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Efflux Activity (efflux + activity)
Selected AbstractsQSAR study of ,-lactam antibiotic efflux by the bacterial multidrug resistance pump AcrB,JOURNAL OF CHEMOMETRICS, Issue 5 2004Márcia M. C. Ferreira Abstract AcrAB-TolC is the most important efflux pump system of Gram-negative bacteria, responsible for their resistance to lipophilic and amphilic drugs. In this work, HCA,PCA studies were performed to investigate the relationship between efflux activities (negative logarithm of minial inhibitor concentration, pMIC) of three strains of S. thypimurium with respect to ,-lactams, and to analyze the relationship between lipophilicity parameters calculated by different methods. The analyses demonstrate that pMICs strongly depend on properties of both bacterial strains and drug molecules, and that lipophilicity parameters do not necessarily contain the same information about the drugs. QSAR studies have shown that the calculated lipophilicities, in some cases, are non linearly related to the pMICs originated by active AcrAB-TolC bacterial pumps, due to the existence of ,-lactams with nitrogen- and sulfur-rich substituents. Among the most important ,-lactam molecular properties quantitatively related to pMICs are lipophilicity and electronic and hydrogen,bonding properties. Parameters describing these properties were included in the QSAR study to obtain parsimonius regression models for MICs. ,-Lactams were classified as good, moderately good and poor AcrAB-TolC substrates. Their stereoelectronic molecular properties, especially the Y-component of the molecular dipole moment and hydrogen binding properties, reflected this classification. Copyright © 2004 John Wiley & Sons, Ltd. [source] Increase in multidrug transport activity is associated with oocyte maturation in sea stars,DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2006Troy A. Roepke In this study, we report on the presence of efflux transporter activity before oocyte maturation in sea stars and its upregulation after maturation. This activity is similar to the multidrug resistance (MDR) activity mediated by ATP binding cassette (ABC) efflux transporters. In sea star oocytes the efflux activity, as measured by exclusion of calcein-am, increased two-fold 3 h post-maturation. Experiments using specific and non-specific dyes and inhibitors demonstrated that the increase in transporter activity involves an ABCB protein, P-glycoprotein (P-gp), and an ABCC protein similar to the MDR-associated protein (MRP)-like transporters. Western blots using an antibody directed against mammalian P-gp recognized a 45 kDa protein in sea star oocytes that increased in abundance during maturation. An antibody directed against sea urchin ABCC proteins (MRP) recognized three proteins in immature oocytes and two in mature oocytes. Experiments using inhibitors suggest that translation and microtubule function are both required for post-maturation increases in transporter activity. Immunolabeling revealed translocation of stored ABCB proteins to the plasma cell membrane during maturation, and this translocation coincided with increased transport activity. These MDR transporters serve protective roles in oocytes and eggs, as demonstrated by sensitization of the oocytes to the maturation inhibitor, vinblastine, by MRP and PGP-specific transporter inhibitors. [source] Formation of cholesterol-enriched structures by aberrant intracellular accumulation of ATP-binding cassette transporter A1GENES TO CELLS, Issue 8 2008Arowu R. Tanaka ATP-binding cassette transporter A1 (ABCA1) is a key transporter associated with excess cellular lipid efflux. Here, we report that in HEK293 cells ABCA1 functions in intracellular compartments along the endocytic pathway. Inhibition of ABCA1-GFP degradation with proteasome inhibitors induced the internalization of ABCA1 and the formation of intracellular round-shaped structures, designated "A1 bodies". Importantly, cholesterol was selectively accumulated in A1 bodies, and this depended on the cholesterol efflux activity of ABCA1. Treatment with either lactacystin or acetylated LDL, which reduces proteasome activity, resulted in internalization of ABCA1 in mouse peritoneal macrophages. By performing array analysis on macrophages treated with these reagents, we identified Rab4 as a key protein involved in the internalization and aberrant accumulation of ABCA1 in HEK cells. Treatment of the cells with proteasome inhibitors inhibited the degradation of Rab4, and Rab4 over-expression induced the formation of small A1 bodies. Furthermore, A1 bodies formation was substantially inhibited by silencing of the endogenous Rab4 gene. Taken together, our findings suggest that the endocytic ABCA1 possesses cholesterol efflux activity, and thus the cellular control of post-endocytic sorting, retention or recycling of functional ABCA1 in the endocytic vesicles, which is in part regulated by Rab4, is important for cholesterol metabolism in living cells. [source] Effluxing ABC transporters in human corneal epitheliumJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 2 2010Kati-Sisko Vellonen Abstract ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1,6 (MRP1,6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed over-expression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:1087,1098, 2010 [source] YB-1 is upregulated during prostate cancer tumor progression and increases P-glycoprotein activityTHE PROSTATE, Issue 3 2004Pepita Giménez-Bonafé Abstract BACKGROUND Currently, the main obstacle to curing advanced prostate cancer is development of androgen independence (AI), where malignant cells acquire the ability to survive in the absence of androgens. Our initial experimental approach used cDNA microarrays to characterize changes in gene expression in the LNCaP human prostate tumor model during progression to AI. The transcription factor Y-box binding protein (YB-1) was shown to be one of the genes upregulated. We focused on increased YB-1 expression during progression in clinical specimens, and further examined one of its downstream targets, P-glycoprotein (P-gp). METHODS Northern blot analysis was performed on LNCaP tumor series, as well as immunohistochemical analyses of human prostate cancer tissue samples. YB-1 was transiently transfected and transport analysis were performed to analyze P-gp efflux activity. RESULTS YB-1 expression is markedly increased during benign to malignant transformation and further following androgen ablation. In addition, increased YB-1 expression after castration in the LNCaP model is linked to upregulation of P-gp. We demonstrate that YB-1 upregulates P-gp activity resulting in a 40% intracellular decrease in the P-gp substrate vinblastine. We have also found that P-gp increases the efflux of the endogenous androgen, dihydrotestosterone (DHT), from prostate cells and leads to decreased androgen regulated gene expression. CONCLUSIONS We hypothesize that early in prostate cancer progression, increased expression of YB-1 may increase P-gp activity which may in turn lower androgen levels in the prostate tumor cells. Suppression of androgen levels may activate cell survival pathways and lead to an adaptive survival advantage of androgen independent prostate cancer cells following androgen ablation therapy. © 2004 Wiley-Liss, Inc. [source] Characterization of efflux proteins in human corneal epithelial cellsACTA OPHTHALMOLOGICA, Issue 2007KS VELLONEN Purpose: Corneal epithelium is the main barrier for absorption of drugs into intraocular tissues after topical administration and part of this barrier may be formed by efflux proteins which translocate molecules from the cell interior to the extracellular space. The aim of this study was to characterize the gene expression and the activity of the efflux transporters in the cell culture model of immortalized human corneal epithelial cells (HCE cells), in primary cell line (HCEpiC), and in the human corneal epithelium. Methods: The mRNA levels of MDR1, MRP1-MRP6, and BCRP were determined by the quantitative RT-PCR. Immunohistochemistry was used to study protein expression and localization of efflux transporters. Functionality of these proteins was assessed with calcein-AM efflux assay and by measuring the efflux of CDCF. Furthermore, bidirectional permeability of rhodamine 123 (Rh123) was studied. Results: The mRNA of MRP1 and MRP5 were detected in the human cornea and in both cell lines. These efflux proteins were found in the cell membranes of the human corneal epithelium. At mRNA level some efflux proteins were over-expressed in the HCE and the primary cell lines. Increased calcein retention and decreased CDCF efflux in the presence of inhibitors suggested efflux protein activity in both primary and HCE cells. Likewise, directionality in Rh123 permeability was diminished in the presence of verapamil in HCE model. Conclusions: Functionality of the efflux proteins was demonstrated in the human corneal epithelial cells. MRP1 and MRP5 proteins may have important protecting role in corneal surface by transporting molecules out from the epithelial cells. It seems that the efflux activity in the HCE model differs from that of the corneal epithelium in vivo [source] | ||||||||||||||||