30 research outputs found

    Hydrogeochemistry, Geothermometry, and Sourcing of High Dissolved Boron, Tungsten, and Chlorine Concentrations in the Trans-Himalayan Hotsprings of Ladakh, India

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    Boron (B) and Tungsten (W) are often found enriched in high-temperature geothermal waters associated with the development of subduction-related mafic to felsic arc magma. However, knowledge about the sourcing and transportation of these elements from such hydrothermal systems is sparse and ambiguous. Being the only active continental collision site in the world, the Trans-Himalaya offers a unique chance to study how continental collision sources the high boron and tungsten concentrations in geothermal fluids. This study investigated the distribution of trace elements, major cations, and anions in three physicochemically distinct hotspring sites in the Ladakh region. The results were integrated with the existing geochemical and isotopic data to address the research problem more effectively. This study exhibits that the extreme concentrations of boron, sodium, chlorine, potassium, and tungsten in the hotspring waters were primarily governed by magmatic fluid inputs. In addition, this study recorded the highest-ever chlorine and boron concentrations for the Trans-Himalayan hotspring waters. The highest-ever boron and chlorine concentrations in the hotspring waters probably represented an increase in magmatic activity in the deeper source zone

    Multiple Transport-Active Binding Sites Are Available for a Single Substrate on Human P-Glycoprotein (ABCB1)

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    <div><p>P-glycoprotein (Pgp, ABCB1) is an ATP-Binding Cassette (ABC) transporter that is associated with the development of multidrug resistance in cancer cells. Pgp transports a variety of chemically dissimilar amphipathic compounds using the energy from ATP hydrolysis. In the present study, to elucidate the binding sites on Pgp for substrates and modulators, we employed site-directed mutagenesis, cell- and membrane-based assays, molecular modeling and docking. We generated single, double and triple mutants with substitutions of the Y307, F343, Q725, F728, F978 and V982 residues at the proposed drug-binding site with cys in a cysless Pgp, and expressed them in insect and mammalian cells using a baculovirus expression system. All the mutant proteins were expressed at the cell surface to the same extent as the cysless wild-type Pgp. With substitution of three residues of the pocket (Y307, Q725 and V982) with cysteine in a cysless Pgp, QZ59S-<i>SSS</i>, cyclosporine A, tariquidar, valinomycin and FSBA lose the ability to inhibit the labeling of Pgp with a transport substrate, [<sup>125</sup>I]-Iodoarylazidoprazosin, indicating these drugs cannot bind at their primary binding sites. However, the drugs can modulate the ATP hydrolysis of the mutant Pgps, demonstrating that they bind at secondary sites. In addition, the transport of six fluorescent substrates in HeLa cells expressing triple mutant (Y307C/Q725C/V982C) Pgp is also not significantly altered, showing that substrates bound at secondary sites are still transported. The homology modeling of human Pgp and substrate and modulator docking studies support the biochemical and transport data. In aggregate, our results demonstrate that a large flexible pocket in the Pgp transmembrane domains is able to bind chemically diverse compounds. When residues of the primary drug-binding site are mutated, substrates and modulators bind to secondary sites on the transporter and more than one transport-active binding site is available for each substrate.</p> </div

    BIRB796, the inhibitor of p38 mitogen-activated protein kinase, enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cells.

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    ATP-binding-cassette family membrane proteins play an important role in multidrug resistance. In this study, we investigated BIRB796, an orally active inhibitor of p38 mitogen-activated protein kinase, reversed MDR induced by ABCB1, ABCG2 and ABCC1. Our results showed that BIRB796 could reverse ABCB1-mediated MDR in both the drug selected and transfected ABCB1-overexpressing cell models, but did not enhance the efficacy of substrate-chemotherapeutical agents in ABCC1 or ABCG2 overexpression cells and their parental sensitive cells. Furthermore, BIRB796 increased the intracellular accumulation of the ABCB1 substrates, such as rhodamine 123 and doxorubicin. Moreover, BIRB796 bidirectionally mediated the ATPase activity of ABCB1, stimulating at low concentration, inhibiting at high concentration. However, BIRB796 did not alter the expression of ABCB1 both at protein and mRNA level. The down-regulation of p38 by siRNA neither affected the expression of ABCB1 nor the cytotoxic effect of paclitaxel on KBV200. The binding model of BIRB796 within the large cavity of the transmembrane region of ABCB1 may form the basis for future lead optimization studies. Importantly, BIRB796 also enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBV200 cell xenografts in nude mice. Overall, we conclude that BIRB796 reverses ABCB1-mediated MDR by directly inhibiting its transport function. These findings may be useful for cancer combinational therapy with BIRB796 in the clinic

    The Phosphodiesterase-5 Inhibitor Vardenafil Is a Potent Inhibitor of ABCB1/P-Glycoprotein Transporter

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    One of the major causes of chemotherapy failure in cancer treatment is multidrug resistance (MDR) which is mediated by the ABCB1/P-glycoprotein. Previously, through the use of an extensive screening process, we found that vardenafil, a phosphodiesterase 5 (PDE-5) inhibitor significantly reverses MDR in ABCB1 overexpressing cancer cells, and its efficacy was greater than that of tadalafil, another PDE-5 inhibitor. The present study was designed to determine the reversal mechanisms of vardenafil and tadalafil on ABC transporters-mediated MDR. Vardenafil or tadalafil alone, at concentrations up to 20 µM, had no significant toxic effects on any of the cell lines used in this study, regardless of their membrane transporter status. However, vardenafil when used in combination with anticancer substrates of ABCB1, significantly potentiated their cytotoxicity in ABCB1 overexpressing cells in a concentration-dependent manner, and this effect was greater than that of tadalafil. The sensitivity of the parenteral cell lines to cytotoxic anticancer drugs was not significantly altered by vardenafil. The differential effects of vardenafil and tadalafil appear to be specific for the ABCB1 transporter as both vardenafil and tadalafil had no significant effect on the reversal of drug resistance conferred by ABCC1 (MRP1) and ABCG2 (BCRP) transporters. Vardenafil significantly increased the intracellular accumulation of [3H]-paclitaxel in the ABCB1 overexpressing KB-C2 cells. In addition, vardenafil significantly stimulated the ATPase activity of ABCB1 and inhibited the photolabeling of ABCB1 with [125I]-IAAP. Furthermore, Western blot analysis indicated the incubation of cells with either vardenafil or tadalafil for 72 h did not alter ABCB1 protein expression. Overall, our results suggest that vardenafil reverses ABCB1-mediated MDR by directly blocking the drug efflux function of ABCB1

    XP-Glide predicted binding mode of BIRB796 with homology modeled ABCB1.

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    <p>A, Binding mode of BIRB796 within the drug binding site-1 of human ABCB1. Important amino acids are depicted as sticks with the atoms colored as carbon – green, hydrogen – white, nitrogen – blue, oxygen – red, sulfur – yellow, whereas the BIRB796 is shown as ball and stick model with the same color scheme as above except carbon atoms are represented in orange. Dotted red line indicates distances. Human ABCB1 (B) and p38 MAP kinase (C) are represented as Macromodel surface as per the residue charge (electropositive charge: blue, electronegative charge: red, neutral (hydrophobic): yellow) as implemented in Maestro. Docked conformation of BIRB796 within the drug binding site-1 of human ABCB1 and bound BIRB796 within the allosteric site of p38 MAP kinase is also shown as ball and stick model.</p

    Effect of BIRB796 on reversing ABCG2- and ABCC1 -mediated drug resistance.

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    <p>Note: cell survival was determined by MTT assay as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054181#s2" target="_blank">Materials and Methods</a>”. Data are the means ± SD of at least three independent experiments performed in triplicate. The fold-reversal of MDR was calculated by dividing the IC<sub>50</sub> for cells with the anticancer drug in the absence of inhibitor by that obtained in the presence of inhibitor. a represent <i>P</i><0.01, for values versus that obtained in the absence of inhibitor.</p

    Inhibition of photocrosslinking of mutant Pgps with IAAP by cyclosporine A and tariquidar.

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    <p>Inhibition of IAAP labeling for single mutants Q725C, Y307C, F728C and V982C (upper graphs) and for double Q725C/V982C, Y307C/V982C, F728C/V982C, and triple Y307C/Q725C/V982C (lower graphs) mutants at different concentrations of (A) CsA and (B) tariquidar, are shown. Inhibition of IAAP labeling for cysless WT is included in all graphs, as a reference. Autoradiograms corresponding to cysless, V982C and Y307C/Q725C/V982C, as representative examples of complete inhibition, partial inhibition and no inhibition of IAAP-labeling, respectively, are shown at the top of the figure. Crude membranes containing Pgp (60-80 µg protein) were treated with increasing concentrations of CsA and tariquidar in 100 µL buffer containing 50 mM MES-Tris pH 6.8 for 10 min at 37°C. Then, samples were photocrosslinked with IAAP at 4°C as described in the Materials and Methods section. Mean values from at least three independent experiments are plotted and error bars depict SD.</p
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