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Deciphering the Role of RND Efflux Transporters in Burkholderia cenocepacia  [PDF]
Silvia Bazzini,Claudia Udine,Andrea Sass,Maria Rosalia Pasca,Francesca Longo,Giovanni Emiliani,Marco Fondi,Elena Perrin,Francesca Decorosi,Carlo Viti,Luciana Giovannetti,Livia Leoni,Renato Fani,Giovanna Riccardi,Eshwar Mahenthiralingam,Silvia Buroni
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0018902
Abstract: 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.
Identification and Analysis of the Putative Pentose Sugar Efflux Transporters in Escherichia coli  [PDF]
Khushnuma Koita, Christopher V. Rao
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0043700
Abstract: Escherichia coli possesses a number of proteins that transport sugars out of the cell. We identified 31 candidate sugar efflux transporters based on their similarity to known sugar efflux transporters. We then tested whether these transporters affect arabinose and xylose metabolism. We identified 13 transporters – setC, cmr, ynfM, mdtD, yfcJ, yhhS, emrD, ydhC, ydeA, ybdA, ydeE, mhpT, and kgtP – that appeared to increase or decrease intracellular arabinose concentrations when respectively deleted or over-expressed. None of the candidate transporters affected xylose concentrations. These results indicate that E. coli possesses multiple arabinose efflux transporters. They also provide a novel target for future metabolic engineering.
Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance
Silvia Buroni, Maria R Pasca, Ronald S Flannagan, Silvia Bazzini, Anna Milano, Iris Bertani, Vittorio Venturi, Miguel A Valvano, Giovanna Riccardi
BMC Microbiology , 2009, DOI: 10.1186/1471-2180-9-200
Abstract: To investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed.Two of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of phenotypically similar Gram-negative bacteria [1] that are opportunistic pathogens and sometimes cause serious life-threatening infections in cystic fibrosis (CF) patients [2,3]. Infection in CF patients may result in asymptomatic carriage, but often leads to a rapid decline of the lung function and in some cases to the "cepacia syndrome", characterized by necrotizing pneumonia and sepsis [4].B. cenocepacia and other members of the Bcc demonstrate high-levels of intrinsic resistance to most cl
Protection against Oxidative Stress in Beta Thalassemia/Hemoglobin E Erythrocytes by Inhibitors of Glutathione Efflux Transporters  [PDF]
Chatchai Muanprasat, Chokdee Wongborisuth, Nutthapoom Pathomthongtaweechai, Saravut Satitsri, Suradej Hongeng
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0055685
Abstract: In beta thalassemia/hemoglobin E (Hb E), abnormally high levels of oxidative stress account for accelerated senescence and increased destruction of erythrocytes. The present study aimed to investigate the role of glutathione efflux transporters, namely cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein 1 (MRP1), in the control of glutathione levels and protection against oxidative challenges in beta thalassemia/Hb E erythrocytes. We found that CFTR protein was expressed in the erythrocytes of beta thalassemia/Hb E patients. Treatments with GlyH-101 (50 μM), a small molecule CFTR inhibitor, and MK571 (50 μM), an MRP1 inhibitor, reduced H2O2-induced free radical generation in the erythrocytes by ~80% and 50%, respectively. Furthermore, combined treatment with GlyH-101 and MK571 completely abolished the induction of reactive oxygen radicals. Increased oxidative stress in the erythrocytes following H2O2 challenges was accompanied by a decrease in intracellular level of reduced glutathione (GSH), which was prevented by treatments with GlyH-101 and MK571. CMFDA-based assays revealed that GlyH-101 and MK571 reduced H2O2-induced glutathione efflux from the erythrocytes by 87% and 66%, respectively. Interestingly, H2O2-induced osmotic tolerance of erythrocytes, a sign of erythrocyte aging, was ameliorated by treatment with GlyH-101. Our study indicates that oxidative stress induces glutathione efflux via CFTR and MRP1 in beta thalassemia/Hb E erythrocytes. Pharmacological inhibition of glutathione efflux represents a potential therapy to delay aging and premature destruction of erythrocytes in beta thalassemia/Hb E.
Expression of ABC Efflux Transporters in Placenta from Women with Insulin-Managed Diabetes  [PDF]
Gregory J. Anger, Alex M. Cressman, Micheline Piquette-Miller
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0035027
Abstract: Drug efflux transporters in the placenta can significantly influence the materno-fetal transfer of a diverse array of drugs and other xenobiotics. To determine if clinically important drug efflux transporter expression is altered in pregnancies complicated by gestational diabetes mellitus (GDM-I) or type 1 diabetes mellitus (T1DM-I), we compared the expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2) and the breast cancer resistance protein (BCRP) via western blotting and quantitative real-time polymerase chain reaction in samples obtained from insulin-managed diabetic pregnancies to healthy term-matched controls. At the level of mRNA, we found significantly increased expression of MDR1 in the GDM-I group compared to both the T1DM-I (p<0.01) and control groups (p<0.05). Significant changes in the placental protein expression of MDR1, MRP2, and BCRP were not detected (p>0.05). Interestingly, there was a significant, positive correlation observed between plasma hemoglobin A1c levels (a retrospective marker of glycemic control) and both BCRP protein expression (r = 0.45, p<0.05) and BCRP mRNA expression (r = 0.58, p<0.01) in the insulin-managed DM groups. Collectively, the data suggest that the expression of placental efflux transporters is not altered in pregnancies complicated by diabetes when hyperglycemia is managed; however, given the relationship between BCRP expression and plasma hemoglobin A1c levels it is plausible that their expression could change in poorly managed diabetes.
Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters  [PDF]
Walid Fayad, M?rten Frykn?s, Slavica Brnjic, Maria H?gg Olofsson, Rolf Larsson, Stig Linder
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0007238
Abstract: 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.
In Vitro Drug Response and Efflux Transporters Associated with Drug Resistance in Pediatric High Grade Glioma and Diffuse Intrinsic Pontine Glioma  [PDF]
Susanna J. E. Veringa, Dennis Biesmans, Dannis G. van Vuurden, Marc H. A. Jansen, Laurine E. Wedekind, Ilona Horsman, Pieter Wesseling, William Peter Vandertop, David P. Noske, GertJan J. L. Kaspers, Esther Hulleman
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0061512
Abstract: 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.
Transcriptional Elongation and mRNA Export Are Coregulated Processes  [PDF]
Maria Micaela Molina-Navarro,Celia Pilar Martinez-Jimenez,Susana Rodriguez-Navarro
Genetics Research International , 2011, DOI: 10.4061/2011/652461
Abstract: Chromatin structure complexity requires the interaction and coordinated work of a multiplicity of factors at different transcriptional regulation stages. Transcription control comprises a set of processes that ensures proper balance in the gene expression under different conditions, such as signals, metabolic states, or development. We could frame those steps from epigenetic marks to mRNA stability to support the holistic view of a fine-tune balance of final mRNA levels through mRNA transcription, export, stability, translation, and degradation. Transport of mRNA from the nucleus to the cytoplasm is a key process in regulated gene expression. Transcriptional elongation and mRNA export are coregulated steps that determine the mature mRNA levels in the cytoplasm. In this paper, recent insights into the coordination of these processes in eukaryotes will be summarised. 1. Introduction Gene expression is a coordinated multistep process that begins with transcription and RNA processing in the nucleus, followed by mRNA export to the cytoplasm for translation. In eukaryotes, it depends on the orchestrated action of several multiprotein complexes which regulate the gene expression by RNA polymerase II (RNAP-II) at multiple levels. Given the association of DNA with nucleosomes, they become a physical barrier for the transcription elongation by RNAP-II, where histone chaperones play an essential role in facilitating transcription in vitro and in vivo, and by establishing a bridge between RNAP-II and elongation factors (reviewed in [1]). Interestingly, recent genome-wide profiling has provided a partial picture of the chromatin landscape, including a wide variety of epigenetic information about posttranslational modifications (PTMs), histone variants, DNA methylation patterns, and nucleosome occupancy (reviewed in [2]). In this scenario, histone turnover determines continuous access to sequence-specific DNA-binding proteins; therefore, nucleosome positioning is actively involved in gene-expression regulation. Mechanisms to export mRNA are integrated into the biogenesis of messenger ribonucleoparticles (mRNPs). In this context, the nuclear pore complex (NPC) provides a molecular environment that regulates gene tethering and mRNA export. Moreover, a functional connection between transcription and RNA export has been reinforced by human genetic disorders caused by mutation in mRNA export factors and adaptors [3, 4]. mRNA biogenesis requires the transport of competent mRNPs to the cytoplasm, coupling nuclear export, and formation of mature RNA. This process is complex
Quantification of Protoporphyrin IX Accumulation in Glioblastoma Cells: A New Technique  [PDF]
Johnathan E. Lawrence,Ashish S. Patel,Richard A. Rovin,Robert J. Belton Jr.,Catherine E. Bammert,Christopher J. Steele,Robert J. Winn
ISRN Surgery , 2014, DOI: 10.1155/2014/405360
Abstract: Introduction. 5-Aminolevulinic Acid (5-ALA) is a precursor of heme synthesis. A metabolite, protoporphyrin IX (PpIX), selectively accumulates in neoplastic tissue including glioblastoma. Presurgical administration of 5-ALA forms the basis of fluorescence-guided resection (FGR) of glioblastoma (GBM) tumors. However, not all gliomas accumulate sufficient quantities of PpIX to fluoresce, thus limiting the utility of FGR. We therefore developed an assay to determine cellular and pharmacological factors that impact PpIX fluorescence in GBM. This assay takes advantage of a GBM cell line engineered to express yellow fluorescent protein. Methods. The human GBM cell line U87MG was transfected with a YFP expression vector. After treatment with a series of 5-ALA doses, both PpIX and YFP fluorescence were measured. The ratio of PpIX to YFP fluorescence was calculated. Results. YFP fluorescence permitted the quantification of cell numbers and did not interfere with 5-ALA metabolism. The PpIX/YFP fluorescence ratio provided accurate relative PpIX levels, allowing for the assessment of PpIX accumulation in tissue. Conclusion. Constitutive YFP expression strongly correlates with cell number and permits PpIX quantification. Absolute PpIX fluorescence alone does not provide information regarding PpIX accumulation within the cells. Our research indicates that our PpIX/YFP ratio assay may be a promising model for in vitro 5-ALA testing and its interactions with other compounds during FGR surgery. 1. Introduction Glioblastoma multiforme (GBM) is the most common and aggressive primary malignant brain tumor. With the current standard of care, recurrence is inevitable and less than 5% of GBM patients will survive five years after diagnosis [1]. Subtotal surgical resection may contribute to recurrence as tumor initiating cells are left in place. A valuable tool to achieve gross total resection and thereby increase progression-free survival is the use of 5-Aminolevulinic Acid (5-ALA) for fluorescence-guided resection (FGR) [2]. 5-ALA is a substrate of the heme biosynthesis pathway and is the source of carbon for porphyrin synthesis [3]. Malignant gliomas metabolize 5-ALA and accumulate the fluorescent compound, protoporphyrin IX (PpIX) [4]. The presence of PpIX fluorescence within the tumor bed allows for discrimination between neoplastic and nonneoplastic cells [4]. Yet, not all brain tumors accumulate sufficient quantities of PpIX to make the use of 5-ALA advantageous during surgery [5, 6], and it is not fully understood how 5-ALA might affect other physiological processes. At
13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages
Ines K?mmerer, Robert Ringseis, Ronald Biemann, Gaiping Wen, Klaus Eder
Lipids in Health and Disease , 2011, DOI: 10.1186/1476-511x-10-222
Abstract: RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux.Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells.13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.Although dietary consumption of oxidized fats (OF) is known to cause some unfavourable effects (e.g., oxidative stress, depletion of antioxidants; [1-3]), experiments in laboratory animals and pigs consistently demonstrated that administration of OF reduces lipid concentrations (triacylglycerols and cholesterol) in liver and plasma (reviewed in [4]). Recent evidence suggests that activation of the peroxisome proliferator-activated receptor α (PPARα) pathway in the liver is largely responsible for the lipid lowering action of OF [5-7]. PPARα is a ligand-activated transcription factor which controls a comprehensive set of genes involved in most aspects of lipid catabolism [8,9]. Thus, targeting PPARα by the administration of pharmacological PPARα activators, e.g., fenofibrate, bezafibrate, gemfibrozil, is an effective approach for the treatment of hyperlipidemia [10].Besides targeting lipid catabolism in the liver and regulating plasma lipid concentrations, synthetic PPAR
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