in Arabidopsis hypocotyls. Until now, however, the underlying regulatory mechanism has remained poorly understood. Here, we show that loss of function of clathrin light chain CLC2 and CLC3 genes enhanced auxin maxima and thereby hook curvature, alleviated the inhibitory effect of auxin overproduction on auxin maxima and hook curvature, and delayed blue light-stimulated auxin maxima reduction and hook opening. Moreover, pharmacological experiments revealed that auxin maxima formation and hook curvature in clc2 clc3 were sensitive to auxin efflux inhibitors 1-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid but not to the auxin influx inhibitor 1-naphthoxyacetic acid. Live-cell imaging analysis further uncovered that loss of CLC2 and CLC3 function impaired PIN3 endocytosis and promoted its lateralization in the cortical cells but did not affect AUX1 localization. Taken together, these results suggest that clathrin regulates auxin maxima and thereby hook formation through modulating PIN3 localization and auxin efflux, providing a novel mechanism that integrates developmental signals and environmental cues to regulate plant skotomorphogenesis and photomorphogenesis. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Effects of sodium ions on rat thyrocyte (FRTL-5 cells) swelling- and thyrotropin-activated taurine efflux dependent on cAMP and Epac.

Science.gov (United States)

Fugelli, Kjell

2016-03-01

Cellular osmolyte release is important in preventing water accumulation and swelling. However, the signaling pathways that detect volume increase and activate solute efflux are still not fully understood. We investigated efflux activation of the osmolyte taurine which is actively accumulated in rat thyrocytes (FRTL-5). Efflux of accumulated [(3)H]taurine was stimulated by cellular swelling and thyrotropin (TSH). These effects were significantly diminished in cells having reduced TSH receptor concentrations. Phosphodiesterase inhibitors (IBMX, Rolipram) enhanced both responses. An analog of forskolin (FSK; 7-deacetyl-7-[O-(N-methylpiperazino)-γ-butyryl] dihydrochloride) and an analog of cAMP, specific for activating exchange protein activated directly by cAMP (Epac; 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, acetoxymethyl ester), significantly stimulated [(3)H]taurine efflux. A cAMP analog specific for activating protein kinase A (PKA; N6-benzoyladenosine-3',5'-cyclic monophosphate, acetoxymethyl ester) had no significant stimulatory effect on [(3)H]taurine efflux rate. The amiloride analog, 5-(N-ethyl-N-isopropyl)-amiloride, which inhibits a TSH-stimulated Na(+)/H(+) exchanger, enhanced (100 %) and ouabain inhibited (50 %) the TSH-stimulated [(3)H]taurine efflux rate. The effect of FSK on efflux was strongly potentiated by Na(+)-free iso-osmotic conditions and by osmolality/cell volume that affected also the db-cAMP-stimulated efflux. The TSH receptors and downstream elements of the signaling pathway comprising adenylyl cyclase, cAMP and Epac appeared to mediate the hormone-induced signal for [(3)H]taurine efflux from FRTL-5 cells. With less evidence, the cell volume/osmolality-induced [(3)H]taurine efflux cascade appeared to share some of the hormone signaling elements and to modulate the hormone signaling pathway at two levels through cellular Na(+).

Potent and selective mediators of cholesterol efflux

Energy Technology Data Exchange (ETDEWEB)

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.

Interplay of Drug-Metabolizing Enzymes and Transporters in Drug Absorption and Disposition.

Science.gov (United States)

Shi, Shaojun; Li, Yunqiao

2014-01-01

In recent years, the functional interplay between drug-metabolizing enzymes (DMEs) and drug transporters (DTs) in drug absorption and disposition, as well as the complex drug interactions (DIs), has become an intriguing contention, which has also been termed the "transport-metabolism interplay". The current mechanistic understanding for this interplay is first discussed. In the present article, studies investigating the interplay between cytochrome P450 enzymes (CYPs) and efflux transporters have been systematically reviewed in vitro, in situ, in silico, in animals and humans, followed by CYPs-uptake transporters, CYPs-uptake transporters-efflux transporters, and phase II metabolic enzymes-transporters interplay studies. Although several cellular, isolated organ and whole animal studies, in conjunction with simulation and modelling, have addressed the issue that DMEs and DTs can work cooperatively to affect the bioavailability of shared substrate drugs, convincing evidences in human studies are still lacking. Furthermore, the functional interplay between DMEs and DTs will be highly substrate- and dose- dependent. Additionally, we review recent studies to evaluate the influence of genetic variations in the interplay between DMEs and DTs, which might be helpful for the prediction of pharmacokinetics (PK) and possible DIs in human more correctly. There is strong evidence of coordinately regulated DEMs and DTs gene expression and protein activity (e.g. nuclear receptors). Taken together, further investigations and analysis are urgently needed to explore the functional interplay of DMEs and DTs and to delineate the underlying mechanisms.

Interleukin-10 increases reverse cholesterol transport in macrophages through its bidirectional interaction with liver X receptor α

International Nuclear Information System (INIS)

Halvorsen, Bente; Holm, Sverre; Yndestad, Arne; Scholz, Hanne; Sagen, Ellen Lund; Nebb, Hilde; Holven, Kirsten B.; Dahl, Tuva B.; Aukrust, Pål

2014-01-01

Highlights: • IL-10 promotes reverse cholesterol efflux from lipid loaded macrophages. • IL-10 increases the expression of ABCA-1 and ABCG-1. • IL-10 exhibits cross-talk with the nuclear receptor LXRα. - Abstract: Interleukin (IL)-10 is a prototypical anti-inflammatory cytokine that has been shown to attenuate atherosclerosis development. In addition to its anti-inflammatory properties, the anti-atherogenic effect of IL-10 has recently also been suggested to reflect a complex effect of IL-10 on lipid metabolism in macrophages. In the present study we examined the effects of IL-10 on cholesterol efflux mechanism in lipid-loaded THP-1 macrophages. Our main findings were: (i) IL-10 significantly enhanced cholesterol efflux induced by fetal-calf serum, high-density lipoprotein (HDL) 2 and apolipoprotein A-1. (ii) The IL-10-mediated effects on cholesterol efflux were accompanied by an increased IL-10-mediated expression of the ATP-binding cassette transporters ABCA1 and ABCG1, that was further enhanced when the cells were co-activated with the liver X receptor (LXR)α agonist (22R)-hydroxycholesterol. (iii) The effect of LXRα activation on the IL-10-mediated effects on the ATP-binding cassette transporters seems to include enhancing effects on the IL-10 receptor 1 (IL10R1) expression and interaction with STAT-3 signaling. (iv) These enhancing effects on ABCA1 and ABCG1 was not seen when the cells were stimulated with the IL-10 family members IL-22 and IL-24. This study suggests that the anti-atherogenic properties of IL-10 may include enhancing effects on cholesterol efflux mechanism that involves cross-talk with LXRα activation

Interleukin-10 increases reverse cholesterol transport in macrophages through its bidirectional interaction with liver X receptor α

Energy Technology Data Exchange (ETDEWEB)

Halvorsen, Bente, E-mail: Bente.Halvorsen@rr-research.no [Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo (Norway); Institute of Clinical Medicine, University of Oslo, Oslo (Norway); K.G. Jebsen Inflammation Research Center, University of Oslo, Oslo (Norway); Holm, Sverre [Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo (Norway); Yndestad, Arne [Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo (Norway); Institute of Clinical Medicine, University of Oslo, Oslo (Norway); K.G. Jebsen Inflammation Research Center, University of Oslo, Oslo (Norway); Scholz, Hanne [Section for Transplantation, Institute for Surgical Research, Oslo University Hospital Rikshospitalet, Oslo (Norway); Sagen, Ellen Lund [Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo (Norway); Nebb, Hilde [Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, Oslo (Norway); Institute of Clinical Medicine, University of Oslo, Oslo (Norway); Holven, Kirsten B. [Department of Nutrition, Institute for Basic Medical Sciences, University of Oslo, Oslo (Norway); Dahl, Tuva B. [Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo (Norway); Institute of Clinical Medicine, University of Oslo, Oslo (Norway); Aukrust, Pål [Research Institute of Internal Medicine, Oslo University Hospital Rikshospitalet, Oslo (Norway); Section of Clinical Immunology and Infectious Diseases, Oslo University Hospital Rikshospitalet, Oslo (Norway); Institute of Clinical Medicine, University of Oslo, Oslo (Norway); K.G. Jebsen Inflammation Research Center, University of Oslo, Oslo (Norway)

2014-08-08

Highlights: • IL-10 promotes reverse cholesterol efflux from lipid loaded macrophages. • IL-10 increases the expression of ABCA-1 and ABCG-1. • IL-10 exhibits cross-talk with the nuclear receptor LXRα. - Abstract: Interleukin (IL)-10 is a prototypical anti-inflammatory cytokine that has been shown to attenuate atherosclerosis development. In addition to its anti-inflammatory properties, the anti-atherogenic effect of IL-10 has recently also been suggested to reflect a complex effect of IL-10 on lipid metabolism in macrophages. In the present study we examined the effects of IL-10 on cholesterol efflux mechanism in lipid-loaded THP-1 macrophages. Our main findings were: (i) IL-10 significantly enhanced cholesterol efflux induced by fetal-calf serum, high-density lipoprotein (HDL){sub 2} and apolipoprotein A-1. (ii) The IL-10-mediated effects on cholesterol efflux were accompanied by an increased IL-10-mediated expression of the ATP-binding cassette transporters ABCA1 and ABCG1, that was further enhanced when the cells were co-activated with the liver X receptor (LXR)α agonist (22R)-hydroxycholesterol. (iii) The effect of LXRα activation on the IL-10-mediated effects on the ATP-binding cassette transporters seems to include enhancing effects on the IL-10 receptor 1 (IL10R1) expression and interaction with STAT-3 signaling. (iv) These enhancing effects on ABCA1 and ABCG1 was not seen when the cells were stimulated with the IL-10 family members IL-22 and IL-24. This study suggests that the anti-atherogenic properties of IL-10 may include enhancing effects on cholesterol efflux mechanism that involves cross-talk with LXRα activation.

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport.

Science.gov (United States)

Szczerba, Mark W; Britto, Dev T; Balkos, Konstantine D; Kronzucker, Herbert J

2008-01-01

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.

Tannic acid affects the phenotype of Staphylococcus aureus resistant to tetracycline and erythromycin by inhibition of efflux pumps.

Science.gov (United States)

Tintino, Saulo R; Morais-Tintino, Cícera D; Campina, Fábia F; Costa, Maria do S; Menezes, Irwin R A; de Matos, Yedda Maria L S; Calixto-Júnior, João T; Pereira, Pedro S; Siqueira-Junior, José P; Leal-Balbino, Teresa C; Coutinho, Henrique D M; Balbino, Valdir Q

2017-10-01

The widespread use of antibiotics created selective pressure for the emergence of strains that would persist despite antibiotic toxicity. The bacterial resistance mechanisms are several, with efflux pumps being one of the main ones. These pumps are membrane proteins with the function of removing antibiotics from the cell cytoplasm. Due to this importance, the aim of this work was to evaluate the inhibitory effect of tannic acid against efflux pumps expressed by the Staphylococcus aureus RN4220 and IS-58 strains. The efflux pump inhibition was assayed using a sub-inhibitory concentration of efflux pump standard inhibitors and tannic acid (MIC/8), observing their capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due the possible inhibitory effect of these substances. The MICs of EtBr and antibiotics were significantly different in the presence of tannic acid, indicating the inhibitory effect of this product against efflux pumps of both strains. These results indicate the possible usage of tannic acid asan inhibitor and an adjuvant in the antibiotic therapy against multidrug resistant bacteria (MDR). Copyright © 2017 Elsevier Inc. All rights reserved.

Coregulation of soybean vegetative storage protein gene expression by methyl jasmonate and soluble sugars.

Science.gov (United States)

Mason, H S; Dewald, D B; Creelman, R A; Mullet, J E

1992-03-01

The soybean vegetative storage protein genes vspA and vspB are highly expressed in developing leaves, stems, flowers, and pods as compared with roots, seeds, and mature leaves and stems. In this paper, we report that physiological levels of methyl jasmonate (MeJA) and soluble sugars synergistically stimulate accumulation of vsp mRNAs. Treatment of excised mature soybean (Glycine max Merr. cv Williams) leaves with 0.2 molar sucrose and 10 micromolar MeJA caused a large accumulation of vsp mRNAs, whereas little accumulation occurred when these compounds were supplied separately. In soybean cell suspension cultures, the synergistic effect of sucrose and MeJA on the accumulation of vspB mRNA was maximal at 58 millimolar sucrose and was observed with fructose or glucose substituted for sucrose. In dark-grown soybean seedlings, the highest levels of vsp mRNAs occurred in the hypocotyl hook, which also contained high levels of MeJA and soluble sugars. Lower levels of vsp mRNAs, MeJA, and soluble sugars were found in the cotyledons, roots, and nongrowing regions of the stem. Wounding of mature soybean leaves induced a large accumulation of vsp mRNAs when wounded plants were incubated in the light. Wounded plants kept in the dark or illuminated plants sprayed with dichlorophenyldimethylurea, an inhibitor of photosynthetic electron transport, showed a greatly reduced accumulation of vsp mRNAs. The time courses for the accumulation of vsp mRNAs induced by wounding or sucrose/MeJA treatment were similar. These results strongly suggest that vsp expression is coregulated by endogenous levels of MeJA (or jasmonic acid) and soluble carbohydrate during normal vegetative development and in wounded leaves.

Phosphorylation of conserved PIN motifs directs Arabidopsis PIN1 polarity and auxin transport

NARCIS (Netherlands)

Huang, F.; Kemel Zago, M.; Abas, L.; van Marion, A.; Galván-Ampudia, C.S.; Offringa, R.

2010-01-01

Polar cell-to-cell transport of auxin by plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers generates auxin gradients that provide positional information for various plant developmental processes. The apical-basal polar localization of the PIN proteins that determines the direction of

SOIL CO2 EFFLUX IN FOUR DIFFERENT LAND USE SYSTEMS IN RIO POMBA, MINAS GERAIS/BRAZIL

Directory of Open Access Journals (Sweden)

Joel Marques de Oliveira

2014-07-01

Full Text Available Functioning and sustainability of agricultural systems depend directly on the soil biological activity. Soil respiration, or CO2 efflux, is a sensible indicator of biological activity, revealing fast and accurately whether changes in environment affect soil community. In this context, soil respiration can be used to evaluate soil organisms behavior after an environmental change revealing the capacity of a soil in it normal functioning after a disturb event. The objective of this work was to study seasonal variation in soil CO 2 efflux in Rio Pomba/MG and its relation with typical land uses of Zona da Mata region of Minas Gerais. Fluctuation on soil CO2 efflux was observed in all areas throughout the period of the study, from September 2010 to August 2011, as a result of climatic variation. We have also reported specific patterns on CO 2 efflux that can be associated with land use. It was observed that the area under annual crops presented the highest amplitude of changes in respiratory rates, while forest and guava plantation presented the lowest. The principal component analysis revealed that the area cultivated with guava presented pattern of CO 2 efflux similar to forest, and the area intensively cultivated with annual crops showed behavior opposite to the forest. We conclude that variation in soil respiration rates is higher in intensive cropped areas. Additionally, total soil respiration can be used as a methodology to assess the interference of cropping on soil biota.

[Effect of bamboo leaf biochar addition on soil CO2 efflux and labile organic carbon pool in a Chinese chestnut plantation].

Science.gov (United States)

Wang, Zhan-Lei; Li, Yong-Fu; Jiang, Pei-Kun; Zhou, Guo-Mo; Liu, Juan

2014-11-01

Effect of biochar addition on soil CO2 efflux in a typical Chinese chestnut (Castanea mollissima) plantation in Lin'an, Zhejiang Province, China was investigated from July 2012 to July 2013 by the static closed chamber-GC technique. Soil temperature, soil moisture, WSOC and MBC concentrations were determined as well. Results showed that soil CO2 efflux exhibited a strong sea- sonal pattern. Compared with the control (without biochar application), the biochar treatment increased the soil CO2 efflux only in the first month since application, and then the effect diminished thereafter. There were no significant differences in the annual cumulative value of soil CO2 efflux between the biochar and control treatments. The annual mean value in soil MBC concentration (362 mg · kg(-1)) in the biochar treatment was higher than that (322 mg · kg(-1)) in the control. However, no significant difference in the soil WSOC concentration was found between the biochar and control treatments. Strong exponential relationships between soil temperature and soil CO2 efflux were observed regardless of the treatment and soil layer. The apparent temperature sensitivity (Q10) of soil CO2 efflux in the biochar treatment was higher than that in the control. Soil CO2 efflux was related to soil WSOC concentration but not with soil MBC or moisture content. To conclude, the application of bamboo leaf biochar did not affect the annual cumulative CO2 emission in the Chinese chestnut plantation but increased the Q10, and the CO2 efflux was predominantly controlled by the soil temperature and soil WSOC level.

Cytotoxicity and Efflux Pump Inhibition Induced by Molybdenum Disulfide and Boron Nitride Nanomaterials with Sheetlike Structure.

Science.gov (United States)

Liu, Su; Shen, Zhuoyan; Wu, Bing; Yu, Yue; Hou, Hui; Zhang, Xu-Xiang; Ren, Hong-Qiang

2017-09-19

Sheetlike molybdenum disulfide (MoS 2 ) and boron nitride (BN) nanomaterials have attracted attention in the past few years due to their unique material properties. However, information on adverse effects and their underlying mechanisms for sheetlike MoS 2 and BN nanomaterials is rare. In this study, cytotoxicities of sheetlike MoS 2 and BN nanomaterials on human hepatoma HepG2 cells were systematically investigated at different toxic end points. Results showed that MoS 2 and BN nanomaterials decreased cell viability at 30 μg/mL and induced adverse effects on intracellular ROS generation (≥2 μg/mL), mitochondrial depolarization (≥4 μg/mL), and membrane integrity (≥8 μg/mL for MoS 2 and ≥2 μg/mL for BN). Furthermore, this study first found that low exposure concentrations (0.2-2 μg/mL) of MoS 2 and BN nanomaterials could increase plasma membrane fluidity and inhibit transmembrane ATP binding cassette (ABC) efflux transporter activity, which make both nanomaterials act as a chemosensitizer (increasing arsenic toxicity). Damage to plasma membrane and release of soluble Mo or B species might be two reasons that both nanomaterials inhibit efflux pump activities. This study provides a systematic understanding of the cytotoxicity of sheetlike MoS 2 and BN nanomaterials at different exposure levels, which is important for their safe use.

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

Directory of Open Access Journals (Sweden)

Jody L. Andersen

2015-01-01

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

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

Science.gov (United States)

Fernández, Lucía

2012-01-01

Summary: The substantial use of antibiotics in the clinic, combined with a dearth of new antibiotic classes, has led to a gradual increase in the resistance of bacterial pathogens to these compounds. Among the various mechanisms by which bacteria endure the action of antibiotics, those affecting influx and efflux are of particular importance, as they limit the interaction of the drug with its intracellular targets and, consequently, its deleterious effects on the cell. This review evaluates the impact of porins and efflux pumps on two major types of resistance, namely, mutational and adaptive types of resistance, both of which are regarded as key phenomena in the global rise of antibiotic resistance among pathogenic microorganisms. In particular, we explain how adaptive and mutational events can dramatically influence the outcome of antibiotic therapy by altering the mechanisms of influx and efflux of antibiotics. The identification of porins and pumps as major resistance markers has opened new possibilities for the development of novel therapeutic strategies directed specifically against these mechanisms. PMID:23034325

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

Science.gov (United States)

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

2015-01-01

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

Transporter-mediated biofuel secretion.

Science.gov (United States)

Doshi, Rupak; Nguyen, Tuan; Chang, Geoffrey

2013-05-07

Engineering microorganisms to produce biofuels is currently among the most promising strategies in renewable energy. However, harvesting these organisms for extracting biofuels is energy- and cost-intensive, limiting the commercial feasibility of large-scale production. Here, we demonstrate the use of a class of transport proteins of pharmacological interest to circumvent the need to harvest biomass during biofuel production. We show that membrane-embedded transporters, better known to efflux lipids and drugs, can be used to mediate the secretion of intracellularly synthesized model isoprenoid biofuel compounds to the extracellular milieu. Transporter-mediated biofuel secretion sustainably maintained an approximate three- to fivefold boost in biofuel production in our Escherichia coli test system. Because the transporters used in this study belong to the ubiquitous ATP-binding cassette protein family, we propose their use as "plug-and-play" biofuel-secreting systems in a variety of bacteria, cyanobacteria, diatoms, yeast, and algae used for biofuel production. This investigation showcases the potential of expressing desired membrane transport proteins in cell factories to achieve the export or import of substances of economic, environmental, or therapeutic importance.

Landscape structure control on soil CO2 efflux variability in complex terrain: Scaling from point observations to watershed scale fluxes

Science.gov (United States)

Diego A. Riveros-Iregui; Brian L. McGlynn

2009-01-01

We investigated the spatial and temporal variability of soil CO2 efflux across 62 sites of a 393-ha complex watershed of the northern Rocky Mountains. Growing season (83 day) cumulative soil CO2 efflux varied from ~300 to ~2000 g CO2 m-2, depending upon landscape position, with a median of 879.8 g CO2 m-2. Our findings revealed that highest soil CO2 efflux rates were...

Co-regulation of water and K(+) transport in sunflower plants during water stress recovery.

Science.gov (United States)

Benlloch, Manuel; Benlloch-González, María

2016-06-01

16-day-old sunflower (Helianthus annuus L.) plants were subjected to deficit irrigation for 12 days. Following this period, plants were rehydrated for 2 days to study plant responses to post-stress recovery. The moderate water stress treatment applied reduced growth in all plant organs and the accumulation of K(+) in the shoot. After the rehydration period, the stem recovered its growth and reached a similar length to the control, an effect which was not observed in either root or leaves. Moreover, plant rehydration after water stress favored the accumulation of K(+) in the apical zone of the stem and expanding leaves. In the roots of plants under water stress, watering to field capacity, once the plants were de- topped, rapidly favored K(+) and water transport in the excised roots. This quick and short-lived response was not observed in roots of plants recovered from water stress for 2 days. These results suggest that the recovery of plant growth after water stress is related to coordinated water and K(+) transport from the root to the apical zone of the ​​stem and expanding leaves. This stimulation of K(+) transport in the root and its accumulation in the cells of the growing zones of the ​​stem must be one of the first responses induced in the plant during water stress recovery. Copyright © 2016 Elsevier GmbH. All rights reserved.

Structure, mechanism and cooperation of bacterial multidrug transporters.

Science.gov (United States)

Du, Dijun; van Veen, Hendrik W; Murakami, Satoshi; Pos, Klaas M; Luisi, Ben F

2015-08-01

Cells from all domains of life encode energy-dependent trans-membrane transporters that can expel harmful substances including clinically applied therapeutic agents. As a collective body, these transporters perform as a super-system that confers tolerance to an enormous range of harmful compounds and consequently aid survival in hazardous environments. In the Gram-negative bacteria, some of these transporters serve as energy-transducing components of tripartite assemblies that actively efflux drugs and other harmful compounds, as well as deliver virulence agents across the entire cell envelope. We draw together recent structural and functional data to present the current models for the transport mechanisms for the main classes of multi-drug transporters and their higher-order assemblies. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Antibacterial Efficacy of Polysaccharide Capped Silver Nanoparticles Is Not Compromised by AcrAB-TolC Efflux Pump

Directory of Open Access Journals (Sweden)

Mitali Mishra

2018-05-01

Full Text Available Antibacterial therapy is of paramount importance in treatment of several acute and chronic infectious diseases caused by pathogens. Over the years extensive use and misuse of antimicrobial agents has led to emergence of multidrug resistant (MDR and extensive drug resistant (XDR pathogens. This drastic escalation in resistant phenotype has limited the efficacy of available therapeutic options. Thus, the need of the hour is to look for alternative therapeutic approaches to mitigate healthcare concerns caused due to MDR bacterial infections. Nanoparticles have gathered much attention as potential candidates for antibacterial therapy. Equipped with advantages of, wide spectrum bactericidal activity at very low dosage, inhibitor of biofilm formation and ease of permeability, nanoparticles have been considered as leading therapeutic candidates to curtail infections resulting from MDR bacteria. However, substrate non-specificity of efflux pumps, particularly those belonging to resistance nodulation division super family, have been reported to reduce efficacy of many potent antibacterial therapeutic drugs. Previously, we had reported antibacterial activity of polysaccharide-capped silver nanoparticles (AgNPs toward MDR bacteria. We showed that AgNPs inhibits biofilm formation and alters expression of cytoskeletal proteins FtsZ and FtsA, with minimal cytotoxicity toward mammalian cells. In the present study, we report no reduction in antibacterial efficacy of silver nanoparticles in presence of AcrAB-TolC efflux pump proteins. Antibacterial tests were performed according to CLSI macrobroth dilution method, which revealed that both silver nanoparticles exhibited bactericidal activity at very low concentrations. Further, immunoblotting results indicated that both the nanoparticles modulate the transporter AcrB protein expression. However, expression of the membrane fusion protein AcrA did show a significant increase after exposure to AgNPs. Our results

Kinetics of lactate and pyruvate transport in cultured rat myotubes

DEFF Research Database (Denmark)

von Grumbckow, Lena; Elsner, Peter; Hellsten, Ylva

1999-01-01

, respectively. Furthermore, it was observed that the two monocarboxylate transporter isoforms present in mature skeletal muscles, MCT1 and MCT4 (formerly called MCT3 (M.C. Wilson, V.N. Jackson, C. Heddle, N.T. Price, H. Pilegaard, C. Juel, A. Bonen, I. Montgomery, O.F. Hutter, A.P. Halestrap, Lactic acid efflux...... from white skeletal muscle is catalyzed by the monocarboxylate transporter isoform MCT3, J. Biol. Chem. 273 (1998) 15920-15926)), were also expressed in primary culture of myotubes....

Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr.

OpenAIRE

Klyachko, K A; Schuldiner, S; Neyfakh, A A

1997-01-01

The Bacillus subtilis multidrug transporter Bmr, a member of the major facilitator superfamily of transporters, causes the efflux of a number of structurally unrelated toxic compounds from cells. We have shown previously that the activity of Bmr can be inhibited by the plant alkaloid reserpine. Here we demonstrate that various substitutions of residues Phe143 and Phe306 of Bmr not only reduce its sensitivity to reserpine inhibition but also significantly change its substrate specificity. Cros...

Dexamethasone attenuates grain sorghum dust extract-induced increase in macromolecular efflux in vivo.

Science.gov (United States)

Akhter, S R; Ikezaki, H; Gao, X P; Rubinstein, I

1999-05-01

The purpose of this study was to determine whether dexamethasone attenuates grain sorghum dust extract-induced increase in macromolecular efflux from the in situ hamster cheek pouch and, if so, whether this response is specific. By using intravital microscopy, we found that an aqueous extract of grain sorghum dust elicited significant, concentration-dependent leaky site formation and increase in clearance of FITC-labeled dextran (FITC-dextran; mol mass, 70 kDa) from the in situ hamster cheek pouch (P grain sorghum dust extract- and substance P-induced increases in macromolecular efflux from the in situ hamster cheek pouch in a specific fashion.

Transport of cysteate by synaptosomes isolated from rat brain

International Nuclear Information System (INIS)

Wilson, D.F.; Pastuszko, A.

1986-01-01

Synaptosomes isolated from rat brain were observed to take up cysteic acid by a high affinity transport system (K/sub M = 12.3 +/- 2.1 μM; V/sub m/ = 2.5 n mole/mg protein/minute). This uptake was competitively inhibited by aspartate (K/sub i/ = 13.3 +/- 1.8 μM) and cysteine sulfinate (K/sub i/ = 13.3 +/- 3.3 μM). Addition of extrasynaptosomal cysteate, aspartate or cysteine sulfinate to synaptosomes loaded with [ 35 S] cysteate induced rapid efflux of the cysteate. This efflux was via stoichiometric exchange of amino acids with half maximal rates at 5.0 +/- 1.1 μM aspartate or 8.0 +/- 1.3 μM cysteine sulfinate. Conversely, added extrasynaptosomal cysteate exchanged for endogenous aspartate and glutamate with half maximal rates at 5.0 +/- 0.4 μM cysteate. In the steady state after maximal accumulation of cysteate, the intrasynaptosomal cysteate concentrations exceeded the extrasynaptosomal concentrations by up to 10,000 fold. The measured concentration ratios were the same, within experimental error, as those for aspartate and glutamate. Depolarization, with either high K + or veratridine, of the plasma membrane of synaptosomes loaded with cysteate caused parallel release of cysteate, aspartate and glutamate. It is concluded that neurons transport cysteate, cysteine sulfinate, aspartate and glutamate with the same transport system. This transport system catalyzes homoexchange and heteroexchange as well as net uptake and release of all these amino acids

Missense dopamine transporter mutations associate with adult parkinsonism and ADHD

DEFF Research Database (Denmark)

Hansen, Freja H; Skjørringe, Tina; Yasmeen, Saiqa

2014-01-01

experiments suggested that the disrupted function of the DAT-Asp421Asn mutant is the result of compromised sodium binding, in agreement with Asp421 coordinating sodium at the second sodium site. For DAT-Asp421Asn, substrate efflux experiments revealed a constitutive, anomalous efflux of dopamine......Parkinsonism and attention deficit hyperactivity disorder (ADHD) are widespread brain disorders that involve disturbances of dopaminergic signaling. The sodium-coupled dopamine transporter (DAT) controls dopamine homeostasis, but its contribution to disease remains poorly understood. Here, we......-deoxy-glucose-PET/MRI (FDG-PET/MRI) scan, the patient suffered from progressive dopaminergic neurodegeneration. In heterologous cells, both DAT variants exhibited markedly reduced dopamine uptake capacity but preserved membrane targeting, consistent with impaired catalytic activity. Computational simulations and uptake...

Sodium content and sodium efflux of mononuclear leucocytes from young subjects at increased risk of developing essential hypertension

DEFF Research Database (Denmark)

Pedersen, K E; Nielsen, J R; Klitgaard, N A

1990-01-01

Mononuclear leucocytes were used as a cellular model for the in vitro measurements of volume, sodium and potassium content, sodium efflux rate constants and absolute sodium efflux in order to assess any cellular changes in young men at increased risk of developing essential hypertension...

Involvement of the efflux pumps in chloramphenicol selected strains of Burkholderia thailandensis: proteomic and mechanistic evidence.

Directory of Open Access Journals (Sweden)

Fabrice V Biot

Full Text Available Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections.

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics

Science.gov (United States)

Bailly, Aurelien; Zwiewka, Marta; Sovero, Valpuri; Ge, Pei; Aryal, Bibek; Hao, Pengchao; Linnert, Miriam; Burgardt, Noelia Inés; Lücke, Christian; Weiwad, Matthias; Michel, Max; Weiergräber, Oliver H.; Pollmann, Stephan; Azzarello, Elisa; Fukao, Yoichiro; Hoffmann, Céline; Wedlich-Söldner, Roland

2016-01-01

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity. PMID:27053424

Amyloid-beta transporter expression at the blood-CSF barrier is age-dependent

Directory of Open Access Journals (Sweden)

Pascale Crissey L

2011-07-01

Full Text Available Abstract Background Age is the major risk factor for many neurodegenerative diseases, including Alzheimer's disease (AD. There is an accumulation of amyloid-beta peptides (Aβ in both the AD brain and the normal aging brain. Clearance of Aβ from the brain occurs via active transport at the blood-brain barrier (BBB and blood-cerebrospinal fluid barrier (BCSFB. With increasing age, the expression of the Aβ efflux transporters is decreased and the Aβ influx transporter expression is increased at the BBB, adding to the amyloid burden in the brain. Expression of the Aβ transporters at the choroid plexus (CP epithelium as a function of aging was the subject of this study. Methods This project investigated the changes in expression of the Aβ transporters, the low density lipoprotein receptor-related protein-1 (LRP-1, P-glycoprotein (P-gp, LRP-2 (megalin and the receptor for advanced glycation end-products (RAGE at the BCSFB in Brown-Norway/Fischer rats at ages 3, 6, 9, 12, 20, 30 and 36 months, using real time RT-PCR to measure transporter mRNA expression, and immunohistochemistry (IHC to measure transporter protein in isolated rat CP. Results There was an increase in the transcription of the Aβ efflux transporters, LRP-1 and P-gp, no change in RAGE expression and a decrease in LRP-2, the CP epithelium influx transporter, at the BCSFB with aging. Decreased Aβ42 concentration in the CP, as measured by quantitative IHC, was associated with these Aβ transporter alterations. Conclusions Age-dependent alterations in the CP Aβ transporters are associated with a decrease in Aβ42 accumulation in the CP, and are reciprocal to the changes seen in these transporters at the BBB, suggesting a possible compensatory role for the BCSFB in Aβ clearance in aging.

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

Science.gov (United States)

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 (...

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

International Nuclear Information System (INIS)

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

1988-01-01

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

Human skeletal muscle drug transporters determine local exposure and toxicity of statins.

Science.gov (United States)

Knauer, Michael J; Urquhart, Bradley L; Meyer zu Schwabedissen, Henriette E; Schwarz, Ute I; Lemke, Christopher J; Leake, Brenda F; Kim, Richard B; Tirona, Rommel G

2010-02-05

The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, or statins, are important drugs used in the treatment and prevention of cardiovascular disease. Although statins are well tolerated, many patients develop myopathy manifesting as muscle aches and pain. Rhabdomyolysis is a rare but severe toxicity of statins. Interindividual differences in the activities of hepatic membrane drug transporters and metabolic enzymes are known to influence statin plasma pharmacokinetics and risk for myopathy. Interestingly, little is known regarding the molecular determinants of statin distribution into skeletal muscle and its relevance to toxicity. We sought to identify statin transporters in human skeletal muscle and determine their impact on statin toxicity in vitro. We demonstrate that the uptake transporter OATP2B1 (human organic anion transporting polypeptide 2B1) and the efflux transporters, multidrug resistance-associated protein (MRP)1, MRP4, and MRP5 are expressed on the sarcolemmal membrane of human skeletal muscle fibers and that atorvastatin and rosuvastatin are substrates of these transporters when assessed using a heterologous expression system. In an in vitro model of differentiated, primary human skeletal muscle myoblast cells, we demonstrate basal membrane expression and drug efflux activity of MRP1, which contributes to reducing intracellular statin accumulation. Furthermore, we show that expression of human OATP2B1 in human skeletal muscle myoblast cells by adenoviral vectors increases intracellular accumulation and toxicity of statins and such effects were abrogated when cells overexpressed MRP1. These results identify key membrane transporters as modulators of skeletal muscle statin exposure and toxicity.

Combustion of a high-velocity hydrogen microjet effluxing in air

Science.gov (United States)

Kozlov, V. V.; Grek, G. R.; Korobeinichev, O. P.; Litvinenko, Yu. A.; Shmakov, A. G.

2016-09-01

This study is devoted to experimental investigation of hydrogen-combustion modes and the structure of a diffusion flame formed at a high-velocity efflux of hydrogen in air through round apertures of various diameters. The efflux-velocity range of the hydrogen jet and the diameters of nozzle apertures at which the flame is divided in two zones with laminar and turbulent flow are found. The zone with the laminar flow is a stabilizer of combustion of the flame as a whole, and in the zone with the turbulent flow the intense mixing of fuel with an oxidizer takes place. Combustion in these two zones can occur independently from each other, but the steadiest mode is observed only at the existence of the flame in the laminar-flow zone. The knowledge obtained makes it possible to understand more deeply the features of modes of microjet combustion of hydrogen promising for various combustion devices.

Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation

DEFF Research Database (Denmark)

Henrichs, Sina; Wang, Bangjun; Fukao, Yoichiro

2012-01-01

Polar transport of the plant hormone auxin is controlled by PIN-and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-I...

The Role of ATP-Binding Cassette Transporters in Neuro-Inflammation: Relevance for Bioactive Lipids

NARCIS (Netherlands)

Kooij, G.; van Horssen, J.; Bandaru, V.V.R.; Haughey, N.J.; de Vries, H.E.

2012-01-01

ATP-binding cassette (ABC) transporters are highly expressed by brain endothelial cells that form the blood-brain barrier (BBB). These efflux pumps play an important role in maintaining brain homeostasis as they actively hinder the entry of unwanted blood-derived compounds into the central nervous

Membrane transporter engineering in industrial biotechnology and whole cell biocatalysis.

Science.gov (United States)

Kell, Douglas B; Swainston, Neil; Pir, Pınar; Oliver, Stephen G

2015-04-01

Because they mainly do not involve chemical changes, membrane transporters have been a Cinderella subject in the biotechnology of small molecule production, but this is a serious oversight. Influx transporters contribute significantly to the flux towards product, and efflux transporters ensure the accumulation of product in the much greater extracellular space of fermentors. Programmes for improving biotechnological processes might therefore give greater consideration to transporters than may have been commonplace. Strategies for identifying important transporters include expression profiling, genome-wide knockout studies, stress-based selection, and the use of inhibitors. In addition, modern methods of directed evolution and synthetic biology, especially those effecting changes in energy coupling, offer huge opportunities for increasing the flux towards extracellular product formation by transporter engineering. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evidence for a role of transporter-mediated currents in the depletion of brain serotonin induced by serotonin transporter substrates.

Science.gov (United States)

Baumann, Michael H; Bulling, Simon; Benaderet, Tova S; Saha, Kusumika; Ayestas, Mario A; Partilla, John S; Ali, Syed F; Stockner, Thomas; Rothman, Richard B; Sandtner, Walter; Sitte, Harald H

2014-05-01

Serotonin (5-HT) transporter (SERT) substrates like fenfluramine and 3,4-methylenedioxymethamphetamine cause long-term depletion of brain 5-HT, while certain other substrates do not. The 5-HT deficits produced by SERT substrates are dependent upon transporter proteins, but the exact mechanisms responsible are unclear. Here, we compared the pharmacology of several SERT substrates: fenfluramine, d-fenfluramine, 1-(m-chlorophenyl)piperazine (mCPP) and 1-(m-trifluoromethylphenyl)piperainze (TFMPP), to establish relationships between acute drug mechanisms and the propensity for long-term 5-HT depletions. In vivo microdialysis was carried out in rat nucleus accumbens to examine acute 5-HT release and long-term depletion in the same subjects. In vitro assays were performed to measure efflux of [(3)H]5-HT in rat brain synaptosomes and transporter-mediated ionic currents in SERT-expressing Xenopus oocytes. When administered repeatedly to rats (6 mg/kg, i.p., four doses), all drugs produce large sustained elevations in extracellular 5-HT (>5-fold) with minimal effects on dopamine. Importantly, 2 weeks after dosing, only rats exposed to fenfluramine and d-fenfluramine display depletion of brain 5-HT. All test drugs evoke fluoxetine-sensitive efflux of [(3)H]5-HT from synaptosomes, but d-fenfluramine and its bioactive metabolite d-norfenfluramine induce significantly greater SERT-mediated currents than phenylpiperazines. Our data confirm that drug-induced 5-HT release probably does not mediate 5-HT depletion. However, the magnitude of transporter-mediated inward current may be a critical factor in the cascade of events leading to 5-HT deficits. This hypothesis warrants further study, especially given the growing popularity of designer drugs that target SERT.

Efflux pump induction by quaternary ammonium compounds and fluoroquinolone resistance in bacteria.

Science.gov (United States)

Buffet-Bataillon, Sylvie; Tattevin, Pierre; Maillard, Jean-Yves; Bonnaure-Mallet, Martine; Jolivet-Gougeon, Anne

2016-01-01

Biocides, primarily those containing quaternary ammonium compounds (QAC), are heavily used in hospital environments and various industries (e.g., food, water, cosmetic). To date, little attention has been paid to potential implications of QAC use in the emergence of antibiotic resistance, especially fluoroquinolone-resistant bacteria in patients and in the environment. QAC-induced overexpression of efflux pumps can lead to: cross resistance with fluoroquinolones mediated by multidrug efflux pumps; stress response facilitating mutation in the Quinolone Resistance Determining Region; and biofilm formation increasing the risk of transfer of mobile genetic elements carrying fluoroquinolone or QAC resistance determinants. By following the European Biocidal Product Regulation, manufacturers of QAC are required to ensure that their QAC-based biocidal products are safe and will not contribute to emerging bacterial resistance.

Effect of phosphorus limiting on phytase activity, proton efflux and ...

African Journals Online (AJOL)

This work intended to measure the nodulated-roots oxygen consumption, proton efflux and phytase activity in 2 lines of common bean (Phaseolus vulgaris) (115, 147) at 2 levels of P supply. Rooted seedlings were inoculated with Rhizobium tropici CIAT 899 in hydroaeroponic cultivation under glasshouse. Phosphorus was ...

Investigating the Role of the Host Multidrug Resistance Associated Protein Transporter Family in Burkholderia cepacia Complex Pathogenicity Using a Caenorhabditis elegans Infection Model.

Science.gov (United States)

Tedesco, Pietro; Visone, Marco; Parrilli, Ermenegilda; Tutino, Maria Luisa; Perrin, Elena; Maida, Isabel; Fani, Renato; Ballestriero, Francesco; Santos, Radleigh; Pinilla, Clemencia; Di Schiavi, Elia; Tegos, George; de Pascale, Donatella

2015-01-01

This study investigated the relationship between host efflux system of the non-vertebrate nematode Caenorhabditis elegans and Burkholderia cepacia complex (Bcc) strain virulence. This is the first comprehensive effort to profile host-transporters within the context of Bcc infection. With this aim, two different toxicity tests were performed: a slow killing assay that monitors mortality of the host by intestinal colonization and a fast killing assay that assesses production of toxins. A Virulence Ranking scheme was defined, that expressed the toxicity of the Bcc panel members, based on the percentage of surviving worms. According to this ranking the 18 Bcc strains were divided in 4 distinct groups. Only the Cystic Fibrosis isolated strains possessed profound nematode killing ability to accumulate in worms' intestines. For the transporter analysis a complete set of isogenic nematode single Multidrug Resistance associated Protein (MRP) efflux mutants and a number of efflux inhibitors were interrogated in the host toxicity assays. The Bcc pathogenicity profile of the 7 isogenic C. elegans MRP knock-out strains functionality was classified in two distinct groups. Disabling host transporters enhanced nematode mortality more than 50% in 5 out of 7 mutants when compared to wild type. In particular mrp-2 was the most susceptible phenotype with increased mortality for 13 out 18 Bcc strains, whereas mrp-3 and mrp-4 knock-outs had lower mortality rates, suggesting a different role in toxin-substrate recognition. The use of MRP efflux inhibitors in the assays resulted in substantially increased (>40% on average) mortality of wild-type worms.

Efflux Pumps Might Not Be the Major Drivers of QAC Resistance in Methicillin-Resistant Staphylococcus aureus.

Science.gov (United States)

Jennings, Megan C; Forman, Megan E; Duggan, Stephanie M; Minbiole, Kevin P C; Wuest, William M

2017-08-17

Quaternary ammonium compounds (QACs) are commonly used antiseptics that are now known to be subject to bacterial resistance. The prevalence and mechanisms of such resistance, however, remain underexplored. We investigated a variety of QACs, including those with multicationic structures (multiQACs), and the resistance displayed by a variety of Staphylococcus aureus strains with and without genes encoding efflux pumps, the purported main driver of bacterial resistance in MRSA. Through minimum inhibitory concentration (MIC)-, kinetic-, and efflux-based assays, we found that neither the qacR/qacA system present in S. aureus nor another efflux pump system is the main reason for bacterial resistance to QACs. Our findings suggest that membrane composition could be the predominant driver that allows CA-MRSA to withstand the assault of conventional QAC antiseptics. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inactivation of a putative efflux pump (LmrB) in Streptococcus mutans results in altered biofilm structure and increased exopolysaccharide synthesis: implications for biofilm resistance.

Science.gov (United States)

Liu, Jia; Zhang, Jianying; Guo, Lihong; Zhao, Wei; Hu, Xiaoli; Wei, Xi

2017-07-01

Efflux pumps are a mechanism associated with biofilm formation and resistance. There is limited information regarding efflux pumps in Streptococcus mutans, a major pathogen in dental caries. The aim of this study was to investigate potential roles of a putative efflux pump (LmrB) in S. mutans biofilm formation and susceptibility. Upon lmrB inactivation and antimicrobial exposure, the biofilm structure and expression of other efflux pumps were examined using confocal laser scanning microscopy (CLSM) and qRT-PCR. lmrB inactivation resulted in biofilm structural changes, increased EPS formation and EPS-related gene transcription (p < 0.05), but no improvement in susceptibility was observed. The expression of most efflux pump genes increased upon lmrB inactivation when exposed to antimicrobials (p < 0.05), suggesting a feedback mechanism that activated the transcription of other efflux pumps to compensate for the loss of lmrB. These observations imply that sole inactivation of lmrB is not an effective solution to control biofilms.

Do Phytotropins Inhibit Auxin Efflux by Impairing Vesicle Traffic?

Czech Academy of Sciences Publication Activity Database

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

2003-01-01

Roč. 131, č. 1 (2003), s. 254-263 ISSN 0032-0889 R&D Projects: GA MŠk LN00A081 Grant - others:EU INCO COPERNICUS(XE) ERBIC15 CT98 0118 Institutional research plan: CEZ:AV0Z5038910 Keywords : 1-N-naphthylphthalamic acid * BY-2 tobacco * auxin efflux Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.634, year: 2003

Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2).

Science.gov (United States)

Edwards, Noel; Anderson, Catriona M H; Gatfield, Kelly M; Jevons, Mark P; Ganapathy, Vadivel; Thwaites, David T

2011-01-01

The H(+)-coupled amino acid transporter PAT2 (SLC36A2) transports the amino acids proline, glycine, alanine and hydroxyproline. A physiological role played by PAT2 in amino acid reabsorption in the renal proximal tubule is demonstrated by mutations in SLC36A2 that lead to an iminoglycinuric phenotype (imino acid and glycine uria) in humans. A number of proline, GABA and tryptophan derivatives were examined to determine if they function either as transported substrates or non-transported inhibitors of PAT2. The compounds were investigated following heterologous expression of rat PAT2 in Xenopus laevis oocytes. PAT2 function was characterised by: radiotracer uptake and competition (cis-inhibition) studies; radiotracer efflux and trans-stimulation; and measurement of substrate-induced positive inward current by two-electrode voltage-clamp. In general, the proline derivatives appeared to be transported substrates and the relative ability to induce current flow was closely related to the inhibitory effects on PAT2-mediated l-[(3)H]proline uptake. In contrast, certain heterocyclic GABA derivatives (e.g. l-pipecolic acid) were translocated only slowly. Finally, the tryptophan derivatives inhibited PAT2 function but did not undergo transport. l-Proline uptake was inhibited by 5-hydroxy-l-tryptophan (IC(50) 1.6±0.4mM), α-methyl-d,l-tryptophan (3.5±1.5mM), l-tryptophan, 1-methyl-l-tryptophan and indole-3-propionic acid. Although neither 5-hydroxy-l-tryptophan nor α-methyl-d,l-tryptophan were able to elicit inward current in PAT2-expressing oocytes both reduced the current evoked by l-proline. 5-Hydroxy-l-tryptophan and α-methyl-d,l-tryptophan were unable to trans-stimulate l-proline efflux from PAT2-expressing oocytes, confirming that the two compounds act as non-transported blockers of PAT2. These two tryptophan derivatives should prove valuable experimental tools in future investigations of the physiological roles of PAT2. Copyright © 2010 Elsevier B.V. All rights

In vivo evaluation of thiolated poly(acrylic acid) as a drug absorption modulator for MRP2 efflux pump substrates.

Science.gov (United States)

Greindl, Melanie; Föger, Florian; Hombach, Juliane; Bernkop-Schnürch, Andreas

2009-08-01

Recently, several polymers have been reported to modulate drug absorption by inhibition of intestinal efflux pumps such as multidrug resistance proteins (MRPs) and P-glycoprotein (P-gp). The aim of the present study was to evaluate the efficiency of thiolated poly(acrylic acid) (PAA-Cys) to act as a drug absorption modulator for MRP2 efflux pump substrates in vivo, using sulforhodamine 101 as representative MRP2 substrate. In vitro, the permeation-enhancing effect of unmodified PAA and PAA(250)-Cys(,) displaying 580 micromol free thiol groups per gram polymer, was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to that of the buffer control, the sulforhodamine 101 transport in the presence of 0.5% unmodified PAA(250) and 0.5% (w/v) PAA(250)-Cys was 1.3- and 4.0-fold improved, respectively. In vivo, sulforhodamine 101 solutions containing 4% (w/v) unmodified PAA(250) or 4% (w/v) thiolated PAA(250) were orally given to rats. The PAA(250)-Cys solution increased the area under the plasma concentration-time curve (AUC(0-12)) of sulforhodamine 101 3.8-fold in comparison to control and 2.2-fold in comparison to unmodified PAA(250). This in vivo study revealed that PAA(250)-Cys significantly increased the oral bioavailability of MRP2 substrate sulforhodamine 101.

Transport of curcumin derivatives in Caco-2 cell monolayers.

Science.gov (United States)