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Sample records for atp-binding cassette transporters

  1. Regulatory pathways for ATP-binding cassette transport proteins in kidney proximal tubules

    NARCIS (Netherlands)

    Masereeuw, R.; Russel, F.G.M.

    2012-01-01

    The ATP-binding cassette transport proteins (ABC transporters) represent important determinants of drug excretion. Protective or excretory tissues where these transporters mediate substrate efflux include the kidney proximal tubule. Regulation of the transport proteins in this tissue requires elabor

  2. Atovaquone and quinine anti-malarials inhibit ATP binding cassette transporter activity

    NARCIS (Netherlands)

    Rijpma, S.R.; Heuvel, J.J.; Velden, M. van der; Sauerwein, R.W.; Russel, F.G.; Koenderink, J.B.

    2014-01-01

    BACKGROUND: Therapeutic blood plasma concentrations of anti-malarial drugs are essential for successful treatment. Pharmacokinetics of pharmaceutical compounds are dependent of adsorption, distribution, metabolism, and excretion. ATP binding cassette (ABC) transport proteins are particularly involve

  3. Multidrug transport by ATP binding cassette transporters : a proposed two-cylinder engine mechanism

    NARCIS (Netherlands)

    van Veen, HW; Higgins, CF; Konings, WN

    2001-01-01

    The elevated expression of ATP binding cassette (ABC) multidrug transporters in multidrug-resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. This review summarizes current insi

  4. Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast

    DEFF Research Database (Denmark)

    Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D.;

    2014-01-01

    Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been i...

  5. Transport in technicolor: mapping ATP-binding cassette transporters in sea urchin embryos.

    Science.gov (United States)

    Gökirmak, Tufan; Shipp, Lauren E; Campanale, Joseph P; Nicklisch, Sascha C T; Hamdoun, Amro

    2014-09-01

    One quarter of eukaryotic genes encode membrane proteins. These include nearly 1,000 transporters that translocate nutrients, signaling molecules, and xenobiotics across membranes. While it is well appreciated that membrane transport is critical for development, the specific roles of many transporters have remained cryptic, in part because of their abundance and the diversity of their substrates. Multidrug resistance ATP-binding cassette (ABC) efflux transporters are one example of cryptic membrane proteins. Although most organisms utilize these ABC transporters during embryonic development, many of these transporters have broad substrate specificity, and their developmental functions remain incompletely understood. Here, we review advances in our understanding of ABC transporters in sea urchin embryos, and methods developed to spatially and temporally map these proteins. These studies reveal that multifunctional transporters are required for signaling, homeostasis, and protection of the embryo, and shed light on how they are integrated into ancestral developmental pathways recapitulated in disease.

  6. Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes.

    Science.gov (United States)

    Schneider, Erwin; Eckey, Viola; Weidlich, Daniela; Wiesemann, Nicole; Vahedi-Faridi, Ardeshir; Thaben, Paul; Saenger, Wolfram

    2012-04-01

    ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium.

  7. ATP-binding cassette transporters in tumor endothelial cells and resistance to metronomic chemotherapy.

    Science.gov (United States)

    Hida, Kyoko; Kikuchi, Hiroshi; Maishi, Nako; Hida, Yasuhiro

    2017-02-16

    Drug resistance is a major problem in anticancer therapy. ATP-binding cassette (ABC) transporters have a role in the multidrug resistance. A new regimen of chemotherapy has been proposed, called "metronomic chemotherapy". Metronomic chemotherapy is the frequent, regular administration of drug doses designed to maintain low, but active, concentrations of chemotherapeutic drugs over prolonged periods of time, without causing serious toxicities. Metronomic chemotherapy regimens were developed to optimize the antitumor efficacy of agents that target the tumor vasculature instead of tumor cells, and to reduce toxicity of antineoplastic drugs [1]. Nevertheless, recent studies revealed that ABC transporters are expressed at a higher level in the endothelium in the tumor. To avoid resistance to metronomic anti-angiogenic chemotherapy, ABC transporter inhibition of tumor endothelial cells may be a promising strategy. In this mini-review, we discuss the possible mechanism of resistance to metronomic chemotherapy from the viewpoint of tumor endothelial cell biology, focusing on ABC transporters.

  8. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter

    NARCIS (Netherlands)

    Yang, Nuan; Driessen, Arnold J. M.

    2015-01-01

    Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporte

  9. Selective and ATP-dependent translocation of peptides by the homodimeric ATP binding cassette transporter TAP-like (ABCB9)

    NARCIS (Netherlands)

    Wolters, Justina Clarinda; Abele, Rupert; Tampé, Robert

    2005-01-01

    The transporter associated with antigen processing (TAP)-like (TAPL, ABCB9) belongs to the ATP-binding cassette transporter family, which translocates a vast variety of solutes across membranes. The function of this half-size transporter has not yet been determined. Here, we show that TAPL forms a h

  10. Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

    Science.gov (United States)

    Rice, Austin J; Harrison, Alistair; Alvarez, Frances J D; Davidson, Amy L; Pinkett, Heather W

    2014-05-23

    Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.

  11. Protection against chemotherapy-induced alopecia: targeting ATP-binding cassette transporters in the hair follicle?

    Science.gov (United States)

    Haslam, Iain S; Pitre, Aaron; Schuetz, John D; Paus, Ralf

    2013-11-01

    Currently, efficacious treatments for chemotherapy-induced alopecia (hair loss) are lacking, and incidences of permanent hair loss following high-dose chemotherapy are on the increase. In this article, we describe mechanisms by which the pharmacological defense status of the hair follicle might be enhanced, thereby reducing the accumulation of cytotoxic cancer drugs and preventing or reducing hair loss and damage. We believe this could be achieved via the selective increase in ATP-binding cassette (ABC) transporter expression within the hair follicle epithelium, following application of topical agonists for regulatory nuclear receptors. Clinical application would require the development of hair follicle-targeted formulations, potentially utilizing nanoparticle technology. This novel approach has the potential to yield entirely new therapeutic options for the treatment and management of chemotherapy-induced alopecia, providing significant psychological and physical benefit to cancer patients.

  12. The role of ATP-binding cassette (ABC) transporters in pathogenesis and multidrug resistance of the wheat pathogen Mycosphaerella graminicola

    NARCIS (Netherlands)

    Stergiopoulos, I.

    2003-01-01

    ATP-binding cassette (ABC) transporters are membrane proteins that utilise the energy derived from the hydrolysis of ATP to drive the transport of compounds over biological membranes. They are members of one of the largest protein families to date, present in both pro- and eukaryotic

  13. Functional analysis of the ATP-binding cassette (ABC transporter gene family of Tribolium castaneum

    Directory of Open Access Journals (Sweden)

    Broehan Gunnar

    2013-01-01

    Full Text Available Abstract Background The ATP-binding cassette (ABC transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. Results We have identified 73 ABC transporter genes in the genome of T. castaneum, which group into eight subfamilies (ABCA-H. This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. Conclusions The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

  14. The role of ATP-binding cassette transporters in neuro-inflammation: relevance for bioactive lipids

    Directory of Open Access Journals (Sweden)

    Gijs eKooij

    2012-04-01

    Full Text Available 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 system (CNS. Consequently, their high activity at the BBB has been a major hurdle for the treatment of several brain diseases, as they prevent numerous drugs to reach their site of action within the brain. Importantly, recent data indicate that endogenous substrates for ABC transporters may include inflammatory mediators, such as prostaglandins, leukotrienes, cytokines, chemokines and bioactive lipids, suggesting a potential role for ABC transporters in immunological responses, and more specifically in inflammatory brain disorders, such as multiple sclerosis (MS. In this review, we will give a comprehensive overview of recent findings that illustrate this novel role for ABC transporters in neuro-inflammatory processes. Moreover, we will provide first insights into underlying mechanisms and focus on the importance for bioactive lipids, in particular platelet-activating factor (PAF, herein. A thorough understanding of these events may form the basis for the development for selective treatment modalities to dampen the neuro-inflammatory attack in MS and thereby reducing tissue damage.

  15. A conserved mitochondrial ATP-binding cassette transporter exports glutathione polysulfide for cytosolic metal cofactor assembly.

    Science.gov (United States)

    Schaedler, Theresia A; Thornton, Jeremy D; Kruse, Inga; Schwarzländer, Markus; Meyer, Andreas J; van Veen, Hendrik W; Balk, Janneke

    2014-08-22

    An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe(2+) alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.

  16. Serum albumin promotes ATP-binding cassette transporter-dependent sterol uptake in yeast.

    Science.gov (United States)

    Marek, Magdalena; Silvestro, Daniele; Fredslund, Maria D; Andersen, Tonni G; Pomorski, Thomas G

    2014-12-01

    Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans.

  17. Caveolin-1 and ATP binding cassette transporter A1 and G1-mediated cholesterol efflux.

    Science.gov (United States)

    Wang, Faqi; Gu, Hong-mei; Zhang, Da-wei

    2014-01-01

    Atherosclerosis is one major cause of cardiovascular diseases, the leading cause of death in industrialized countries. Reverse cholesterol transport (RCT) is thought to be one primary pathway to protect against atherosclerosis. The first and rate-limiting step of RCT is ATP-binding cassette transport A1 (ABCA1) and ABCG1-mediated cholesterol efflux from the cells. Recently, caveolin-1 (CAV1), a scaffolding protein that organizes and concentrates certain caveolin-interacting signaling molecules and receptors within caveolae membranes, has been shown to regulate ABCA1 and ABCG1-mediated cholesterol efflux probably via interacting with them. In the present review, we summarize the current knowledge and views on the regulatory role of CAV1 on the cholesterol homeostasis with emphasis on the association of CAV1 with ABCA1 and ABCG1. We conclude that the dominance of the positive regulation by CAV1 on the ABCA1 and ABCG1-mediated cholesterol efflux is depending on the species, cell types, as well as the levels of CAV1 expression.

  18. ATP Binding Cassette Transporter Mediates Both Heme and Pesticide Detoxification in Tick Midgut Cells.

    Directory of Open Access Journals (Sweden)

    Flavio Alves Lara

    Full Text Available In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA, a well-known inhibitor of ATP binding cassette (ABC transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may

  19. ATP-Binding Cassette Transporters Modulate Both Coelenterazine- and D-Luciferin-Based Bioluminescence Imaging

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    Ruimin Huang

    2011-05-01

    Full Text Available Bioluminescence imaging (BLI of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI readout intensity from intact living cells. To investigate the effect of ATP-binding cassette (ABC transporters on BLI readout, we generated click beetle (cLuc, firefly (fLuc, Renilla (rLuc, and Gaussia (gLuc luciferase HEK-293 reporter cells that overexpressed different ABC transporters (ABCB1, ABCC1, and ABCG2. In vitro studies showed a significant BLI intensity decrease in intact cells compared to cell lysates, when ABCG2 was overexpressed in HEK-293/cLuc, fLuc, and rLuc cells. Selective ABC transporter inhibitors were also applied. Inhibition of ABCG2 activity increased the BLI intensity more than two-fold in HEK-293/cLuc, fLuc, and rLuc cells; inhibition of ABCB1 elevated the BLI intensity two-fold only in HEK-293/rLuc cells. BLI of xenografts derived from HEK-293/ABC transporter/luciferase reporter cells confirmed the results of inhibitor treatment in vivo. These findings demonstrate that coelenterazine-based rLuc-BLI intensity can be modulated by ABCB1 and ABCG2. ABCG2 modulates d-luciferin-based BLI in a luciferase type–independent manner. Little ABC transporter effect on gLuc-BLI intensity is observed because a large fraction of gLuc is secreted. The expression level of ABC transporters is one key factor affecting BLI intensity, and this may be particularly important in luciferase-based applications in stem cell research.

  20. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance

    OpenAIRE

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting fr...

  1. ATP-binding cassette-like transporters are involved in the transport of lignin precursors across plasma and vacuolar membranes

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Y.C.; Liu, C.

    2010-12-28

    Lignin is a complex biopolymer derived primarily from the condensation of three monomeric precursors, the monolignols. The synthesis of monolignols occurs in the cytoplasm. To reach the cell wall where they are oxidized and polymerized, they must be transported across the cell membrane. However, the molecular mechanisms underlying the transport process are unclear. There are conflicting views about whether the transport of these precursors occurs by passive diffusion or is an energized active process; further, we know little about what chemical forms are required. Using isolated plasma and vacuolar membrane vesicles prepared from Arabidopsis, together with applying different transporter inhibitors in the assays, we examined the uptake of monolignols and their derivatives by these native membrane vesicles. We demonstrate that the transport of lignin precursors across plasmalemma and their sequestration into vacuoles are ATP-dependent primary-transport processes, involving ATP-binding cassette-like transporters. Moreover, we show that both plasma and vacuolar membrane vesicles selectively transport different forms of lignin precursors. In the presence of ATP, the inverted plasma membrane vesicles preferentially take up monolignol aglycones, whereas the vacuolar vesicles are more specific for glucoconjugates, suggesting that the different ATP-binding cassette-like transporters recognize different chemical forms in conveying them to distinct sites, and that glucosylation of monolignols is necessary for their vacuolar storage but not required for direct transport into the cell wall in Arabidopsis.

  2. The saci_2123 gene of the hyperthermoacidophile Sulfolobus acidocaldarius encodes an ATP-binding cassette multidrug transporter.

    Science.gov (United States)

    Yang, Nuan; Driessen, Arnold J M

    2015-01-01

    Multidrug resistance (MDR) transporters are capable of secreting structurally and functionally unrelated toxic compounds from the cell. Among this group are ATP-binding cassette (ABC) transporters. These membrane proteins are typically arranged as either hetero- or homo-dimers of ABC half-transporters with each subunit consisting of a membrane domain fused at the C-terminus to an ATP-binding domain, or as full transporters in which the two subunits are fused into a single polypeptide. The saci_2123 gene of the thermoacidophilic archaeon Sulfolobus acidocaldarius is the only gene in the genome that encodes an ATP-binding cassette half-transporter, while a homologous gene is present in the genomes of S. solfataricus, S. tokodaii and S islandicus. Saci_2123 shares homology with well-characterized bacterial and mammalian MDR transporters. The saci_2132 gene is up-regulated when cells are exposed to drugs. A deletion mutant of saci_2132 was found to be more vulnerable to a set of toxic compounds, including detergents, antibiotics and uncouplers as compared to the wild-type strain, while the drug resistance could be restored through the plasmid-based expression of saci_2132. These data demonstrate that Saci_2132 is an archaeal ABC-MDR transporter and therefore it was termed Smr1 (Sulfolobus multidrug resistance transporter 1).

  3. Oxidized LDL upregulated ATP binding cassette transporter-1 in THP-1 macrophages

    Institute of Scientific and Technical Information of China (English)

    Chao-ke TANG; Guang-hui YI; Jun-hao YANG; Lu-shan LIU; Zuo WANG; Chang-geng RUAN; Yong-zong YANG

    2004-01-01

    AIM: To study the effect of oxidized low density lipoprotein (ox-LDL) on ATP binding cassette transporter A1 (ABCA1) in THP-1 macrophages. METHODS: After exposing the cultured THP-1 macrophages to ox-LDL for different periods, cholesterol efflux was determined by FJ-2107P type liquid scintillator. ABCA1 mRNA and protein level were determined by reverse trancriptase-polymerase chain reaction (RT-PCR) and Western blot, respectively.The cholesterol level in THP-1 macrophage foam cells was detected by high performance liquid chromatography.RESULTS: ox-LDL elevated AB CA1 in both protein and mRNA levels and increased apolipoprotein (apo) A-I-mediated cholesterol efflux in a time- and dose-dependent manner. 22(R)-hydroxyeholesterol and 9-cis-retinoic acid did significantly increase cholesterol efflux in THP-1 macrophage foam cells (P<0.05), respectively. Both of them further promoted cholesterol efflux (P<0.01). As expected, liver X receptor (LXR) agonist decreased content of esterified cholesterol in the macrophage foam cells compared with control, whereas only a slight decrease of free cholesterol was observed. LXR activity was slightly increased by oxidized LDL by 12 % at 12 h compared with 6 h.However, LXR activity was increased about 1.8 times at 24 h, and oxidized LDL further increased LXR activity by about 2.6 times at 48 h. CONCLUSION: ABCA1 gene expression was markedly increased in cholesterol-loaded cells as a result of activation of LXR/RXR. ABCA1 plays an important role in the homeostasis of cholesterol in the macrophages.

  4. Molecular Events Involved in a Single Cycle of Ligand Transfer from an ATP Binding Cassette Transporter, LolCDE, to a Molecular Chaperone, LolA*

    OpenAIRE

    Taniguchi, Naohiro; Tokuda, Hajime

    2008-01-01

    An ATP binding cassette transporter LolCDE complex releases lipoproteins from the inner membrane of Escherichia coli in an ATP-dependent manner, leading to the formation of a complex between a lipoprotein and a periplasmic chaperone, LolA. LolA is proposed to undergo a conformational change upon the lipoprotein binding. The lipoprotein is then transferred from the LolA-lipoprotein complex to the outer membrane via LolB. Unlike most ATP binding cassette transporters med...

  5. ABCC4与人类肿瘤%ATP-binding cassette transporter family class C4 and human cancer

    Institute of Scientific and Technical Information of China (English)

    石妮; 赵晓航

    2011-01-01

    ATP-binding cassette transporter family class C4 (ABCC4) is known as a member of the ATP-binding cassette transporter super-family, involved in the active transport of endogenous anions and xenobiotic, which is not normally produced or expected to be present in human, such as antibiotics. Recently it has been found that the copy number variations of Abcc4 gene and overexpression of ABCC4 protein in many kinds of human cancers, which might involved in tumorigenesis, progress and chemotherapeutic response. This review will focus on the ectopic expression of Abcc4 in human cancer and the potential role of ABCC4 in tumorigenesis and progress.%ABCC4(ATP-binding cassette transporter family class C4,ABCC4)是ABC蛋白家族成员,主要参与转运机体物质代谢中产生的有机阴离子和一些异型生物质等生物学功能.近年研究发现某些人类肿瘤存在Abcc4基因的拷贝数变异,主要表现为Abcc4基因拷贝数增加和ABCC4蛋白过表达,这些改变与肿瘤发生发展、耐药,以及治疗疗效具有相关性.该文综述了Abcc4基因的拷贝数变异和异常表达与肿瘤生物学特性的关系,探讨ABCC4在肿瘤发生发展中的作用机制.

  6. Repositioning of Tyrosine Kinase Inhibitors as Antagonists of ATP-Binding Cassette Transporters in Anticancer Drug Resistance

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    Yi-Jun Wang

    2014-09-01

    Full Text Available The phenomenon of multidrug resistance (MDR has attenuated the efficacy of anticancer drugs and the possibility of successful cancer chemotherapy. ATP-binding cassette (ABC transporters play an essential role in mediating MDR in cancer cells by increasing efflux of drugs from cancer cells, hence reducing the intracellular accumulation of chemotherapeutic drugs. Interestingly, small-molecule tyrosine kinase inhibitors (TKIs, such as AST1306, lapatinib, linsitinib, masitinib, motesanib, nilotinib, telatinib and WHI-P154, have been found to have the capability to overcome anticancer drug resistance by inhibiting ABC transporters in recent years. This review will focus on some of the latest and clinical developments with ABC transporters, TKIs and anticancer drug resistance.

  7. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2

    KAUST Repository

    Weng, Jingwei

    2017-02-23

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called

  8. LrABCF1, a GCN-type ATP-binding cassette transporter from Lilium regale, is involved in defense responses against viral and fungal pathogens

    Science.gov (United States)

    ATP-binding cassette (ABC) transporters are essential for membrane translocation in diverse biological processes, such as plant development and defense response. Here, a general control non-derepressible (GCN)-type ABC transporter gene, designated LrABCF1, was identified from Cucumber mosaic virus (...

  9. The ABCC6 transporter: what lessons can be learnt from other ATP-binding cassette transporters?

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    Olivier M. Vanakker

    2013-10-01

    Full Text Available ABC transporters represent a large family of ATP-driven transmembrane transporters involved in uni- or bidirectional transfer of a variety of substrates. Divided in 7 families, they represent 48 transporter proteins, several of which are associated with human disease. Among the latter is ABCC6, a unidirectional exporter protein primarily expressed in liver and kidney. ABCC6 deficiency causes pseudoxanthoma elasticum (PXE, characterised by calcification and fragmentation of elastic fibres, resulting in oculocutaneous and cardiovascular symptoms. Unique among connective tissue disorders, the relation between the ABCC6 transporter and ectopic mineralization in PXE remains enigmatic, not in the least because of lack of knowledge on the substrate(s of ABCC6 and its unusual expression pattern. Because many features, including structure and transport mechanism, are shared by ABC transporters, it is worthwhile to evaluate if and to what extent the knowledge on the (pathophysiology of other transporters may provide useful clues towards understanding the (pathophysiological role of ABCC6. In this paper, we summarize relevant knowledge and methods for analysis on ABC transporters which may be useful for the further study of ABCC6.

  10. Secretion of natural and synthetic toxic compounds from filamentous fungi by membrane transporters of the ATP-binding cassette and major facilitator superfamily

    NARCIS (Netherlands)

    Stergiopoulos, I.; Zwiers, L.H.; Waard, De M.A.

    2002-01-01

    This review provides an overview of members of the ATP-binding cassette (ABC) and major facilitator superfamily (MFS) of transporters identified in filamentous fungi. The most common function of these membrane proteins is to provide protection against natural toxic compounds present in the environme

  11. An ATP Binding Cassette Transporter Mediates the Uptake of α-(1,6)-Linked Dietary Oligosaccharides in Bifidobacterium and Correlates with Competitive Growth on These Substrates

    DEFF Research Database (Denmark)

    Hansen, Morten Ejby; Fredslund, Folmer; Andersen, Joakim Mark

    2016-01-01

    and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds -(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide...

  12. Control of Mycosphaerella graminicola on wheat seedlings by medical drugs known to modulate the activity of ATP-binding cassette transporters

    NARCIS (Netherlands)

    Roohparvar, R.; Huser, A.; Zwiers, L.H.; Waard, de M.A.

    2007-01-01

    Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due

  13. Substrate Specificity and Ionic Regulation of GlnPQ from Lactococcus lactis. An ATP-Binding Cassette Transporter with Four Extracytoplasmic Substrate-Binding Domains

    NARCIS (Netherlands)

    Schuurman-Wolters, Gea K.; Poolman, Bert

    2005-01-01

    We report on the functional characterization of GlnPQ, an ATP-binding cassette transporter with four extracytoplasmic substrate-binding domains. The first predicted transmembrane helix of GlnP was cleaved off in the mature protein and most likely serves as the signal sequence for the extracytoplasmi

  14. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

    Science.gov (United States)

    Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

    2015-11-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.

  15. Whole-genome survey of the putative ATP-binding cassette transporter family genes in Vitis vinifera.

    Science.gov (United States)

    Çakır, Birsen; Kılıçkaya, Ozan

    2013-01-01

    The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 "full-size," 41 "half-size," and 15 "soluble" putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.

  16. Whole-genome survey of the putative ATP-binding cassette transporter family genes in Vitis vinifera.

    Directory of Open Access Journals (Sweden)

    Birsen Çakır

    Full Text Available The ATP-binding cassette (ABC protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 "full-size," 41 "half-size," and 15 "soluble" putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog and ABCC (multidrug resistance-associated protein. We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera.

  17. The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism.

    Science.gov (United States)

    Davis, Warren

    2014-01-01

    The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

  18. High glucose decreases the expression of ATP-binding cassette transporter G1 in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Jiahong Xue; Zuyi Yuan; Yue Wu; Yan Zhao; Zhaofei Wan

    2008-01-01

    Objective:ATP-binding cassette transporters(ABC) A1 and G1 play an important role in mediating cholesterol efflux and preventing macrophage foam cell formation. In this study, we examined the regulation of ABC transporters by high glucose in human vascular smooth muscle cells(VSMCs), the other precursor of foam cells. Methods:Incubation of human VSMCs with D-ghicose(5 to 30 mM) for 1 to 7 days in the presence or absence of antioxidant and nuclear factor(NF)-kB inhibitors, the expressions of ABCA1 and ABCG1 were analyzed by real time PCR and Western blotting. Results:High glucose decreased ABCG1 mRNA and protein expression in cultured VSMCs, whereas the expression of ABCA1 was not significantly decreased. Down-regulation of ABCG1 mRNA expression by high glucose was abolished by antioxidant N-acetyl-L-cysteine(NAC) and NF-kB inhibitors, BAY 11-7085 and tosyl-phenylalanine chloromethyl-ketone(TPCK). Conclusion:High glucose suppresses the expression of ABCG1 in VSMCs, which is the possible mechanism of VSMC derived foam cell transformation.

  19. AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans.

    Science.gov (United States)

    Handali, Melody; Roychowdhury, Hridindu; Neupane, Durga P; Yukl, Erik T

    2015-12-11

    Bacterial ATP-binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of Gram-negative bacteria, including human pathogens, have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase, and solute-binding protein (SBP) genes of the AztABC zinc transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties, and interaction with the SBP (AztC). Transcription of the aztD gene is significantly up-regulated under conditions of zinc starvation. Recombinantly expressed AztD purifies with slightly substoichiometric zinc from the periplasm of Escherichia coli and is capable of binding up to three zinc ions with high affinity. Size exclusion chromatography and a simple intrinsic fluorescence assay were used to determine that AztD as isolated is able to transfer bound zinc nearly quantitatively to apo-AztC. Transfer occurs through a direct, associative mechanism that prevents loss of metal to the solvent. These results indicate that AztD is a zinc chaperone to AztC and likely functions to maintain zinc homeostasis through interaction with the AztABC system. This work extends our understanding of periplasmic zinc trafficking and the function of chaperones in this process.

  20. Biotin uptake in prokaryotes by solute transporters with an optional ATP-binding cassette-containing module.

    Science.gov (United States)

    Hebbeln, Peter; Rodionov, Dmitry A; Alfandega, Anja; Eitinger, Thomas

    2007-02-20

    BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one-third of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the alpha-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters.

  1. The ATP-binding Cassette Transporter OsABCG15 is Required for Anther Development and Pollen Fertility in Rice

    Institute of Scientific and Technical Information of China (English)

    Bai-Xiao Niu; Fu-Rong He; Ming He; Ding Ren; Le-Tian Chen; Yao-Guang Liu

    2013-01-01

    Plant male reproductive development is a complex biological process,but the underlying mechanism is not well understood.Here,we characterized a rice (Oryza sativa L.) male sterile mutant.Based on mapbased cloning and sequence analysis,we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene,OsABCG15,causing abnormal anthers and male sterility.Therefore,we named this mutant osabcg15.Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis Ⅱ stage) to stage 10 (late microspore stage).Two genes CYP704B2 and WDA1,involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine,were downregulated in osabcg15 mutant,suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules.Consistently,histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration,leading to rapid degredation of young microspores.The results suggest that OsABCG15 plays a critical role in exine formation and pollen development,similar to the homologous gene of AtABCG26 in Arabidopsis.This work is helpful to understand the regulatory network in rice anther development.

  2. Computational characterization of TTHA0379: A potential glycerophosphocholine binding protein of Ugp ATP-binding cassette transporter.

    Science.gov (United States)

    Chandravanshi, Monika; Gogoi, Prerana; Kanaujia, Shankar Prasad

    2016-11-05

    For the de novo biosynthesis of phospholipids, byproducts such as sn-glycerol-3-phosphate (G3P) and glycerophosphocholine (GPC) of glycerophospholipid metabolic pathway are imported inside the cell by an ATP-binding cassette (ABC) transporter known as UgpABCE. Of which, UgpA and UgpE constitutes the transmembrane domains (TMDs), UgpC forms the dimer of ATP-hydrolyzing component and UgpB is the periplasmic substrate binding protein. Structurally, UgpABCE transporter displays similarity to the maltose ABC transporter of Escherichia coli; thus, has been grouped into the CUT1 (Carbohydrate Uptake Transporter-1) family of bacterial ABC transporters. Being a member of CUT1 family, several Ugp (Uptake glycerol phosphate) protein sequences in biological database(s) exhibit sequence and structure similarity to sugar ABC transporters and have been annotated as sugar binding proteins; one of such proteins is TTHA0379 from Thermus thermophilus HB8. Here, in this study, we used computational method(s) to distinguish UgpB and sugar binding proteins based on their primary and tertiary structure features. A comprehensive analysis of these proteins indicates that they are evolutionarily related to each other having common conserved features at their primary and tertiary structure levels. However, they display differences at their active sites owing to the dissimilarity in their ligand preferences. In addition, phylogenetic analysis of TTHA0379 along with UgpB and sugar binding proteins reveals that both the groups of proteins forms two distinct clades and TTHA0379 groups with UgpB proteins. Furthermore, analysis of the ligand binding pocket shows that all the essential features of glycerophosphocholine binding protein i.e. UgpB, are conserved in TTHA0379 as well. Combining these features, here, we designate TTHA0379 to be a GPC binding protein.

  3. Polymorphisms in ATP-binding cassette transporter genes and interaction with diet and life style factors in relation to colorectal cancer in a Danish prospective case-cohort study

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Andersen, Vibeke; Tjonneland, Anne;

    2015-01-01

    The ATP-binding cassette (ABC) transporter family transports various molecules across the enterocytes in the gut protecting the intestine against potentially harmful substances. Moreover, ABC transporters are involved in mucosal immune defence through interaction with cytokines. The study aimed...

  4. Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo*

    OpenAIRE

    Raichaudhuri, Ayan; Peng, Mingsheng; Naponelli, Valeria; Chen, Sixue; Sánchez-Fernández, Rocío; Gu, Honglan; Gregory, Jesse F.; Hanson, Andrew D; Rea, Philip A.

    2009-01-01

    The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuol...

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

    Science.gov (United States)

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

    2010-02-01

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

  6. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance

    Science.gov (United States)

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting from articles published inreputable journals from1994 to date and experiments newly performed by our group, a comprehensive review is written about ABCA2 and its role in MDR regarding childhood ALL. ABCA2 transports drugs from the cytoplasm into the lysosomal compartment, where they may become degraded and exported from the cell. The aforementioned mechanism may contribute to MDR. It has been reported that ABCA2 may induce resistance to mitoxantrone, estrogen derivatives and estramustine. It is resistant to the aforementioned compounds. Furthermore, the overexpression ofABCA2 in methotrexate, vinblastine and/or doxorubicin treated Jurkat cells are observed in several publications. The recent study of our group showsthatthe overexpression ofABCA2 gene in children with ALL increases the risk of MDR by 15 times. ABCA2 is the second identified member of the ABCA; ABC transporters' subfamily. ABCA2 gene expression profile is suggested to be an unfavorable prognostic factor in ALL treatment. Better understanding of the MDR mechanisms and the factors involved may improve the therapeutic outcome of ALL by modifying the treatment protocols. PMID:25254091

  7. The role of ATP-binding cassette transporter A2 in childhood acute lymphoblastic leukemia multidrug resistance.

    Science.gov (United States)

    Aberuyi, N; Rahgozar, S; Moafi, A

    2014-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most prevalent hematologic malignancies in children. Although the cure rate of ALL has improved over the past decades, the most important reason for ALL treatment failure is multidrug resistance (MDR) phenomenon. The current study aims to explain the mechanisms involved in multidrug resistance of childhood ALL, and introduces ATP-binding cassette transporterA2 (ABCA2) as an ABC transporter gene which may have a high impact on MDR. Benefiting from articles published inreputable journals from1994 to date and experiments newly performed by our group, a comprehensive review is written about ABCA2 and its role in MDR regarding childhood ALL. ABCA2 transports drugs from the cytoplasm into the lysosomal compartment, where they may become degraded and exported from the cell. The aforementioned mechanism may contribute to MDR. It has been reported that ABCA2 may induce resistance to mitoxantrone, estrogen derivatives and estramustine. It is resistant to the aforementioned compounds. Furthermore, the overexpression ofABCA2 in methotrexate, vinblastine and/or doxorubicin treated Jurkat cells are observed in several publications. The recent study of our group showsthatthe overexpression ofABCA2 gene in children with ALL increases the risk of MDR by 15 times. ABCA2 is the second identified member of the ABCA; ABC transporters' subfamily. ABCA2 gene expression profile is suggested to be an unfavorable prognostic factor in ALL treatment. Better understanding of the MDR mechanisms and the factors involved may improve the therapeutic outcome of ALL by modifying the treatment protocols.

  8. Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

    Directory of Open Access Journals (Sweden)

    Yu Lan

    2012-08-01

    Full Text Available Abstract Background The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production. Results In order to investigate OtrC’s function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877 and 1.4-fold in SR16 (P = 0.00973 duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively. Conclusions The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.

  9. The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption

    NARCIS (Netherlands)

    Meszaros, Peter; Hummel, Ina; Klappe, Karin; Draghiciu, Oana; Hoekstra, Dick; Kok, Jan W.

    2013-01-01

    Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding

  10. ATP binding cassette transporters modulate both coelenterazine- and D-luciferin- based bioluminescence imaging

    OpenAIRE

    Huang, Ruimin; Vider, Jelena; Serganova, Inna; Blasberg, Ronald G.

    2011-01-01

    Bioluminescence imaging (BLI) of luciferase reporters provides a cost-effective and sensitive means to image biological processes. However, transport of luciferase substrates across the cell membrane does affect BLI-readout-intensity from intact living cells.

  11. Disruption of lolCDE, Encoding an ATP-Binding Cassette Transporter, Is Lethal for Escherichia coli and Prevents Release of Lipoproteins from the Inner Membrane

    OpenAIRE

    Narita, Shin-ichiro; Tanaka, Kimie; Matsuyama, Shin-ichi; Tokuda, Hajime

    2002-01-01

    ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli. Mutations resulting in defective LolD were previously shown to be lethal for E. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstituti...

  12. ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus.

    Science.gov (United States)

    Yu, Fang; De Luca, Vincenzo

    2013-09-24

    The Madagascar periwinkle (Catharanthus roseus) is highly specialized for the biosynthesis of many different monoterpenoid indole alkaloids (MIAs), many of which have powerful biological activities. Such MIAs include the commercially important chemotherapy drugs vinblastine, vincristine, and other synthetic derivatives that are derived from the coupling of catharanthine and vindoline. However, previous studies have shown that biosynthesis of these MIAs involves extensive movement of metabolites between specialized internal leaf cells and the leaf epidermis that require the involvement of unknown secretory processes for mobilizing catharanthine to the leaf surface and vindoline to internal leaf cells. Spatial separation of vindoline and catharanthine provides a clear explanation for the low levels of dimers that accumulate in intact plants. The present work describes the molecular cloning and functional identification of a unique catharanthine transporter (CrTPT2) that is expressed predominantly in the epidermis of young leaves. CrTPT2 gene expression is activated by treatment with catharanthine, and its in planta silencing redistributes catharanthine to increase the levels of catharanthine-vindoline drug dimers in the leaves. Phylogenetic analysis shows that CrTPT2 is closely related to a key transporter involved in cuticle assembly in plants and that may be unique to MIA-producing plant species, where it mediates secretion of alkaloids to the plant surface.

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

    Science.gov (United States)

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

    2014-01-01

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

  14. ATP-binding cassette transporter enhances tolerance to DDT in Tetrahymena.

    Science.gov (United States)

    Ning, YingZhi; Dang, Huai; Liu, GuangLong; Xiong, Jie; Yuan, DongXia; Feng, LiFang; Miao, Wei

    2015-03-01

    The reuse of dichlorodiphenyltrichloroethane (DDT) as an indoor residual spray was permitted by the World Health Organization in 2007, and approximately 14 countries still use DDT to control disease vectors. The extensive exposure of insects to DDT has resulted in the emergence of DDT resistance, especially in mosquitoes, and the mechanism for this resistance in mosquitoes has been widely reported. Spraying can also introduce DDT directly into surface water, and DDT can subsequently accumulate in microorganisms, but the mechanism for the resistance to DDT degradation in microorganisms is unclear. Using whole-genome microarray analysis, we detected an abcb15 gene that was up-regulated in a specific manner by DDT treatment in T. thermophile. The deduced ABCB15 peptide sequence had two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs) to form the structure TMD-NBD-TMD-NBD, and each NBD contained three conserved motifs: Walker-A, C-loop, and Walker-B, which indicated the T. thermophila abcb15 was a typical ABC transporter gene. The expression of ABCB15 fused with a C-terminal green fluorescent protein was found to be on the periphery of the cell, suggesting that ABCB15 was a membrane pump protein. In addition, cells with abcb15 partially knocked down (abcb15-KD) grew slower than wild-type cells in the presence of 256 mg L(-1) DDT, indicating the tolerance of abcb15-KD strain to DDT exposure was decreased. Thus, we suggest that in Tetrahymena, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance to DDT and protect cells from this exogenous toxin by efficiently pumping it to the extracellular space.

  15. Retinoic acid receptor agonists regulate expression of ATP-binding cassette transporter G1 in macrophages.

    Science.gov (United States)

    Ayaori, Makoto; Yakushiji, Emi; Ogura, Masatsune; Nakaya, Kazuhiro; Hisada, Tetsuya; Uto-Kondo, Harumi; Takiguchi, Shunichi; Terao, Yoshio; Sasaki, Makoto; Komatsu, Tomohiro; Iizuka, Maki; Yogo, Makiko; Uehara, Yoshinari; Kagechika, Hiroyuki; Nakanishi, Tsuyoshi; Ikewaki, Katsunori

    2012-04-01

    ABC transporter G1 (ABCG1) plays a pivotal role in HDL-mediated cholesterol efflux and atherogenesis. We investigated whether, and how, retinoic acid receptors (RARs) regulate ABCG1 expression in macrophages. All-trans retinoic acid (ATRA), an RAR ligand, increased ABCG1 protein levels and apoA-I/HDL-mediated cholesterol efflux from the macrophages. Both ATRA and other RAR agonists, TTNPB and Am580, increased major transcripts driven by promoter B upstream of exon 5, though minor transcripts driven by promoter A upstream of exon 1 were only increased by ATRA. The stimulatory effects of ATRA on ABCG1 expression were completely abolished in the presence of RAR/RXR antagonists but were only partially canceled in the presence of an LXR antagonist. Adenovirus with overexpressed oxysterol sulfotransferase abolished the LXR pathway, as previously reported, and ATRA-responsiveness in ABCA1/ABCG1 expressions were respectively attenuated by 38 and 22% compared to the control virus. Promoter assays revealed that ABCG1 levels were regulated more by promoter B than promoter A, and ATRA activated promoter B in a liver X receptor-responsive element (LXRE)-dependent manner. Further, LXRE-B in intron 7, but not LXRE-A in intron 5, enhanced ATRA responsiveness under overexpression of all RAR isoforms-RARα/β/γ. In contrast, the activation of promoter B by TTNPB depended on LXRE-B and RARα, but not on RARβ/γ. Finally, chromatin immunoprecipitation and gel-shift assays revealed a specific and direct repeat 4-dependent binding of RARα to LXRE-B. In conclusion, RAR ligands increase ABCA1/G1 expression and apoA-I/HDL-mediated cholesterol efflux from macrophages, and modulate ABCG1 promoter activity via LXRE-dependent mechanisms.

  16. Genome-wide identification and characterization of ATP-binding cassette transporters in the silkworm, Bombyx mori

    Directory of Open Access Journals (Sweden)

    Zhang Jianzhen

    2011-10-01

    Full Text Available Abstract Background The ATP-binding cassette (ABC transporter superfamily is the largest transporter gene family responsible for transporting specific molecules across lipid membranes in all living organisms. In insects, ABC transporters not only have important functions in molecule transport, but also play roles in insecticide resistance, metabolism and development. Results From the genome of the silkworm, Bombyx mori, we have identified 51 putative ABC genes which are classified into eight subfamilies (A-H by phylogenetic analysis. Gene duplication is very evident in the ABCC and ABCG subfamilies, whereas gene numbers and structures are well conserved in the ABCD, ABCE, ABCF, and ABCH subfamilies. Microarray analysis revealed that expression of 32 silkworm ABC genes can be detected in at least one tissue during different developmental stages, and the expression patterns of some of them were confirmed by quantitative real-time PCR. A large number of ABC genes were highly expressed in the testis compared to other tissues. One of the ABCG genes, BmABC002712, was exclusively and abundantly expressed in the Malpighian tubule implying that BmABC002712 plays a tissue-specific role. At least 5 ABCG genes, including BmABC005226, BmABC005203, BmABC005202, BmABC010555, and BmABC010557, were preferentially expressed in the midgut, showing similar developmental expression profiles to those of 20-hydroxyecdysone (20E-response genes. 20E treatment induced the expression of these ABCG genes in the midgut and RNA interference-mediated knockdown of USP, a component of the 20E receptor, decreased their expression, indicating that these midgut-specific ABCG genes are 20E-responsive. Conclusion In this study, a genome-wide analysis of the silkworm ABC transporters has been conducted. A comparison of ABC transporters from 5 insect species provides an overview of this vital gene superfamily in insects. Moreover, tissue- and stage-specific expression data of the

  17. Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

    Directory of Open Access Journals (Sweden)

    Simone Bocchetta

    Full Text Available Hepatitis C virus (HCV establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1 mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts. The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis

  18. Evaluation of the role of ATP-binding cassette transporters as a defence mechanism against temephos in populations of Aedes aegypti

    Directory of Open Access Journals (Sweden)

    Estelita Pereira Lima

    2014-11-01

    Full Text Available The role of ATP-binding cassette (ABC transporters in the efflux of the insecticide, temephos, was assessed in the larvae of Aedes aegypti. Bioassays were conducted using mosquito populations that were either susceptible or resistant to temephos by exposure to insecticide alone or in combination with sublethal doses of the ABC transporter inhibitor, verapamil (30, 35 and 40 μM. The best result in the series was obtained with the addition of verapamil (40 μM, which led to a 2x increase in the toxicity of temephos, suggesting that ABC transporters may be partially involved in conferring resistance to the populations evaluated.

  19. Genetic Analysis of the Mode of Interplay between an ATPase Subunit and Membrane Subunits of the Lipoprotein-Releasing ATP-Binding Cassette Transporter LolCDE†

    OpenAIRE

    Ito, Yasuko; Matsuzawa, Hitomi; Matsuyama, Shin-ichi; Narita, Shin-ichiro; Tokuda, Hajime

    2006-01-01

    The LolCDE complex, an ATP-binding cassette (ABC) transporter, releases lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane of Escherichia coli. The LolCDE complex is composed of two copies of an ATPase subunit, LolD, and one copy each of integral membrane subunits LolC and LolE. LolD hydrolyzes ATP on the cytoplasmic side of the inner membrane, while LolC and/or LolE recognize and release lipoproteins anchored to the periplasmic leaflet of the i...

  20. Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori.

    Science.gov (United States)

    Atsumi, Shogo; Miyamoto, Kazuhisa; Yamamoto, Kimiko; Narukawa, Junko; Kawai, Sawako; Sezutsu, Hideki; Kobayashi, Isao; Uchino, Keiro; Tamura, Toshiki; Mita, Kazuei; Kadono-Okuda, Keiko; Wada, Sanae; Kanda, Kohzo; Goldsmith, Marian R; Noda, Hiroaki

    2012-06-19

    Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis.

  1. Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells

    DEFF Research Database (Denmark)

    Robey, R W; Medina-Pérez, W Y; Nishiyama, K

    2001-01-01

    microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold......We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines...... resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated...

  2. Cholesterol efflux via ATP-binding cassette transporter A1 (ABCA1) and cholesterol uptake via the LDL receptor influences cholesterol-induced impairment of beta cell function in mice

    NARCIS (Netherlands)

    Kruit, J. K.; Kremer, P. H. C.; Dai, L.; Tang, R.; Ruddle, P.; de Haan, W.; Brunham, L. R.; Verchere, C. B.; Hayden, M. R.

    2010-01-01

    Cellular cholesterol accumulation is an emerging mechanism for beta cell dysfunction in type 2 diabetes. Absence of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) results in increased islet cholesterol and impaired insulin secretion, indicating that impaired cholesterol effl

  3. Function and regulation of ATP-binding cassette transport proteins involved in hepatobiliary transport (vol 12, pg 13, 2000)

    NARCIS (Netherlands)

    Hooiveld, GJEJ; van Montfoort, JE; Meijer, DKF; Muller, M

    2001-01-01

    Hepatobiliary transport of endogenous and exogenous compounds is mediated by the coordinated action of multiple transport systems present at the sinusoidal (basolateral) and canalicular (apical) membrane domains of hepatocytes. During the last few years many of these transporters have been cloned an

  4. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells.

    Science.gov (United States)

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning; Watari, Fumio; Liu, Hudan; Chen, Xiao

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  5. Involvement of CjMDR1, a plant multidrug-resistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica

    Science.gov (United States)

    Shitan, Nobukazu; Bazin, Ingrid; Dan, Kazuyuki; Obata, Kazuaki; Kigawa, Koji; Ueda, Kazumitsu; Sato, Fumihiko; Forestier, Cyrille; Yazaki, Kazufumi

    2003-01-01

    Alkaloids comprise one of the largest groups of plant secondary metabolites. Berberine, a benzylisoquinoline alkaloid, is preferentially accumulated in the rhizome of Coptis japonica, a ranunculaceous plant, whereas gene expression for berberine biosynthetic enzymes has been observed specifically in root tissues, which suggests that berberine synthesized in the root is transported to the rhizome, where there is high accumulation. We recently isolated a cDNA encoding a multidrug-resistance protein (MDR)-type ATP-binding cassette (ABC) transporter (Cjmdr1) from berberine-producing cultured C. japonica cells, which is highly expressed in the rhizome. Functional analysis of Cjmdr1 by using a Xenopus oocyte expression system showed that CjMDR1 transported berberine in an inward direction, resulting in a higher accumulation of berberine in Cjmdr1-injected oocytes than in the control. Typical inhibitors of ABC proteins, such as vanadate, nifedipine, and glibenclamide, as well as ATP depletion, clearly inhibited this CjMDR1-dependent berberine uptake, suggesting that CjMDR1 functioned as an ABC transporter. Conventional membrane separation methods showed that CjMDR1 was localized in the plasma membrane of C. japonica cells. In situ hybridization indicated that Cjmdr1 mRNA was expressed preferentially in xylem tissues of the rhizome. These findings strongly suggest that CjMDR1 is involved in the translocation of berberine from the root to the rhizome. PMID:12524452

  6. Distinct alterations in ATP-binding cassette transporter expression in liver, kidney, small intestine, and brain in adjuvant-induced arthritic rats.

    Science.gov (United States)

    Kawase, Atsushi; Norikane, Sari; Okada, Ayaka; Adachi, Mamiko; Kato, Yukio; Iwaki, Masahiro

    2014-08-01

    Pathophysiological changes of infection or inflammation are associated with alterations in the production of numerous absorption, distribution, metabolism and excretion-related proteins. However, little information is available on the effects of inflammation on the expression levels and activities of ATP-binding cassette (ABC) transporters. We examined the effect of acute (on day 7) and chronic (on day 21) inflammation on the expression of ABC transporters in some major tissues in rat. Adjuvant-induced arthritis (AA) in rats was used as an animal model for inflammation. The mRNA levels of mdr1a and mdr1b encoding P-glycoprotein (P-gp) decreased significantly in livers of AA rats on day 21. Hepatic protein levels of P-gp, Mrp2, and Bcrp decreased significantly in membranes but not homogenates of AA rats after 7 days and after 21 days of treatment with adjuvant. Contrary to liver, protein levels of P-gp and Mrp2, but not Bcrp in kidney, increased significantly in membranes. The biliary excretion of rhodamine 123 was decreased in rats with chronic inflammation owing to decreases in efflux activities of P-gp. Our results showed that the expression of transporters in response to inflammation was organ dependent. In particular, hepatic and renal P-gp and Mrp2 exhibited opposite changes in membrane protein levels.

  7. Whole-transcriptome survey of the putative ATP-binding cassette (ABC) transporter family genes in the latex-producing laticifers of Hevea brasiliensis.

    Science.gov (United States)

    Zhiyi, Nie; Guijuan, Kang; Yu, Li; Longjun, Dai; Rizhong, Zeng

    2015-01-01

    The ATP-binding cassette (ABC) proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree). A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 'full-size', 21 'half-size' and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress) stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis.

  8. Seminal Plasma Characteristics and Expression of ATP-binding Cassette Transporter A1 (ABCA1) in Canine Spermatozoa from Ejaculates with Good and Bad Freezability.

    Science.gov (United States)

    Schäfer-Somi, S; Palme, N

    2016-04-01

    The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as 40% morphologically abnormal sperm and/or Bad freezers were older than good freezers (5.3 vs 3.4 years, p bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p good freezers (p bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings.

  9. Whole-transcriptome survey of the putative ATP-binding cassette (ABC transporter family genes in the latex-producing laticifers of Hevea brasiliensis.

    Directory of Open Access Journals (Sweden)

    Nie Zhiyi

    Full Text Available The ATP-binding cassette (ABC proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree. A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 'full-size', 21 'half-size' and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis.

  10. Lipid absorption defects in intestine-specific microsomal triglyceride transfer protein and ATP-binding cassette transporter A1-deficient mice.

    Science.gov (United States)

    Iqbal, Jahangir; Parks, John S; Hussain, M Mahmood

    2013-10-18

    We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92-95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-γ (PPARγ) and carnitine palmitoyltransferase 1α (CPT1α). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations.

  11. Genome-wide identification of ATP-binding cassette (ABC) transporters and their roles in response to polycyclic aromatic hydrocarbons (PAHs) in the copepod Paracyclopina nana.

    Science.gov (United States)

    Jeong, Chang-Bum; Kim, Duck-Hyun; Kang, Hye-Min; Lee, Young Hwan; Kim, Hui-Su; Kim, Il-Chan; Lee, Jae-Seong

    2017-02-01

    The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families.

  12. Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

    Science.gov (United States)

    Keniya, Mikhail V; Holmes, Ann R; Niimi, Masakazu; Lamping, Erwin; Gillet, Jean-Pierre; Gottesman, Michael M; Cannon, Richard D

    2014-10-01

    ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-β mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

  13. The capacity of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette transporters to form biofilms and comparison with the wild type

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2014-02-01

    Full Text Available Listeria monocytogenes (Lm is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877 were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that DLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment.

  14. The Capacity of Listeria Monocytogenes Mutants with In-Frame Deletions in Putative ATP-Binding Cassette Transporters to form Biofilms and Comparison with the Wild Type

    Science.gov (United States)

    Ceruso, Marina; Fratamico, Pina; Chirollo, Claudia; Taglialatela, Rosanna; Cortesi, Maria Luisa

    2014-01-01

    Listeria monocytogenes (Lm) is a food-borne pathogen responsible for human listeriosis, an invasive infection with high mortality rates. Lm has developed efficient strategies for survival under stress conditions such as starvation and wide variations in temperature, pH, and osmolarity. Therefore, Lm can survive in food under multiple stress conditions. Detailed studies to determine the mode of action of this pathogen for survival under stress conditions are important to control Lm in food. It has been shown that genes encoding for ATP-binding cassette (ABC) transporters are induced in Lm in food, in particular under stress conditions. Previous studies showed that these genes are involved in sensitivity to nisin, acids, and salt. The aim of this study was to determine the involvement of some ABC transporters in biofilm formation. Therefore, deletion mutants of ABC transporter genes (LMOf2365_1875 and LMOf2365_1877) were created in Lm F2365, and then were compared to the wild type for their capacity to form biofilms. Lm strain F2365 was chosen as reference since the genome is fully sequenced and furthermore this strain is particularly involved in food-borne outbreaks of listeriosis. Our results showed that ΔLMOf2365_1875 had an increased capacity to form biofilms compared to the wild type, indicating that LMOf2365_1875 negatively regulates biofilm formation. A deeper knowledge on the ability to form biofilms in these mutants may help in the development of intervention strategies to control Lm in food and in the environment. PMID:27800311

  15. Galectin-3 silencing inhibits epirubicin-induced ATP binding cassette transporters and activates the mitochondrial apoptosis pathway via β-catenin/GSK-3β modulation in colorectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Yung-Kuo Lee

    Full Text Available Multidrug resistance (MDR, an unfavorable factor compromising the treatment efficacy of anticancer drugs, involves the upregulation of ATP binding cassette (ABC transporters and induction of galectin-3 signaling. Galectin-3 plays an anti-apoptotic role in many cancer cells and regulates various pathways to activate MDR. Thus, the inhibition of galectin-3 has the potential to enhance the efficacy of the anticancer drug epirubicin. In this study, we examined the effects and mechanisms of silencing galectin-3 via RNA interference (RNAi on the β-catenin/GSK-3β pathway in human colon adenocarcinoma Caco-2 cells. Galectin-3 knockdown increased the intracellular accumulation of epirubicin in Caco-2 cells; suppressed the mRNA expression of galectin-3, β-catenin, cyclin D1, c-myc, P-glycoprotein (P-gp, MDR-associated protein (MRP 1, and MRP2; and downregulated the protein expression of P-gp, cyclin D1, galectin-3, β-catenin, c-Myc, and Bcl-2. Moreover, galectin-3 RNAi treatment significantly increased the mRNA level of GSK-3β, Bax, caspase-3, and caspase-9; remarkably increased the Bax-to-Bcl-2 ratio; and upregulated the GSK-3β and Bax protein expressions. Apoptosis was induced by galectin-3 RNAi and/or epirubicin as demonstrated by chromatin condensation, a higher sub-G1 phase proportion, and increased caspase-3 and caspase-9 activity, indicating an intrinsic/mitochondrial apoptosis pathway. Epirubicin-mediated resistance was effectively inhibited via galectin-3 RNAi treatment. However, these phenomena could be rescued after galectin-3 overexpression. We show for the first time that the silencing of galectin-3 sensitizes MDR cells to epirubicin by inhibiting ABC transporters and activating the mitochondrial pathway of apoptosis through modulation of the β-catenin/GSK-3β pathway in human colon cancer cells.

  16. Polycyclic aromatic hydrocarbons (PAHs) mediate transcriptional activation of the ATP binding cassette transporter ABCB6 gene via the aryl hydrocarbon receptor (AhR).

    Science.gov (United States)

    Chavan, Hemantkumar; Krishnamurthy, Partha

    2012-09-14

    Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

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

    Science.gov (United States)

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

    2015-02-01

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

  18. Effect of Apolipoprotein A-I on ATP Binding Cassette Transporter A1 Degradation and Cholesterol Efflux in THP-1 Macrophage-derived Foam Cells

    Institute of Scientific and Technical Information of China (English)

    Chao-Ke TANG; Chang-Geng RUAN; Yong-Zong YANG; Guo-Hua TANG; Guang-Hui IY; Zuo WANG; Lu-Shan LIU; Shuang WAN; Zhong-Hua YUAN; Xiu-Sheng HE; Jun-Hao YANG

    2004-01-01

    Cholesterol-loaded macrophage foam cells are a central componentof atherosclerotic lesions ATP binding cassette transporter A1(ABCA1),the defective molecule in Tangier disease,mediates the efflux ofphospholipid and cholesterol from cells to apolipoprotein A-I(apoA-I),reversing foam cell formation.This study investigated the effect of apoA-I on ABCA1 degradation and cholesterol efflux in THP-1 macrophagederived foam cells.After exposure of the cultured THP-1 macrophage-derived foam cells to apoA-I for different time,cholesterol efflux,ABCA1 mRNA and protein levels were determined by FJ-2107P type liquid scintillator,RT-PCR and Western blot,respectively.The mean ABCA1 fluorescence intensity on THP-1macrophage-derived foam cells was detected by flow cytometry.Results showed that apoA-I markedly increased ABCAl-mediated cholesterol efflux from THP-1 macrophage-derived foam cells.This was accompanied by an increase in the content of ABCA1.ApoA-I did not alter ABCA 1 mRNA abundance.Significantly,thiol protease inhibitors increased the level ofABCA1 protein and slowed its decay in THP-1macrophage-derived foam cells,whereas none of the proteosome-specific inhibitor lactacystin,other protease inhibitors,or the lysosomal inhibitor NH4Cl showed such effects.The apoA-I-mediated cellular cholesterol efflux was enhanced by thiol protease inhibitors.Our results suggested that thiol protease inhibitors mightprovide an alternative way to upregulate ABCA1 protein.This strategy is especially appealing since it may mimic the stabilizing effect of the natural ligands apoA-I.

  19. Enhancement of avermectin and ivermectin production by overexpression of the maltose ATP-binding cassette transporter in Streptomyces avermitilis.

    Science.gov (United States)

    Li, Meng; Chen, Zhi; Zhang, Xuan; Song, Yuan; Wen, Ying; Li, Jilun

    2010-12-01

    We investigated the function of maltose ABC transporter system encoded by malEFG-a and the effect of its overexpression on antibiotic production in Streptomyces avermitilis. A malEFG-a deletion mutant was unable to grow in a minimal medium with maltose as sole carbon source and produce avermectin. Maltose utilization and avermectin production were restored by introduction of a single copy of malEFG-a. RT-PCR analysis showed that the expression of malE-a was induced by maltose, and was strongly repressed by glucose. When multi-copy, integrative malEFG-a gene expression vectors were introduced into wild-type strain ATCC31267 and ivermectin-producer OI-31, antibiotic production increased by 2.6- to 3.3-fold and the time required for fermentation decreased by about 10%. The overexpression of malEFG-a improved the utilization rate of starch, and thereby enhanced avermectin production. Such an approach would be useful for the improvement of commercial antibiotic production using starch as the main carbon source in the fermentation process.

  20. Host response transcriptional profiling reveals extracellular components and ABC (ATP-binding cassette transporters gene enrichment in typhoid fever-infected Nigerian children

    Directory of Open Access Journals (Sweden)

    Resau James H

    2011-09-01

    bacterial invasion. Distinct gene expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We found novel down-regulation of ABC (ATP-binding cassette transporters genes such as ABCA7, ABCC5, and ABCD4 and ATPase activity as the highest enriched pathway. Conclusions We identified unique extracellular components and ABC transporters gene enrichments in typhoid fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for typhoid fever in Africa.

  1. ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

    Science.gov (United States)

    ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

  2. Advances in research of ATP-binding cassette transporters in drug resistance mechanisms of intractable epilepsy%ATP结合盒式蛋白在难治性癫(痫)耐药性机制的研究进展

    Institute of Scientific and Technical Information of China (English)

    付帅

    2014-01-01

    Epilepsy is one of the common diseases in the nervous system with its complicated pathogenesis still remains unknown.The drug resistance mechanism of intractable epilepsy has always been a key point in the research of neuroscience.A possible cause for the drug resistance is the over expression of efflux drug transporters,e.g.ATP-binding cassette transporters,which may decrease extracellular antiepileptic drugs levels in brains of intractable epilepsy patients.ATP-binding cassette transporters are super family of transporter proteins that require ATP hydrolysis for the transport of substrates across membranes,including P-glycoprotein,multidrug resistance-associated protein,major vault protein and breast cancer resistance associated protein.They are major impediment for the AED successful treatment of many forms of refractory epilepsy in human.This paper reviews the research progress on over-expression of ATP-binding cassette transporters and mechanism of drug resistance in intractable epilepsy.%难治性癫(痫)因其耐药机制的复杂性,迄今尚未清楚,目前探究其对抗癫(痫)药物的多重耐药性的一大热点是外流性药物转运蛋白.ATP结合盒式蛋白是外流性药物转运蛋白的代表,其中包括P糖蛋白、多药耐药蛋白、穹窿体主蛋白、乳腺癌耐药蛋白等,它们可以决定抗癫(痫)药物能否有效地作用于癫(痫)部位,而难治性癫(痫)患者对这些蛋白的高表达普遍存在,但是否与疾病耐药性相关仍需进一步探讨.该文从癫(痫)患者的ATP结合盒式蛋白高表达原因和蛋白对药物转运的作用机制方面对患者耐药性影响方面作一综述.

  3. Crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus.

    Science.gov (United States)

    Manjula, M; Pampa, K J; Kumar, S M; Mukherjee, S; Kunishima, N; Rangappa, K S; Lokanath, N K

    2015-03-27

    The ATP binding cassette (ABC) transporters, represent one of the largest superfamilies of primary transporters, which are very essential for various biological functions. The crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus has been determined at 1.77 Å resolution. The crystal structure revealed that the protomer has two thick arms, (arm I and II), which resemble 'L' shape. The ATP-binding pocket is located close to the end of arm I. ATP molecule is docked into the active site of the protein. The dimeric crystal structure of ATP-binding subunit of ABC transporter from G. kaustophilus has been compared with the previously reported crystal structure of ATP-binding subunit of ABC transporter from Salmonella typhimurium.

  4. Suppression of c-Myc is involved in multi-walled carbon nanotubes' down-regulation of ATP-binding cassette transporters in human colon adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhaojing [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Xu, Yonghong [Institute of Ophthalmological Research, Department of Ophthalmology, Renmin Hospital of Wuhan University, 430060 Wuhan (China); Meng, Xiangning [School of Materials and Metallurgy, Northeastern University, Shenyang 110819 (China); Watari, Fumio [Department of Biomedical, Dental Materials and Engineering, Graduate School of Dental Medicine, Hokkaido University, Sapporo 060-8586 (Japan); Liu, Hudan, E-mail: hudanliu@hust.edu.cn [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China); Chen, Xiao, E-mail: mornsmile@yahoo.com [Department of Pharmacology, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan (China)

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance.

  5. The bovine ATP-binding cassette transporter ABCG2 Tyr581Ser single-nucleotide polymorphism increases milk secretion of the fluoroquinolone danofloxacin.

    Science.gov (United States)

    Otero, Jon A; Real, Rebeca; de la Fuente, Álvaro; Prieto, Julio G; Marqués, Margarita; Álvarez, Ana I; Merino, Gracia

    2013-03-01

    The bovine adenosine triphosphate-binding cassette transporter G2 (ABCG2/breast cancer resistance protein) polymorphism Tyr581Ser (Y581S) has recently been shown to increase in vitro transepithelial transport of antibiotics. Since this transporter has been extensively related to the active secretion of drugs into milk, the potential in vivo effect of this polymorphism on secretion of xenobiotics in livestock could have striking consequences for milk production, the dairy industry, and public health. Our purpose was to study the in vivo effect of this polymorphism on the secretion of danofloxacin, a widely used veterinary antibiotic, into milk. Danofloxacin (1.25 mg/kg) was administered to six Y/Y 581 homozygous and six Y/S 581 heterozygous lactating cows, and plasma and milk samples were collected and analyzed by high-performance liquid chromatography. No differences were found in the pharmacokinetic parameters of danofloxacin in plasma between the two groups of animals. In contrast, Y/S heterozygous cows showed a 2-fold increase in danofloxacin levels in milk. In addition, the pharmacokinetic elimination parameters, mean residence time and elimination half-life, were significantly lower in the milk of the animals carrying the Y/S polymorphism. These in vivo results are in agreement with our previously published in vitro data, which showed a greater capacity of the S581 variant in accumulation assays, and demonstrate, for the first time, an important effect of the Y581S single-nucleotide polymorphism on antibiotic secretion into cow milk. These findings could be extended to other ABCG2 substrates, and may be relevant for the treatment of mastitis and for the design of accurate and novel strategies to handle milk residues.

  6. Binding of PDZ-RhoGEF to ATP-binding cassette transporter A1 (ABCA1) induces cholesterol efflux through RhoA activation and prevention of transporter degradation.

    Science.gov (United States)

    Okuhira, Keiichiro; Fitzgerald, Michael L; Tamehiro, Norimasa; Ohoka, Nobumichi; Suzuki, Kazuhiro; Sawada, Jun-ichi; Naito, Mikihiko; Nishimaki-Mogami, Tomoko

    2010-05-21

    ATP-binding cassette transporter A1 (ABCA1)-mediated lipid efflux to apolipoprotein A1 (apoA-I) initiates the biogenesis of high density lipoprotein. Here we show that the Rho guanine nucleotide exchange factors PDZ-RhoGEF and LARG bind to the C terminus of ABCA1 by a PDZ-PDZ interaction and prevent ABCA1 protein degradation by activating RhoA. ABCA1 is a protein with a short half-life, and apoA-I stabilizes ABCA1 protein; however, depletion of PDZ-RhoGEF/LARG by RNA interference suppressed the apoA-I stabilization of ABCA1 protein in human primary fibroblasts. Exogenous PDZ-RhoGEF expression activated RhoA and increased ABCA1 protein levels and cholesterol efflux activity. Likewise, forced expression of a constitutively active RhoA mutant significantly increased ABCA1 protein levels, whereas a dominant negative RhoA mutant decreased them. The constitutively active RhoA retarded ABCA1 degradation, thus accounting for its ability to increase ABCA1 protein. Moreover, stimulation with apoA-I transiently activated RhoA, and the pharmacological inhibition of RhoA or the dominant negative RhoA blocked the ability of apoA-I to stabilize ABCA1. Finally, depletion of RhoA or RhoGEFs/RhoA reduces the cholesterol efflux when transcriptional regulation via PPARgamma is eliminated. Taken together, our results have identified a novel physical and functional interaction between ABCA1 and PDZ-RhoGEF/LARG, which activates RhoA, resulting in ABCA1 stabilization and cholesterol efflux activity.

  7. An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon

    Science.gov (United States)

    Spivey, Vicky L; Whalan, Rachael H; Hirst, Elizabeth M A; Smerdon, Stephen J; Buxton, Roger S

    2013-01-01

    The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence. PMID:23915284

  8. Localization of the ATP-binding cassette (ABC transport proteins PfMRP1, PfMRP2, and PfMDR5 at the Plasmodium falciparum plasma membrane

    Directory of Open Access Journals (Sweden)

    Luty Adrian JF

    2009-08-01

    Full Text Available Abstract Background The spread of drug resistance has been a major obstacle to the control of malaria. The mechanisms underlying drug resistance in malaria seem to be complex and multigenic. The current literature on multiple drug resistance against anti-malarials has documented PfMDR1, an ATP-binding cassette (ABC protein, as an important determinant of resistance. In the Plasmodium falciparum genome, there are several ABC transporters some of which could be putative drug transporting proteins. In order to understand the molecular mechanisms underlying drug resistance, characterization of these transporters is essential. The aim of this study was to characterize and localize putative ABC transporters. Methods In the plasmoDB database, 16 members of the P. falciparum ABC family can be identified, 11 of which are putative transport proteins. A phylogenetic analysis of the aligned NBDs of the PfABC genes was performed. Antibodies against PfMRP1 (PfABCC1, PfMRP2 (PfABCC2, and PfMDR5 (PfABCB5 were generated, affinity purified and used in immunocytochemistry to localize the proteins in the asexual stages of the parasite. Results The ABC family members of P. falciparum were categorized into subfamilies. The ABC B subfamily was the largest and contained seven members. Other family members that could be involved in drug transport are PfABCC1, PfABCC2, PfABCG1, and PfABCI3. The expression and localization of three ABC transport proteins was determined. PfMRP1, PfMRP2, and PfMDR5 are localized to the plasma membrane in all asexual stages of the parasite. Conclusion In conclusion, 11 of the 16 ABC proteins in the P. falciparum genome are putative transport proteins, some of which might be involved in drug resistance. Moreover, it was demonstrated that three of these proteins are expressed on the parasite's plasma membrane.

  9. Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

    Science.gov (United States)

    Merino, Gracia; Real, Rebeca; Baro, Marta F; Gonzalez-Lobato, Lucia; Prieto, Julio G; Alvarez, Ana I; Marques, Margarita M

    2009-01-01

    ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

  10. Genome-wide identification and evolution of ATP-binding cassette transporters in the ciliate Tetrahymena thermophila: A case of functional divergence in a multigene family

    Directory of Open Access Journals (Sweden)

    Yuan Dongxia

    2010-10-01

    Full Text Available Abstract Background In eukaryotes, ABC transporters that utilize the energy of ATP hydrolysis to expel cellular substrates into the environment are responsible for most of the efflux from cells. Many members of the superfamily of ABC transporters have been linked with resistance to multiple drugs or toxins. Owing to their medical and toxicological importance, members of the ABC superfamily have been studied in several model organisms and warrant examination in newly sequenced genomes. Results A total of 165 ABC transporter genes, constituting a highly expanded superfamily relative to its size in other eukaryotes, were identified in the macronuclear genome of the ciliate Tetrahymena thermophila. Based on ortholog comparisons, phylogenetic topologies and intron characterizations, each highly expanded ABC transporter family of T. thermophila was classified into several distinct groups, and hypotheses about their evolutionary relationships are presented. A comprehensive microarray analysis revealed divergent expression patterns among the members of the ABC transporter superfamily during different states of physiology and development. Many of the relatively recently formed duplicate pairs within individual ABC transporter families exhibit significantly different expression patterns. Further analysis showed that multiple mechanisms have led to functional divergence that is responsible for the preservation of duplicated genes. Conclusion Gene duplications have resulted in an extensive expansion of the superfamily of ABC transporters in the Tetrahymena genome, making it the largest example of its kind reported in any organism to date. Multiple independent duplications and subsequent divergence contributed to the formation of different families of ABC transporter genes. Many of the members within a gene family exhibit different expression patterns. The combination of gene duplication followed by both sequence divergence and acquisition of new patterns of

  11. ATP Binding Cassette Transporter ABCA7 Regulates NKT Cell Development and Function by Controlling CD1d Expression and Lipid Raft Content

    Science.gov (United States)

    Nowyhed, Heba N.; Chandra, Shilpi; Kiosses, William; Marcovecchio, Paola; Andary, Farah; Zhao, Meng; Fitzgerald, Michael L.; Kronenberg, Mitchell; Hedrick, Catherine C.

    2017-01-01

    ABCA7 is an ABC transporter expressed on the plasma membrane, and actively exports phospholipid complexes from the cytoplasmic to the exocytoplasmic leaflet of membranes. Invariant NKT (iNKT) cells are a subpopulation of T lymphocytes that recognize glycolipid antigens in the context of CD1d-mediated antigen presentation. In this study, we demonstrate that ABCA7 regulates the development of NKT cells in a cell-extrinsic manner. We found that in Abca7−/− mice there is reduced expression of CD1d accompanied by an alteration in lipid raft content on the plasma membrane of thymocytes and antigen presenting cells. Together, these alterations caused by absence of ABCA7 negatively affect NKT cell development and function. PMID:28091533

  12. Brucella abortus mutants lacking ATP-binding cassette transporter proteins are highly attenuated in virulence and confer protective immunity against virulent B. abortus challenge in BALB/c mice.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Park, Bo-Kyoung; Hahn, Tae-Wook

    2016-06-01

    Brucella abortus RB51 is an attenuated vaccine strain that has been most frequently used for bovine brucellosis. Although it is known to provide good protection in cattle, it still has some drawbacks including resistance to rifampicin, residual virulence and pathogenicity in humans. Thus, there has been a continuous interest on new safe and effective bovine vaccine candidates. In the present study, we have constructed unmarked mutants by deleting singly cydD and cydC genes, which encode ATP-binding cassette transporter proteins, from the chromosome of the virulent Brucella abortus isolate from Korean cow (referred to as IVK15). Both IVK15ΔcydD and ΔcydC mutants showed increased sensitivity to metal ions, hydrogen peroxide and acidic pH, which are mimic to intracellular environment during host infection. Additionally, the mutants exhibited a significant growth defect in RAW264.7 cells and greatly attenuated in mice. Vaccination of mice with either IVK15ΔcydC or IVK15ΔcydD mutant could elicit an anti-Brucella specific immunoglobulin G (IgG) and IgG subclass responses as well as enhance the secretion of interferon-gamma, and provided better protection against challenge with B. abortus strain 2308 than with the commercial B. abortus strain RB51 vaccine. Collectively, these results suggest that both IVK15ΔcydC and IVK15ΔcydD mutants could be an attenuated vaccine candidate against B. abortus.

  13. Pharmacophore modeling of nilotinib as an inhibitor of ATP-binding cassette drug transporters and BCR-ABL kinase using a three-dimensional quantitative structure-activity relationship approach.

    Science.gov (United States)

    Shukla, Suneet; Kouanda, Abdul; Silverton, Latoya; Talele, Tanaji T; Ambudkar, Suresh V

    2014-07-07

    Nilotinib (Tasigna) is a tyrosine kinase inhibitor approved by the FDA to treat chronic phase chronic myeloid leukemia patients. It is also a transport substrate of the ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-glycoprotein, P-gp) and ABCG2 (BCRP), which may have an effect on the pharmacokinetics and toxicity of this drug. The goal of this study was to identify pharmacophoric features of nilotinib in order to potentially develop specific inhibitors of BCR-ABL kinase with minimal interactions with ABC drug transporters. Three-dimensional pharmacophore modeling and quantitative structure-activity relationship (QSAR) studies were carried out on a series of nilotinib analogues to identify chemical features that contribute to inhibitory activity of nilotinib against BCR-ABL kinase activity, P-gp, and ABCG2. Twenty-five derivatives of nilotinib were synthesized and were then tested to measure their activity to inhibit BCR-ABL kinase and to inhibit the function of ABC drug transporters. A set of in vitro experiments including kinase activity and cell-based transport assays and photolabeling of P-gp and ABCG2 with a transport substrate, [(125)I]-iodoarylazido-prazosin (IAAP), were carried out in isolated membranes to evaluate the potency of the derivatives to inhibit the function of ABC drug transporters and BCR-ABL kinase. Sixteen, fourteen, and ten compounds were selected as QSAR data sets, respectively, to generate PHASE v3.1 pharmacophore models for BCR-ABL kinase, ABCG2, and P-gp inhibitors. The IC50 values of these derivatives against P-gp, ABCG2, or BCR-ABL kinase were used to generate pharmacophore features required for optimal interactions with these targets. A seven-point pharmacophore (AADDRRR) for BCR-ABL kinase inhibitory activity, a six-point pharmacophore (ADHRRR) for ABCG2 inhibitory activity, and a seven-point pharmacophore (AADDRRR) for P-gp inhibitory activity were generated. The derived models clearly demonstrate high predictive power

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

    Science.gov (United States)

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

    2014-08-01

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

  15. Kaempferol suppresses lipid accumulation in macrophages through the downregulation of cluster of differentiation 36 and the upregulation of scavenger receptor class B type I and ATP-binding cassette transporters A1 and G1.

    Science.gov (United States)

    Li, Xiu-Ying; Kong, Ling-Xi; Li, Juan; He, Hai-Xia; Zhou, Yuan-Da

    2013-02-01

    The accumulation of foam cells in atherosclerotic lesions is a hallmark of early-stage atherosclerosis. Kaempferol has been shown to inhibit oxidized low-density lipoprotein (oxLDL) uptake by macrophages; however, the underlying molecular mechanisms are not yet fully investigated. In this study, we shown that treatment with kaempferol markedly suppresses oxLDL-induced macrophage foam cell formation, which occurs due to a decrease in lipid accumulation and an increase in cholesterol efflux from THP-1-derived macrophages. Additionally, the kaempferol treatment of macrophages led to the downregulation of cluster of differentiation 36 (CD36) protein levels, the upregulation of ATP-binding cassette (ABC) transporter A1 (ABCA1), scavenger receptor class B type I (SR-BI) and ABCG1 protein levels, while no effects on scavenger receptor A (SR-A) expression were observed. Kaempferol had similar effects on the mRNA and protein expression of ABCA1, SR-BI, SR-A, CD36 and ABCG1. The reduced CD36 expression following kaempferol treatment involved the inhibition of c-Jun-activator protein-1 (AP-1) nuclear translocation. The inhibition of AP-1 using the inhibitor, SP600125, confirmed this involvement, as the AP-1 inhibition significantly augmented the kaempferol-induced reduction in CD36 expression. Accordingly, the kaempferol-mediated suppression of lipid accumulation in macrophages was also augmented by SP600125. The increased expression of ABCA1, SR-BI and ABCG1 following kaempferol treatment was accompanied by the enhanced protein expression of heme oxygenase-1 (HO-1). This increase was reversed following the knockdown of the HO-1 gene using small hairpin RNA (shRNA). Moreover, the kaempferol-mediated attenuation of lipid accumulation and the promotion of cholesterol efflux was also inhibited by HO-1 shRNA. In conclusion, the c-Jun-AP‑1-dependent downregulation of CD36 and the HO-1-dependent upregulation of ABCG1, SR-BI and ABCA1 may mediate the beneficial effects of

  16. Structure, function, and evolution of bacterial ATP-binding cassette systems

    Energy Technology Data Exchange (ETDEWEB)

    Davidson, A.L.; Dassa, E.; Orelle, C.; Chen, J. (Purdue)

    2010-07-27

    The ATP-binding cassette (ABC) systems constitute one of the largest superfamilies of paralogous sequences. All ABC systems share a highly conserved ATP-hydrolyzing domain or protein (the ABC; also referred to as a nucleotide-binding domain [NBD]) that is unequivocally characterized by three short sequence motifs (Fig. 1): these are the Walker A and Walker B motifs, indicative of the presence of a nucleotide-binding site, and the signature motif, unique to ABC proteins, located upstream of the Walker B motif (426). Other motifs diagnostic of ABC proteins are also indicated in Fig. 1. The biological significance of these motifs is discussed in Structure, Function, and Dynamics of the ABC. ABC systems are widespread among living organisms and have been detected in all genera of the three kingdoms of life, with remarkable conservation in the primary sequence of the cassette and in the organization of the constitutive domains or subunits (203, 420). ABC systems couple the energy of ATP hydrolysis to an impressively large variety of essential biological phenomena, comprising not only transmembrane (TM) transport, for which they are best known, but also several non-transport-related processes, such as translation elongation (62) and DNA repair (174). Although ABC systems deserve much attention because they are involved in severe human inherited diseases (107), they were first discovered and characterized in detail in prokaryotes, as early as the 1970s (13, 148, 238, 468). The most extensively analyzed systems were the high-affinity histidine and maltose uptake systems of Salmonella enterica serovar Typhimurium and Escherichia coli. Over 2 decades ago, after the completion of the nucleotide sequences encoding these transporters in the respective laboratories of Giovanna Ames and Maurice Hofnung, Hiroshi Nikaido and colleagues noticed that the two systems displayed a global similarity in the nature of their components and, moreover, that the primary sequences of MalK and

  17. Molecular Characterization of LjABCG1, an ATP-Binding Cassette Protein in Lotus japonicus.

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    Akifumi Sugiyama

    Full Text Available LjABCG1, a full-size ABCG subfamily of ATP-binding cassette proteins of a model legume, Lotus japonicus, was reported as a gene highly expressed during the early stages of nodulation, but have not been characterized in detail. In this study we showed that the induction of LjABCG1 expression was remarkable by methyl jasmonate treatment, and reporter gene experiments indicated that LjABCG1 was strongly expressed in the nodule parenchyma and cell layers adjacent to the root vascular tissue toward the nodule. LjABCG1 was suggested to be localized at the plasma membrane based on the fractionation of microsomal membranes as well as separation via aqueous two-phase partitioning. The physiological functions of LjABCG1 in symbiosis and pathogenesis were analyzed in homologous and heterologous systems. LjABCG1 knock-down L. japonicus plants did not show clear phenotypic differences in nodule formation, and not in defense against Pseudomonas syringae, either. In contrast, when LjABCG1 was expressed in the Arabidopsis pdr8-1 mutant, the penetration frequency of Phytophthora infestans, a potato late blight pathogen, was significantly reduced in LjABCG1/pdr8-1 than in pdr8-1 plants. This finding indicated that LjABCG1, at least partially, complemented the phenotype of pdr8 in Arabidopsis, suggesting the multiple roles of this protein in plant-microbe interactions.

  18. Lipid raft involved in drug resistance: relationship between multidrug resistance ATP-binding cassette transporters and lipid raft%脂筏参与耐药: 多药耐药相关ABC转运蛋白与脂筏的关系

    Institute of Scientific and Technical Information of China (English)

    王琳; 贾宇; 姜远英

    2011-01-01

    Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. Recently ATP-binding cassette (ABC) transporters, which are associated with multidrug resistance, have been found in lipid rafts; therefore they might be related to drug resistance. Here we introduce the relationship between the localization and functions of three multi-drug related ABC transporters, including two relevant to multidrug resistance in tumor cells(Pgp/ABCB1 and MRP1/ABCC1) and one relevant to multidrug resistance in Candida albicans (Cdrlp). We also discuss the influence of sphingolipids and cholesterol, two major components of lipid rafts, on the localization and function of the above three ABC transporters.%脂筏(lipid raft)和细胞的许多功能,如信号转导、蛋白质和脂类的转运等都相关.近来有研究发现,与多药耐药密切相关的ABC转运蛋白(ATP-binding cassette transporter)定位于脂筏中,因此推测脂筏可能与耐药性有一定关系.本文综述了3种和耐药相关的ABC转运蛋白的定位与其功能之间的联系,分别是和肿瘤细胞多药耐药相关的ABC转运蛋白Pgp/ABCB1、MRP1/ABCC1以及与白假丝酵母菌(白念珠菌)多药耐药相关的ABC转运蛋白Cdr1p;并进一步讨论了脂筏的重要组成成分胆固醇和鞘脂对上述3种ABC转运蛋白的定位和功能的影响.

  19. Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle.

    Science.gov (United States)

    Joseph, Benesh; Jeschke, Gunnar; Goetz, Birke A; Locher, Kaspar P; Bordignon, Enrica

    2011-11-25

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

  20. Modulating the function of ATP-binding cassette subfamily G member 2 (ABCG2) with inhibitor cabozantinib.

    Science.gov (United States)

    Zhang, Guan-Nan; Zhang, Yun-Kai; Wang, Yi-Jun; Barbuti, Anna Maria; Zhu, Xi-Jun; Yu, Xin-Yue; Wen, Ai-Wen; Wurpel, John N D; Chen, Zhe-Sheng

    2017-01-25

    Cabozantinib (XL184) is a small molecule tyrosine kinase receptor inhibitor, which targets c-Met and VEGFR2. Cabozantinib has been approved by the Food and Drug Administration to treat advanced medullary thyroid cancer and renal cell carcinoma. In the present study, we evaluated the ability of cabozantinib to modulate the function of the ATP-binding cassette subfamily G member 2 (ABCG2) by sensitizing cells that are resistant to ABCG2 substrate antineoplastic drugs. We used a drug-selected resistant cell line H460/MX20 and three ABCG2 stable transfected cell lines ABCG2-482-R2, ABCG2-482-G2, and ABCG2-482-T7, which overexpress ABCG2. Cabozantinib, at non-toxic concentrations (3 or 5μM), sensitized the ABCG2-overexpressing cells to mitoxantrone, SN-38, and topotecan. Our results indicate that cabozantinib reverses ABCG2-mediated multidrug resistance by antagonizing the drug efflux function of the ABCG2 transporter instead of downregulating its expression. The molecular docking analysis indicates that cabozantinib binds to the drug-binding site of the ABCG2 transporter. Overall, our findings demonstrate that cabozantinib inhibits the ABCG2 transporter function and consequently enhances the effect of the antineoplastic agents that are substrates of ABCG2. Cabozantinib may be a useful agent in anticancer treatment regimens for patients who are resistant to ABCG2 substrate drugs.

  1. 三磷酸腺苷结合盒转运体A1在巨噬细胞胆固醇流出中的作用%Effects of ATP binding cassette transporter A1 on cholesterol efflux in macrophages

    Institute of Scientific and Technical Information of China (English)

    唐朝克; 严鹏科; 杨永宗

    2003-01-01

    Tangier disease is caused by mutations in ATP binding cassette transporter AI( ABCA1).ABCA1 interacts with lipid-free apolipoproteins, promoting phospholipid and cholesterol ettlux fzom cells and giving rise to HDL particles. ABCA1 may act as a phospholipid translocase facilitating phospholipid binding to apoA-Ⅰ. ABCA1 gene expression is upregulated in cholesterol-loaded cells as a result of activation of IXR/RXR- mediated gene transcription. LXR and RXR coordinately induce a battery of genes mediating cellular cholesterol efllux, centripetal cholesterol tramport, and cholesterol excretion in bile. Small- molecule activators of LXR/RXR or other stimulators of macrophage or intestinal cholesterol efl]ux hold great promise as future treat-ments for atherosclerosis.

  2. Localization of the ATP-binding cassette (ABC) transport proteins PfMRP1, PfMRP2, and PfMDR5 at the Plasmodium falciparum plasma membrane.

    NARCIS (Netherlands)

    Kavishe, R.A.; Heuvel, J.M.W. van den; Vegte-Bolmer, M. van de; Luty, A.J.; Russel, F.G.M.; Koenderink, J.B.

    2009-01-01

    BACKGROUND: The spread of drug resistance has been a major obstacle to the control of malaria. The mechanisms underlying drug resistance in malaria seem to be complex and multigenic. The current literature on multiple drug resistance against anti-malarials has documented PfMDR1, an ATP-binding casse

  3. ATP-Binding Cassette Genes Genotype and Expression: A Potential Association with Pancreatic Cancer Development and Chemoresistance?

    Directory of Open Access Journals (Sweden)

    Li Pang

    2014-01-01

    Full Text Available Genetic polymorphisms in ABC (ATP-binding cassette transporter genes are associated with differential responses to chemotherapy in various cancers including pancreatic cancer. In this study, four SNPs in the ABCB1, ABCC1, and ABCG2 genes were investigated in normal and pancreatic cancerous specimens. The expression of the three transporters was also analyzed. The TT genotypes of G2677T and C3435T in ABCB1 gene were associated with lower risk of developing pancreatic cancer (P=0.013, OR = 0.35 and P=0.015, OR = 0.29, resp.. To our knowledge, this is the first report of the common polymorphisms in the ABCB1 gene affecting the genetic risk of developing pancreatic cancer. Moreover, the expression of ABCB1 in 2677TT and 3435TT carriers was lower compared to the wild-type homozygotes and heterozygotes. A cell viability assay, using standard pancreatic cancer cell lines, revealed that the ABCB1 2677TT-3455TT haplotype was more sensitive than the other haplotypes to gemcitabine. Conclusion. Polymorphisms in ABCB1 G2677T and G3435T were associated with differential susceptibility to pancreatic cancer and may predict responses to chemotherapy.

  4. ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 10 (ABCC10) are not primary resistance factors for cabazitaxel

    Institute of Scientific and Technical Information of China (English)

    Rishil J Kathawala; Yi-Jun Wang; Suneet Shukla; Yun-Kai Zhang; Saeed Alqahtani; Amal Kaddoumi; Suresh V Ambudkar; Charles R Ashby Jr; Zhe-Sheng Chen

    2015-01-01

    Introduction:ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 10 (ABCC10) proteins are efflux transporters that couple the energy derived from ATP hydrolysis to the translocation of toxic substances and chemotherapeutic drugs out of cells. Cabazitaxel is a novel taxane that differs from paclitaxel by its lower affinity for ATP-binding cassette (ABC) transporters. Methods:We determined the effects of cabazitaxel, a novel tubulin-binding taxane, and paclitaxel on paclitaxel-resistant, ABCB1-overexpressing KB-C2 and LLC-MDR1-WT cells and paclitaxel-resistant, ABCC10-overexpressing HEK293/ABCC10 cells by calculating the degree of drug resistance and measuring ATPase activity of the ABCB1 transporter. Results:Decreased resistance to cabazitaxel compared with paclitaxel was observed in KB-C2, LLC-MDR1-WT, and HEK293/ABCC10 cells. Moreover, cabazitaxel had low efficacy, whereas paclitaxel had high efficacy in stimulating the ATPase activity of ABCB1, indicating a direct interaction of both drugs with the transporter. Conclusion:ABCB1 and ABCC10 are not primary resistance factors for cabazitaxel compared with paclitaxel, suggesting that cabazitaxel may have a low affinity for these efflux transporters.

  5. A Survey of the ATP-Binding Cassette (ABC Gene Superfamily in the Salmon Louse (Lepeophtheirus salmonis.

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    Greta Carmona-Antoñanzas

    Full Text Available Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837, are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences, C (11 and G (2. The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance.

  6. Conformational changes of the bacterial type I ATP-binding cassette importer HisQMP2 at distinct steps of the catalytic cycle.

    Science.gov (United States)

    Heuveling, Johanna; Frochaux, Violette; Ziomkowska, Joanna; Wawrzinek, Robert; Wessig, Pablo; Herrmann, Andreas; Schneider, Erwin

    2014-01-01

    Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems.

  7. Lobular Distribution and Variability in Hepatic ATP Binding Cassette Protein B1 (ABCB1, P-gp): Ontogenetic Differences and Potential for Toxicity

    Science.gov (United States)

    Abanda, Ngu Njei; Riches, Zoe; Collier, Abby C.

    2017-01-01

    The ATP Binding Cassette B1 (ABCB1) transporter has critical roles in endo- and xenobiotic efficacy and toxicity. To understand population variability in hepatic transport we determined ABCB1 mRNA and protein levels in total liver lysates sampled from 8 pre-defined sites (n = 24, 18–69 years), and in S9 from randomly acquired samples (n = 87, 7 days–87 years). ABCB1 levels did not differ significantly throughout individual livers and showed 4.4-fold protein variation between subjects. Neither mRNA nor protein levels varied with sex, ethnicity, obesity or triglycerides in lysates or S9 (that showed the same relationships), but protein levels were lower in pediatric S9 (p < 0.0001), with 76% of adult ABCB1 present at birth and predicted to mature in 5 years. Pediatric total liver lysates were not available. In summary, opportunistic collection for studying human hepatic ABCB1 is acceptable. Additionally, ABCB1 may be lower in children, indicating differential potential for toxicity and response to therapy in this special population. PMID:28218636

  8. Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

    Science.gov (United States)

    Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

    2008-01-01

    Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6422, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å3 Da−1, which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan. PMID:18540059

  9. Retinoic acid isomers up-regulate ATP binding cassette A1 and G1 and cholesterol efflux in rat astrocytes: implications for their therapeutic and teratogenic effects.

    Science.gov (United States)

    Chen, Jing; Costa, Lucio G; Guizzetti, Marina

    2011-09-01

    Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (± 0.25), 3.6- (± 0.42), 4.1- (± 0.5), and 1.75- (± 0.43) fold, respectively, and Abcg1 by 2.1- (± 0.26), 2.2- (± 0.33), 2.5- (± 0.23), and 2.2- (± 0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions.

  10. Afatinib reverses multidrug resistance in ovarian cancer via dually inhibiting ATP binding cassette subfamily B member 1.

    Science.gov (United States)

    Wang, Sheng-qi; Liu, Shi-ting; Zhao, Bo-xin; Yang, Fu-heng; Wang, Ya-tian; Liang, Qian-Ying; Sun, Ya-bin; Liu, Yuan; Song, Zhi-hua; Cai, Yun; Li, Guo-feng

    2015-09-22

    ABCB1-mediated multidrug resistance (MDR) remains a major obstacle to successful chemotherapy in ovarian cancer. Herein, afatinib at nontoxic concentrations significantly reversed ABCB1-mediated MDR in ovarian cancer cells in vitro (p afatinib caused tumor regressions and tumor necrosis in A2780T xenografts in vivo. More interestingly, unlike reversible TKIs, afatinib had a distinctive dual-mode action. Afatinib not only inhibited the efflux function of ABCB1, but also attenuated its expression transcriptionally via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB. Furthermore, apart from a substrate binding domain, afatinib could also bind to an ATP binding domain of ABCB1 through forming hydrogen bonds with Gly533, Gly534, Lys536 and Ala560 sites. Importantly, mutations in these four binding sites of ABCB1 and the tyrosine kinase domain of EGFR were not correlated with the reversal activity of afatinib on MDR. Given that afatinib is a clinically approved drug, our results suggest combining afatinib with chemotherapeutic drugs in ovarian cancer. This study can facilitate the rediscovery of superior MDR reversal agents from molecular targeted drugs to provide a more effective and safer way of resensitizing MDR.

  11. Inherited surfactant deficiency due to uniparental disomy of rare mutations in the surfactant protein-B and ATP binding cassette, subfamily A, member 3 genes

    Science.gov (United States)

    Hamvas, Aaron; Nogee, Lawrence M.; Wegner, Daniel J.; DePass, Kelcey; Christodoulou, John; Bennetts, Bruce; McQuade, Leon R.; Gray, Peter H.; Deterding, Robin R.; Carroll, Travis R.; Kammesheidt, Anja; Kasch, Laura M.; Kulkarni, Shashikant; Cole, F. Sessions

    2009-01-01

    Objective To characterize inheritance of homozygous, rare, recessive loss-of-function mutations in the surfactant protein-B (SFTPB) or ATP binding cassette, subfamily A, member 3 (ABCA3) genes in newborns with lethal respiratory failure. Study design We resequenced parents whose infants were homozygous for mutations in SFTPB or ABCA3. For infants with only one heterozygous parent, we performed microsatellite analysis for chromosomes 2 (SFTPB) and 16 (ABCA3). Results We identified one infant homozygous for the c.1549C>GAA mutation (121ins2) in SFTPB for whom only the mother was heterozygous and 3 infants homozygous for mutations in ABCA3 (p.K914R, p.P147L, and c.806_7insGCT) for whom only the fathers were heterozygous. For the SP-B deficient infant, microsatellite markers confirmed maternal heterodisomy with segmental isodisomy. Microsatellite analysis confirmed paternal isodisomy for the three ABCA3 deficient infants. Two ABCA3 deficient infants underwent lung transplantation at 3 and 5 months of age, respectively, and two infants died. None exhibited any non-pulmonary phenotype. Conclusions Uniparental disomy should be suspected in infants with rare homozygous mutations in SFTPB or ABCA3. Confirmation of parental carrier status is important to provide recurrence risk and to monitor expression of other phenotypes that may emerge through reduction to homozygosity of recessive alleles. PMID:19647838

  12. Effects of silencing the ATP-binding cassette protein E1 gene by electroporation on the proliferation and migration of EC109 human esophageal cancer cells.

    Science.gov (United States)

    Li, Xiao-Rui; Yang, Liu-Zhong; Huo, Xiao-Qing; Wang, Ying; Yang, Qing-Hui; Zhang, Qing-Qin

    2015-07-01

    In the present study, the gene expression of ATP-binding cassette protein E1 (ABCE1) in the EC109 human esophageal cancer cell line was silenced using electroporation to examine the effect if the ABCE1 gene on the growth migration and cell cycle of cancer cells. The small interference (si)RNA sequence of ABCE1 was designed and synthesized to transfect the EC109 cells by electroporation. The mRNA and protein expression levels of ABCE1 were then detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The analysis of the cell cycle and apoptosis was performed using flow cytometry. The effect of silencing the ABCE1 gene on the proliferation, migration and invasive ability of the EC109 human esophageal cancer cells were assessed using a Cell counting kit-8 (CCK-8) and with proliferation, wound-healing and cell invasion assays. The mRNA and protein expression levels of ABCE1 were significantly lower in the experimental group compared with the control group (Pmigration capacity of the cells in the experimental group was significantly decreased (Pmigration in esophageal cancer and silencing the ABCE1 gene by electroporation can significantly reduce the proliferation, invasion and migration capacity of EC109 cells in vitro.

  13. Role of NH{sub 2}-terminal hydrophobic motif in the subcellular localization of ATP-binding cassette protein subfamily D: Common features in eukaryotic organisms

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Kostsin, Dzmitry G. [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Academicheskaya Str. 27, Minsk 220072 (Belarus); Kashiwayama, Yoshinori [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Takanashi, Kojiro; Yazaki, Kazufumi [Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoko University, Uji, Kyoto 611-0011 (Japan); Imanaka, Tsuneo, E-mail: imanaka@pha.u-toyama.ac.jp [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Morita, Masashi [Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2014-10-24

    Highlights: • ABCD proteins classifies based on with or without NH{sub 2}-terminal hydrophobic segment. • The ABCD proteins with the segment are targeted peroxisomes. • The ABCD proteins without the segment are targeted to the endoplasmic reticulum. • The role of the segment in organelle targeting is conserved in eukaryotic organisms. - Abstract: In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1–3 possesses the NH{sub 2}-terminal hydrophobic region and are targeted to peroxisomes, while ABCD4 lacking the region is targeted to the endoplasmic reticulum (ER). Based on hydropathy plot analysis, we found that several eukaryotes have ABCD protein homologs lacking the NH{sub 2}-terminal hydrophobic segment (H0 motif). To investigate whether the role of the NH{sub 2}-terminal H0 motif in subcellular localization is conserved across species, we expressed ABCD proteins from several species (metazoan, plant and fungi) in fusion with GFP in CHO cells and examined their subcellular localization. ABCD proteins possessing the NH{sub 2}-terminal H0 motif were localized to peroxisomes, while ABCD proteins lacking this region lost this capacity. In addition, the deletion of the NH{sub 2}-terminal H0 motif of ABCD protein resulted in their localization to the ER. These results suggest that the role of the NH{sub 2}-terminal H0 motif in organelle targeting is widely conserved in living organisms.

  14. Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

    Science.gov (United States)

    Tahiri, Abdelghani; Delporte, Fabienne; Muhovski, Yordan; Ongena, Marc; Thonart, Philippe; Druart, Philippe

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

  15. Prognostic significance and molecular mechanism of ATP-binding cassette subfamily C member 4 in resistance to neoadjuvant radiotherapy of locally advanced rectal carcinoma.

    Directory of Open Access Journals (Sweden)

    Zhiqi Yu

    Full Text Available BACKGROUND: Mechanism of radioresistance in rectal carcinoma remains largely unknown. We aimed to evaluate the predictive role of ATP-binding cassette subfamily C member 4 (ABCC4 in locally advanced rectal carcinoma and explore possible molecular mechanisms by which ABCC4 confers the resistance to neoadjuvant radiotherapy. METHODS: The expression of ABCC4 and P53 mutant in biopsy tissue specimens from 121 locally advanced rectal carcinoma patients was examined using immunohistochemistry. The factors contributing to 3-year overall survival and disease-free survival were evaluated using the Kaplan-Meier method and Cox proportional hazard model. Lentivirus-mediated small hairpin RNA was applied to inhibit ABCC4 expression in colorectal carcinoma cell line RKO, and investigate the radiosensitivity in xenograft model. Intracellular cyclic adenosine monophosphate concentration and cell cycle distribution following irradiation were detected. RESULTS: High expression of ABCC4 and p53 mutant in pretreated tumors, poor pathological response, and high final tumor staging were significant factors independently predicted an unfavorable prognosis of locally advanced rectal carcinoma patients after neoadjuvant radiotherapy. Down-regulation of ABCC4 expression significantly enhanced irradiation-induced suppression of tumor growth in xenograft model. Furthermore, down-regulation of ABCC4 expression enhanced intracellular cyclic adenosine monophosphate production and noticeable deficiency of G1-S phase checkpoint in cell cycle following irradiation. CONCLUSIONS: Our study suggests that ABCC4 serves as a novel predictive biomarker that is responsible for the radioresistance and predicts a poor prognosis for locally advanced rectal carcinoma after neoadjuvant radiotherapy.

  16. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    Science.gov (United States)

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  17. ATP结合盒蛋白E1在真核生物中的作用%Role of ATP-binding cassette protein E1 in eukaryote

    Institute of Scientific and Technical Information of China (English)

    尤锋; 李燕

    2012-01-01

    ATP结合盒(ATP-binding cassette,ABC)蛋白超家族是真核生物进化过程中最为保守的一类蛋白.ABCE1是ABC超家族中E亚家族的唯一成员,除可抑制哺乳动物核糖核酸酶L外,还参与真核生物蛋白合成的翻译起始、终止及核糖体循环,并有可能成为新的抗肿瘤靶点.本文对ABCE1的基本生物学特性及其生物学作用作一介绍.%ATP-binding cassette ( ABC) proteins are one of the most conserved protein families in eukaryote evolution. ABCE1 is the only member in ABC E subfamily. It was found that ABCE1, a ribonuclease L inhibitor in mammals, participates in the protein translation's initiation, termination and ribosome recycling of eukaryote, and may become a new antitumor target. This article reviews the basic biological characteristics and functions of ABCE1.

  18. AtMRP1 gene of Arabidopsis encodes a glutathione S-conjugate pump: Isolation and functional definition of a plant ATP-binding cassette transporter gene

    OpenAIRE

    Lu, Yu-Ping; Li, Ze-Sheng; Rea, Philip A.

    1997-01-01

    Because plants produce cytotoxic compounds to which they, themselves, are susceptible and are exposed to exogenous toxins (microbial products, allelochemicals, and agrochemicals), cell survival is contingent on mechanisms for detoxifying these agents. One detoxification mechanism is the glutathione S-transferase-catalyzed glutathionation of the toxin, or an activated derivative, and transport of the conjugate out of the cytosol. We show here that a transporter responsible for the removal of g...

  19. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    Directory of Open Access Journals (Sweden)

    Shabeesh Balan

    Full Text Available Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS (prototype for AED-resistant epilepsy; juvenile myoclonic epilepsy (JME (prototype for AED-responsive epilepsy; and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004. This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004 and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05 cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency

  20. Genetic association analysis of ATP binding cassette protein family reveals a novel association of ABCB1 genetic variants with epilepsy risk, but not with drug-resistance.

    Science.gov (United States)

    Balan, Shabeesh; Bharathan, Sumitha Prameela; Vellichiramal, Neetha Nanoth; Sathyan, Sanish; Joseph, Vijai; Radhakrishnan, Kurupath; Banerjee, Moinak

    2014-01-01

    Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED)-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype for AED-resistant epilepsy); juvenile myoclonic epilepsy (JME) (prototype for AED-responsive epilepsy); and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T) rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004). This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004) and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05) cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE

  1. ATP-Binding Cassette Systems of Brucella

    Directory of Open Access Journals (Sweden)

    Dominic C. Jenner

    2009-01-01

    Full Text Available Brucellosis is a prevalent zoonotic disease and is endemic in the Middle East, South America, and other areas of the world. In this study, complete inventories of putative functional ABC systems of five Brucella species have been compiled and compared. ABC systems of Brucella melitensis 16M, Brucella abortus 9-941, Brucella canis RM6/66, Brucella suis 1330, and Brucella ovis 63/290 were identified and aligned. High numbers of ABC systems, particularly nutrient importers, were found in all Brucella species. However, differences in the total numbers of ABC systems were identified (B. melitensis, 79; B. suis, 72; B. abortus 64; B. canis, 74; B. ovis, 59 as well as specific differences in the functional ABC systems of the Brucella species. Since B. ovis is not known to cause human brucellosis, functional ABC systems absent in the B. ovis genome may represent virulence factors in human brucellosis.

  2. Role of ATP binding and hydrolysis in the gating of the cystic fibrosis transmembrane conductance regulator

    Directory of Open Access Journals (Sweden)

    Taras Gout

    2012-01-01

    Full Text Available The CFTR gene is unique within the ATP-binding cassette (ABC protein family, predominantly of transporters, by coding a chloride channel. The gating mechanism of ABC proteins has been characterized by the ATP Switch model in terms cycles of dimer formation and dissociation linked to ATP binding and hydrolysis, respectively. It would be of interest to assess the extent that Cystic Fibrosis Transmembrane Conductance Regulator (CFTR, a functional channel, fits the ATP Switch model for ABC transporters. Additional transporter mechanisms, namely those of Pgp and HlyB, are discussed for perspective. Literature search of databases selected key references in comparing and contrasting the gating mechanism. CFTR is a functional chloride channel facilitating transmembrane anion flow down electrochemical gradients. A dysfunctional CFTR protein results in cystic fibrosis, a fatal pleiotropic disease currently managed symptomatically. Understanding the gating mechanism will help target drug development aimed at alleviating and curing the disease.

  3. Linoleic acid suppresses cholesterol efflux and ATP-binding cassette transporters in murine bone marrow-derived macrophages

    Science.gov (United States)

    Individuals with type 2 diabetes mellitus (T2DM) are at increased risk of developing cardiovascular disease (CVD), possibly associated with elevated plasma free fatty acid concentrations. Paradoxically, evidence suggests that unsaturated, compared to saturated fatty acids, suppress macrophage chole...

  4. Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity.

    Science.gov (United States)

    Esser, Lothar; Zhou, Fei; Pluchino, Kristen M; Shiloach, Joseph; Ma, Jichun; Tang, Wai-Kwan; Gutierrez, Camilo; Zhang, Alex; Shukla, Suneet; Madigan, James P; Zhou, Tongqing; Kwong, Peter D; Ambudkar, Suresh V; Gottesman, Michael M; Xia, Di

    2017-01-13

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.

  5. Structures of the Multidrug Transporter P-glycoprotein Reveal Asymmetric ATP Binding and the Mechanism of Polyspecificity*♦

    Science.gov (United States)

    Esser, Lothar; Zhou, Fei; Pluchino, Kristen M.; Shiloach, Joseph; Ma, Jichun; Tang, Wai-kwan; Gutierrez, Camilo; Zhang, Alex; Shukla, Suneet; Madigan, James P.; Zhou, Tongqing; Kwong, Peter D.; Ambudkar, Suresh V.; Gottesman, Michael M.; Xia, Di

    2017-01-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions. PMID:27864369

  6. Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis.

    Science.gov (United States)

    Bompadre, Silvia G; Hwang, Tzyh-Chang

    2007-08-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF.

  7. Equilibrated Atomic Models of Outward-Facing P-glycoprotein and Effect of ATP Binding on Structural Dynamics

    Science.gov (United States)

    Pan, Lurong; Aller, Stephen G.

    2015-01-01

    P-glycoprotein (Pgp) is an ATP-binding cassette (ABC) transporter that alternates between inward- and outward-facing conformations to capture and force substrates out of cells like a peristaltic pump. The high degree of similarity in outward-facing structures across evolution of ABC transporters allowed construction of a high-confidence outward-facing Pgp atomic model based on crystal structures of outward-facing Sav1866 and inward-facing Pgp. The model adhered to previous experimentally determined secondary- and tertiary- configurations during all-atom molecular dynamics simulations in the presence or absence of MgATP. Three long lasting (>100 ns) meta-stable states were apparent in the presence of MgATP revealing new insights into alternating access. The two ATP-binding pockets are highly asymmetric resulting in differential control of overall structural dynamics and allosteric regulation of the drug-binding pocket. Equilibrated Pgp has a considerably different electrostatic profile compared to Sav1866 that implicates significant kinetic and thermodynamic differences in transport mechanisms. PMID:25600711

  8. Variants in the ATP-Binding Cassette Transporter (ABCA7), Apolipoprotein E ε4, and the Risk of Late-Onset Alzheimer Disease in African Americans

    Science.gov (United States)

    Reitz, Christiane; Jun, Gyungah; Naj, Adam; Rajbhandary, Ruchita; Vardarajan, Badri Narayan; Wang, Li-San; Valladares, Otto; Lin, Chiao-Feng; Larson, Eric B.; Graff-Radford, Neill R.; Evans, Denis; De Jager, Philip L.; Crane, Paul K.; Buxbaum, Joseph D.; Murrell, Jill R.; Raj, Towfique; Ertekin-Taner, Nilufer; Logue, Mark; Baldwin, Clinton T.; Green, Robert C.; Barnes, Lisa L.; Cantwell, Laura B.; Fallin, M. Daniele; Go, Rodney C. P.; Griffith, Patrick; Obisesan, Thomas O.; Manly, Jennifer J.; Lunetta, Kathryn L.; Kamboh, M. Ilyas; Lopez, Oscar L.; Bennett, David A.; Hendrie, Hugh; Hall, Kathleen S.; Goate, Alison M.; Byrd, Goldie S.; Kukull, Walter A.; Foroud, Tatiana M.; Haines, Jonathan L.; Farrer, Lindsay A.; Pericak-Vance, Margaret A.; Schellenberg, Gerard D.; Mayeux, Richard

    2013-01-01

    Importance Genetic variants associated with susceptibility to late-onset Alzheimer disease are known for individuals of European ancestry, but whether the same or different variants account for the genetic risk of Alzheimer disease in African American individuals is unknown. Identification of disease-associated variants helps identify targets for genetic testing, prevention, and treatment. Objective To identify genetic loci associated with late-onset Alzheimer disease in African Americans. Design, Setting, and Participants The Alzheimer Disease Genetics Consortium (ADGC) assembled multiple data sets representing a total of 5896 African Americans (1968 case participants, 3928 control participants) 60 years or older that were collected between 1989 and 2011 at multiple sites. The association of Alzheimer disease with genotyped and imputed single-nucleotide polymorphisms (SNPs) was assessed in case-control and in family-based data sets. Results from individual data sets were combined to perform an inverse variance–weighted meta-analysis, first with genome-wide analyses and subsequently with gene-based tests for previously reported loci. Main Outcomes and Measures Presence of Alzheimer disease according to standardized criteria. Results Genome-wide significance in fully adjusted models (sex, age, APOE genotype, population stratification) was observed for a SNP in ABCA7 (rs115550680, allele = G; frequency, 0.09 cases and 0.06 controls; odds ratio [OR], 1.79 [95% CI, 1.47-2.12]; P = 2.2 × 10–9), which is in linkage disequilibrium with SNPs previously associated with Alzheimer disease in Europeans (0.8

  9. ABC转运蛋白提升四膜虫对DDT的耐受能力%ATP-binding cassette transporter enhances Tetrahymena tolerance to DDT

    Institute of Scientific and Technical Information of China (English)

    宁应之; 党怀; 刘光龙; 熊杰; 袁冬霞; 冯立芳; 缪炜

    2014-01-01

    世界卫生组织允许DDT在部分国家和地区使用.大规模频繁使用杀虫剂造成了蚊子的抗药性,大大降低了DDT的防治效果.目前对于DDT的耐受主要集中在蚊子,但对于喷洒的DDT进入水生态系统后,造成食物链底层水生生物体对DDT耐受还未见报道.本研究对DDT胁迫下的四膜虫全基因组中筛选出一个ATP结合盒转运蛋白基因(abcb15),它能够识别DDT作为其转运的底物.绿色荧光蛋白标记显示ABCB 15蛋白定位于细胞膜上,是一个细胞膜泵蛋白.基因敲降技术造成的abcb15基因敲降株在DDT胁迫下表现出更低的生长速率,这表明ABCB15膜泵蛋白能够将进入细胞内的DDT转运至细胞膜外,藉以提高四膜虫对DDT的耐受能力,以减轻DDT对细胞的损伤.

  10. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells

    NARCIS (Netherlands)

    Hinrichs, JWJ; Klappe, K; Hummel, [No Value; Kok, JW

    2004-01-01

    In this study we show that P-glycoprotein in multi-drug-resistant 2780AD human ovarian carcinoma cells and multidrug resistance-associated protein 1 in multi-drug-resistant HT29(col) human colon carcinoma cells are predominantly located in Lubrol-based detergent-insoluble glycosphingolipid-enriched

  11. Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA

    NARCIS (Netherlands)

    Sakamoto, K; Margolles, A; van Veen, HW; Konings, WN

    2001-01-01

    Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino

  12. Cystathionine beta-Synthase (CBS) Domains 1 and 2 Fulfill Different Roles in Ionic Strength Sensing of the ATP-binding Cassette (ABC) Transporter OpuA

    NARCIS (Netherlands)

    Karasawa, Akira; Erkens, Guus B.; Berntsson, Ronnie P. -A.; Otten, Renee; Schuurman-Wolters, Gea K.; Mulder, Frans A. A.; Poolman, Bert

    2011-01-01

    The cystathionine beta-synthase module of OpuA in conjunction with an anionic membrane surface acts as a sensor of internal ionic strength, which allows the protein to respond to osmotic stress. We now show by chemical modification and cross-linking studies that CBS2-CBS2 interface residues are crit

  13. Tiaozhi Tongmai Granules reduce atherogenesis and promote the expression of ATP-binding cassette transporter A1 in rabbit atherosclerotic plaque macrophages and the liver

    Directory of Open Access Journals (Sweden)

    Qing Sun

    2014-07-01

    Conclusions: Tiaozhi Tongmai Granules appear to have an anti-atherogenic effect that is most likely mediated by simultaneously upregulating the protein expression of ABCA1 in rabbit atherosclerotic plaque macrophages and in the liver.

  14. Transcriptional control of hepatocanalicular transporter gene expression

    NARCIS (Netherlands)

    Muller, M

    2000-01-01

    Transport processes for larger organic solutes at the canalicular membrane are mainly driven by members of the superfamily of ATP-binding cassette (ABC) transporters. The funct ions of these transporters range from bile component secretion to xenobiotica and phase II-conjugate export. The transcript

  15. Effect of 5-fluorouracil on the expression of ATP-binding cassette superfamily G member 2 in human colon cancer cell SW480%氟尿嘧啶对人结肠癌SW480细胞ABCG2表达的影响

    Institute of Scientific and Technical Information of China (English)

    瞿金妙; 尤捷; 刘海光; 黄奇迪; 郭贵龙

    2013-01-01

    目的 观察氟尿嘧啶(5-FU)对人结肠癌SW480细胞ABCG2表达的影响.方法 用不同药物浓度的5-FU处理SW480细胞,用CCK8法检测5-FU在SW480中的IC50,流式细胞仪检测SW480细胞ABCG2的阳性表达率,RT-PCR检测ABCG2的mRNA在SW480细胞中的表达差异.结果 5-FU对SW480细胞的IC50随着药物浓度的增加而升高(P<0.05).流式细胞仪检测发现,正常SW480细胞(A组)中ABCG2阳性表达率为(6.26±0.86)%;在药物处理48 h后即刻检测时(B组)的阳性表达率下降至(3.43±1.18)%(P<0.05);在药物处理48 h后的第2代细胞检测时(C组)则升高至(12.91±3.42)%(P<0.05).3组ABCG2 mRNA表达趋势与流式细胞仪检测结果的趋势一致.结论 不同浓度的5-FU可以影响人结肠癌SW480细胞ABCG2的表达.%Objective To investigate the effect of 5-fluorouracil (5-FU) on the expression of ATP-binding cassette superfamily G member 2(ABCG2) in human colon cancer cell SW480.Methods SW480 cells were treated with various concentrations of 5-FU.CCK8 assay was utilized to detect the 5-FU IC50 to SW480 cells.Positive expression of ABCG2 was detected by flow cytometry,and mRNA expression of ABCG2 was detected by real time polymerase chain reaction (RT-PCR).Results The 5-FU IC50 to SW480 cells increased as the drug concentration increased(P<0.05).Flow cytometry revealed that positive expression rate of ABCG2 in normal SW480 cells (group A) was (6.26±0.86)%.Immediately after treatment with 5-FU for 48 hours,the positive expression rate of ABCG2 (group B) was (3.43±1.18)%(P<0.05).In the second passage of cells after treatment with 5-FU for 48 hours,the positive expression rate of ABCG2 (group C) was (12.91±3.42)%(P<0.05).The mRNA expression of ABCG2 detected by RT-PCR was in accordance with the results from flow cytometry.Conclusion Expression of ABCG2 in SW480 cells can be affected by various concentrations of 5-FU.

  16. Drug Transporters in the Intestine

    DEFF Research Database (Denmark)

    Steffansen, Bente

    2016-01-01

    The enterocyte monolayer in the intestinal membrane impacts on the bioavailability (BA) of many orally administered active pharmaceutical ingredients (APIs). The monolayer expresses a multitude of membrane transporters belonging to the solute carrier (SLC) and ATP-binding cassette (ABC) families ...

  17. Structural and mechanistic insights into ABC-type ECF transporters for vitamin uptake

    NARCIS (Netherlands)

    Dosz-Majsnerowska, Maria

    2014-01-01

    Dit proefschrift gaat over de relatie tussen de structuur en het mechanisme van ABC-type ECF transporters voor vitamines, uit de bacterie Lactococcus lactis. Energy-Coupling Factor (ECF) transporters vormen een subgroep van de ATP-binding cassette (ABC) transporters en zijn betrokken bij de opname v

  18. sphingosine-1-phosphate transport and its role in immunology

    NARCIS (Netherlands)

    Reitsema, V.; Bouma, Hjalmar; Kok, Jan

    2014-01-01

    Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite with many important functions in cellular and systemic physiology, including the immune system. As it cannot traverse the membrane, it is exported from cells by transporters. Several members of the ATP-binding cassette (ABC) transporter fami

  19. Lipid dependence of ABC transporter localization and function

    NARCIS (Netherlands)

    Klappe, Karin; Hummel, Ina; Hoekstra, Dick; Kok, Jan Willem

    2009-01-01

    Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and dis

  20. ABC transporter architecture and regulatory roles of accessory domains

    NARCIS (Netherlands)

    Biemans-Oldehinkel, E; Doeven, MK; Poolman, B

    2006-01-01

    We present an overview of the architecture of ATP-binding cassette (ABC) transporters and dissect the systems in core and accessory domains. The ABC transporter core is formed by the transmembrane domains (TMDs) and the nucleotide binding domains (NBDs) that constitute the actual translocator. The a

  1. Biochemical evidence for the presence of two α-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

    NARCIS (Netherlands)

    Koning, Sonja M.; Konings, Wil N.; Driessen, Arnold J.M.

    2002-01-01

    The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of α-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports tr

  2. On the role of the two extracytoplasmic substrate-binding domains in the ABC transporter OpuA

    NARCIS (Netherlands)

    Biemans-Oldehinkel, E; Poolman, B

    2003-01-01

    Members of two transporter families of the ATP-binding cassette (ABC) superfamily use two or even four extracytoplasmic substrate-binding domains (SBDs) for transport. We report on the role of the two SBDs in the translocation cycle of the ABC transporter OpuA from Lactococcus lactis. Heterooligomer

  3. The power stroke driven by ATP binding in CFTR as studied by molecular dynamics simulations.

    Science.gov (United States)

    Furukawa-Hagiya, Tomoka; Furuta, Tadaomi; Chiba, Shuntaro; Sohma, Yoshiro; Sakurai, Minoru

    2013-01-10

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP binding cassette (ABC) protein superfamily. Currently, it remains unclear how ATP binding causes the opening of the channel gate at the molecular level. To clarify this mechanism, we first constructed an atomic model of the inward-facing CFTR using the X-ray structures of other ABC proteins. Molecular dynamics (MD) simulations were then performed to explore the structure and dynamics of the inward-facing CFTR in a membrane environment. In the MgATP-bound state, two nucleotide-binding domains (NBDs) formed a head-to-tail type of dimer, in which the ATP molecules were sandwiched between the Walker A and signature motifs. Alternatively, one of the final MD structures in the apo state was similar to that of a "closed-apo" conformation found in the X-ray analysis of ATP-free MsbA. Principal component analysis for the MD trajectory indicated that NBD dimerization causes significant structural and dynamical changes in the transmembrane domains (TMDs), which is likely indicative of the formation of a chloride ion access path. This study suggests that the free energy gain from ATP binding acts as a driving force not only for NBD dimerization but also for NBD-TMD concerted motions.

  4. ATP-Binding Cassettee Transporter Signals Salt-Induced Stomatal Closure in Arabidopsis thaliana L. by H2S Pathway%ABC转运体位于H2S上游参与盐胁迫诱导的拟南芥气孔关闭

    Institute of Scientific and Technical Information of China (English)

    吴延朋; 李洪旺; 侯丽霞; 张丹丹; 刘新

    2014-01-01

    本文以拟南芥野生型、ABC转运体缺失突变体(Atmrp4、Atmrp5和Atmrp4/5)为材料研究了硫化氢(hydrogen sulfide, H2S)和ABC转运体在盐胁迫诱导拟南芥气孔关闭中的作用及其相互关系。结果表明,盐胁迫能够引起拟南芥叶片AtMRP4及AtMRP5表达量显著升高,诱导野生型拟南芥叶片气孔关闭,但对Atmrp4、Atmrp5及Atmrp4/5气孔开度无显著影响;而ABC转运体抑制剂格列本脲(glibenclamide, Gli)可减弱盐胁迫诱导的拟南芥气孔关闭的作用,表明ABC转运体参与盐胁迫诱导的拟南芥气孔关闭过程。盐胁迫能够引起野生型拟南芥H2S合成相关酶L-/D-半胱氨酸脱巯基酶(L-/D-CDes)活性及H2S含量显著升高,而ABC转运体抑制剂格列本脲处理后则没有这种变化,同时盐胁迫也不能引起Atmrp4、Atmrp5及Atmrp4/5的L-/D-CDes活性及H2S含量显著升高,表明ABC转运体位于H2S上游参与盐胁迫诱导气孔关闭过程。%Using Arabidopsis thaliana wild type, ATP-binding cassette (ABC) transporter deficient mutants Atmrp4, Atmrp5, Atmrp4/5 as materials, the participation of hydrogen sulifde (H2S) and ABC transporter signal pathway in salt-induced stomatal closure were studied. These results showed that AtMRP4 and AtMRP5 transcription rate in Arabidopsis leaves increased dramaticly under salt stress, and salt stress induced stomata closure in wild type, but had no effect on Atmrp4, Atmrp5 and Atmrp4/5. The ABC transporter inhibitor glibenclamide (Gli) inhibited the inducing effects of salt stress on stomata closure in wild type, it could be deduced that ABC transporter participate in salt-induced stomatal closure in Arabidopsis. Salt-induced increase in L-/D-cysteine desulfhydrase (L-/D-CDes, enzymes participate in H2S production) activities and H2S content could be seen in wild type, but had no effect on wild type treated by Gli as well as Atmrp4, Atmrp5, Atmrp4/5. All these results indicated that ABC transporter functions upstream

  5. Construction of deletion mutants in the phosphotransferase transport system and adenosine triphosphate-binding cassette transporters in Listeria monocytogenes and analysis of their growth under different stress conditions

    Directory of Open Access Journals (Sweden)

    Marina Ceruso

    2013-10-01

    Full Text Available Functional genomics approaches enable us to investigate the biochemical, cellular, and physiological properties of each gene product and are nowadays applied to enhance food safety by understanding microbial stress responses in food and host-pathogen interactions. Listeria monocytogenes is a food-borne pathogen that causes listeriosis and is difficult to eliminate this pathogen since it can survive under multiple stress conditions such as low pH and low temperature. Detailed studies are needed to determine its mode of action and to understand the mechanisms that protect the pathogen when it is subjected to stress. In this study, deletion mutants of phosphotransferase transport system genes (PTS and adenosine triphosphate(ATP-binding cassette transporters (ABC of Listeria monocytogenes F2365 were created using molecular techniques. These mutants and the wild-type were tested under different stress conditions, such as in solutions with different NaCl concentration, pH value and for nisin resistance. Results demonstrate that the behaviour of these deletion mutants is different from the wild type. In particular, deleted genes may be involved in L. monocytogenes resistance to nisin and to acid and salt concentrations. Functional genomics research on L. monocytogenes allows a better understanding of the genes related to stress responses and this knowledge may help in intervention strategies to control this food-borne pathogen. Furthermore, specific gene markers can be used to identify and subtype L. monocytogenes. Thus, future development of this study will focus on additional functional analyses of important stress response-related genes, as well as on methods for rapid and sensitive detection of L. monocytogenes such as using DNA microarrays.

  6. Pharmacological functions of multidrug transporters: studies employing combination transporter knockout mice

    NARCIS (Netherlands)

    Lagas, J.S.

    2009-01-01

    ATP-binding cassette (ABC) multidrug transporters are drug efflux pumps located in the plasma membrane that utilize the energy of ATP hydrolysis to extrude a wide spectrum of endogenous and exogenous compounds from cells, including numerous (anticancer) drugs and/or their metabolites. The studies de

  7. Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays.

    NARCIS (Netherlands)

    Krumpochova, P; Sapthu, S.; Brouwers, J.F.H.M.; de Haas, M.; de Vos, R.; Borst, P.; van de Wetering, K.

    2013-01-01

    ABSTRACT The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum o

  8. Thermodynamics of the ATPase cycle of GlcV, the nucleotide-binding domain of the glucose ABC transporter of Sulfolobus solfataricus

    NARCIS (Netherlands)

    Pretz, Monika G.; Albers, Sonja-Verena; Schuurman-Wolters, Gea; Tampe, Robert; Driessen, Arnold J. M.; van der Does, Chris

    2006-01-01

    ATP-binding cassette transporters drive the transport of substrates across the membrane by the hydrolysis of ATP. They typically have a conserved domain structure with two membrane-spanning domains that form the transport channel and two cytosolic nucleotide-binding domains ( NBDs) that energize the

  9. Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein

    NARCIS (Netherlands)

    Konings, WN; Poelarends, GJ

    2002-01-01

    Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural a

  10. Are lipid rafts involved in ABC transporter-mediated drug resistance of tumor cells?

    NARCIS (Netherlands)

    Kok, Jan Willem; Klappe, Karin; Hummel, Ina; Kroesen, Bart-Jan; Sietsma, Hannie; Meszaros, Peter

    2008-01-01

    Since their discovery, lipid rafts have been implicated in several cellular functions, including protein transport in polarized cells and signal transduction. Also in multidrug resistance lipid rafts may be important with regard to the localization of ATP-binding cassette (ABC) transporters in these

  11. Functional Diversity of Tandem Substrate-Binding Domains in ABC Transporters from Pathogenic Bacteria

    NARCIS (Netherlands)

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Vujicic - Zagar, Andreja; Guskov, Albert; Slotboom, Dirk-Jan; Poolman, Bert

    2013-01-01

    The ATP-binding cassette (ABC) transporter GInPQ is an essential uptake system for amino acids in gram-positive pathogens and related nonpathogenic bacteria. The transporter has tandem substrate-binding domains (SBDs) fused to each transmembrane domain, giving rise to four SBDs per functional transp

  12. ABC transporters from Botrytis cinerea in biotic and abiotic interactions

    NARCIS (Netherlands)

    Schoonbeek, H.

    2004-01-01

    Botrytis cinereais the causal agent of grey mould disease on a wide variety of crop plants. It is relatively insensitive to natural and synthetic fungitoxic compounds. This thesis describes how ABC (ATP-binding cassette) transporters contribute to protection by actively secre

  13. Functional analysis of ABC transporter genes from Botrytis cinerea identifies BcatrB as a transporter of eugenol

    NARCIS (Netherlands)

    Schoonbeek, H.; Nistelrooy, van J.G.M.; Waard, de M.A.

    2003-01-01

    The role of multiple ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporter genes from the plant pathogenic fungus Botrytis cinerea in protection against natural fungitoxic compounds was studied by expression analysis and phenotyping of gene-replacement mutants. The expressio

  14. The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel a-glucosides through reverse phosphorolysis by maltose phosphorylase

    NARCIS (Netherlands)

    Nakai, H.; Baumann, M.J.; Petersen, B.O.; Westphal, Y.; Schols, H.A.; Dilokpimol, A.; Hachem, M.A.; Lathinen, S.J.; Duus, J.O.; Svensson, B.

    2009-01-01

    A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regula

  15. The purified and functionally reconstituted multidrug transporter LmrA of Lactococcus lactis mediates the transbilayer movement of specific fluorescent phospholipids

    NARCIS (Netherlands)

    Margolles, A; Putman, M; van Veen, HW; Konings, WN

    1999-01-01

    Lactococcus lactis possesses an ATP-binding cassette transporter, LmrA, which is a homolog of the mammalian multidrug resistance (MDR) P-glycoprotein, and is able to transport a broad range of structurally unrelated amphiphilic drugs. A histidine tag was introduced at the N-terminus of LmrA to facil

  16. Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli

    NARCIS (Netherlands)

    Gul, Nadia; Poolman, Bert

    2013-01-01

    The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., th

  17. Beauvericin counteracted multi-drug resistant Candida albicans by blocking ABC transporters

    DEFF Research Database (Denmark)

    Tong, Yaojun; Liu, Mei; Zhang, Yu

    2016-01-01

    screening and whole-cell based mechanism study, identified a natural product, beauvericin (BEA) as a drug efflux pump modulator, which can reverse the multi-drug resistant phenotype of Candida albicans by specifically blocking the ATP-binding cassette (ABC) transporters; meantime, BEA alone has fungicidal...

  18. the role of the actin cytoskeleton and lipid rafts in the localization and function of the ABCC1 transporter

    NARCIS (Netherlands)

    Kok, Jan; Klappe, Katharina; Hummel, Ina

    2014-01-01

    ATP-binding cassette (ABC) transporters are known to be important factors in multidrug resistance of tumor cells. Lipid rafts have been implicated in their localization in the plasma membrane, where they function as drug efflux pumps. This specific localization in rafts may support the activity of A

  19. Inhibition of multixenobiotic resistance transporters (MXR) by silver nanoparticles and ions in vitro and in Daphnia magna

    NARCIS (Netherlands)

    Georgantzopoulou, Anastasia; Cambier, Sébastien; Serchi, Tommaso; Kruszewski, Marcin; Balachandran, Yekkuni L.; Grysan, Patrick; Audinot, Jean Nicolas; Ziebel, Johanna; Guignard, Cédric; Gutleb, Arno C.; Murk, Tinka

    2016-01-01

    The P-glycoprotein (P-gp, ABCB1) and multidrug resistance associated protein 1 (MRP1), important members of the ABC (ATP-binding cassette) transporters, protect cells and organisms via efflux of xenobiotics and are responsible for the phenomenon of multidrug or multixenobiotic resistance (MXR). I

  20. Human breast cancer resistance protein : Interactions with steroid drugs, hormones, the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine, and transport of cimetidine

    NARCIS (Netherlands)

    Pavek, P; Merino, G; Wagenaar, E; Bolscher, E; Novotna, M; Jonker, JW; Schinkel, AH

    2005-01-01

    The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells, mediating drug resistance and affecting the pharmacological behavior of many compounds. To study the interaction of human wild-type BCRP with steroid drugs, hormo

  1. Inhibiting NF-K B increases cholesterol efflux from THP-1 derived- foam cells treated with Angll via up-regulating the expression of ATP-binding cassette transporter A1

    Institute of Scientific and Technical Information of China (English)

    Kun Liu; Yanfu Wang; Zhijian Chen; Yuhua Liao; Xiang Gao; Jian Chen

    2008-01-01

    Objective:To study the role of nuclear factor-kappa B(NF- K B) in cholesterol efflux from THP-I derived-foam cells treated with Angiotensin Ⅱ (Ang Ⅱ ). Methods:Cultured THP-l derived-foam cells were treated with Ang Ⅱ or preincubated with tosyl-phenylalan inechloromethyl-ketone(TPCK) NF-K B inhibitor. The levels of activated NF-K B in the cells were examined by sandwich ELISA. Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol efflux was detected by scintillation counting technique. ABCAI mRNA and protein were quantified by RT-PCR and Western blotting. Results:Addition of TPCK to the cells before Ang Ⅱ stimulation attenuated the response of NF- K B p65 nuclear translocation induced by Ang Ⅱ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol effiux by 24.1%(P < 0.05) and 41.1%(P < 0.05) respectively, when compared with Ang Ⅱ group. In accordance, the ABCAl mRNA and protein were increased by 30% and 19%(P< 0.05) respectively, when compared with Ang Ⅱ group. Conclusion:Ang Ⅱ can down- regulate ABCAI in THP-l derived-foam cells via NF- K B, which leads to less cholesterol effiux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.

  2. EFFECTS OF ANTHOCYANIN ON ATP BINDING CASSETTE TRANSPORTER G1 EXPRESSION AND ANALYSIS OF THE MECHANISMS%花色苷对THP-1细胞中ABCG1表达的影响及机制分析

    Institute of Scientific and Technical Information of China (English)

    张英慧; 董华强; 钟希琼; 黄剑波

    2011-01-01

    目的 研究花色苷矢车菊素-3-O-β-葡萄糖苷(C3G)对人巨噬细胞THP-1中ABCG1表达的影响,并分析相关作用机制.方法 以C3G干预培养诱导分化的THP-1细胞,荧光实时定量RT-PCR检测ATP结合盒式转运蛋白G1(ABCG1)以及其直接调节因子肝脏X受体α(liver X receptotα,LXRα)的mRNA表达,时间分辨荧光共振能量转移(time-resolved fluorescence resonance energy transfer,TR-FRET)方法检测C3G与LXRα配体结合域的结合能力.结果 100μ mol/L C3G处理THP-1细胞16 h显著增加了ABCG1 mRNA的表达(为对照组的1.98倍,P<0.05);尽管在TR-FRET试验中,C3G并未呈现典型的配体结合曲线,而50 μ mol/L和100μmol/L C3G显著增加了THP-1细胞中LXRαmRNA的表达(分别为对照组的2.21倍和2.83倍,P<0.05).结论 C3G促进了ABCG1表达,该机制可能通过增加核受体LXRαmRNA表达来实现.

  3. Effects of ATP-binding cassette exporters on virulence factors in Streptococcus mutans%三磷酸腺苷结合盒外排子对变异链球菌毒力因子影响的研究进展

    Institute of Scientific and Technical Information of China (English)

    曾荟荟; 凌均棨

    2015-01-01

    ABC transporters have been proved to be integral membrane proteins that actively transported a diverse range of substrates across cell membranes. ABC transporters had varied functions, and took part in gene competence, (p)ppGpp accumulation, bacteriocin secretion and immunity in Streptococcus mutans. The structures, functions, mechanisms and inhibitors of the known ABC exporters in Streptococcus mutans were summarized.%三磷酸腺苷结合盒(ABC)转运子是膜蛋白的一部分,透过细胞膜转运各种生物分子,参与多种生理功能。在变异链球菌中,ABC外排子与基因感受态、四(五)磷酸鸟苷[(p)ppGpp]累积、细菌素分泌与免疫密切相关。本文就变异链球菌ABC外排子的结构、生理功能、作用机制和抑制剂作一综述。

  4. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Svenningsen, Katrine; Almind Knudsen, Lina;

    2015-01-01

    AIM: To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development. METHODS: Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette....../Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes...... translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters...

  5. Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models.

    Science.gov (United States)

    Real, R; González-Lobato, L; Baro, M F; Valbuena, S; de la Fuente, A; Prieto, J G; Alvarez, A I; Marques, M M; Merino, G

    2011-12-01

    In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0

  6. Automated cassette-to-cassette substrate handling system

    Science.gov (United States)

    Kraus, Joseph Arthur; Boyer, Jeremy James; Mack, Joseph; DeChellis, Michael; Koo, Michael

    2014-03-18

    An automated cassette-to-cassette substrate handling system includes a cassette storage module for storing a plurality of substrates in cassettes before and after processing. A substrate carrier storage module stores a plurality of substrate carriers. A substrate carrier loading/unloading module loads substrates from the cassette storage module onto the plurality of substrate carriers and unloads substrates from the plurality of substrate carriers to the cassette storage module. A transport mechanism transports the plurality of substrates between the cassette storage module and the plurality of substrate carriers and transports the plurality of substrate carriers between the substrate carrier loading/unloading module and a processing chamber. A vision system recognizes recesses in the plurality of substrate carriers corresponding to empty substrate positions in the substrate carrier. A processor receives data from the vision system and instructs the transport mechanism to transport substrates to positions on the substrate carrier in response to the received data.

  7. ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

    Science.gov (United States)

    Müller, Pavel; Bouly, Jean-Pierre; Hitomi, Kenichi; Balland, Véronique; Getzoff, Elizabeth D.; Ritz, Thorsten; Brettel, Klaus

    2014-06-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH. radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD.-, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396-. Its negative charge could trigger conformational changes necessary for signal transduction.

  8. Metabolism and transport of tamoxifen in relation to its effectiveness: new perspectives on an ongoing controversy

    OpenAIRE

    Cronin-Fenton, Deirdre P.; Damkier, Per; Lash, Timothy L

    2014-01-01

    Tamoxifen reduces the rate of breast cancer recurrence by approximately a half. Tamoxifen is metabolized to more active metabolites by enzymes encoded by polymorphic genes, including cytochrome P450 2D6 (CYP2D6). Tamoxifen is a substrate for ATP-binding cassette transporter proteins. We review tamoxifen's clinical pharmacology and use meta-analyses to evaluate the clinical epidemiology studies conducted to date on the association between CYP2D6 inhibition and tamoxifen effectiveness. Our find...

  9. Sterol transporter adenosine triphosphate-binding cassette transporter G8, gallstones, and biliary cancer in 62,000 individuals from the general population

    DEFF Research Database (Denmark)

    Stender, Stefan; Frikke-Schmidt, Ruth; Nordestgaard, Børge G;

    2011-01-01

    Gallstone disease, a risk factor for biliary cancer, has a strong heritable component, but the underlying genes are largely unknown. To test the hypothesis that ABCG8 (adenosine triphosphate-binding cassette transporter G8) Asp19His (D19H) genotype predicted risk of gallstones and biliary cancer ...

  10. Role of ABC transporters in cancer chemotherapy

    Institute of Scientific and Technical Information of China (English)

    Yue-Li Sun; Atish Patel; Priyank Kumar; Zhe-Sheng Chen

    2012-01-01

    Multidrug resistance (MDR) in cancer cells can significantly attenuate the response to chemotherapy and increase the likelihood of mortality.The major mechanism involved in conferring MDR is the overexpression of ATP-binding cassette (ABC) transporters,which can increase efflux of drugs from cancer cells,thereby decreasing intracellular drug concentration.Modulators of ABC transporters have the potential to augment the efficacy of anticancer drugs.This editorial highlights some major findings related to ABC transporters and current strategies to overcome MDR.

  11. Drug transporters in breast cancer

    DEFF Research Database (Denmark)

    Kümler, Iben; Stenvang, Jan; Moreira, José

    2015-01-01

    Despite the advances that have taken place in the past decade, including the development of novel molecular targeted agents, cytotoxic chemotherapy remains the mainstay of cancer treatment. In breast cancer, anthracyclines and taxanes are the two main chemotherapeutic options used on a routine...... basis. Although effective, their usefulness is limited by the inevitable development of resistance, a lack of response to drug-induced cancer cell death. A large body of research has resulted in the characterization of a plethora of mechanisms involved in resistance; ATP-binding cassette transporter...

  12. Cystic fibrosis transmembrane conductance regulator: a chloride channel gated by ATP binding and hydrolysis%囊性纤维化跨膜电导调节体:ATP结合和水解门控Cl-通道

    Institute of Scientific and Technical Information of China (English)

    BOMPADRE; Silvia; G; HWANG; Tzyh-Chang

    2007-01-01

    囊性纤维化跨膜电导调节体(cystic fibrosis transmembrane conductance regulator,CFTR)是一种Cl-通道,属于ATP结合(ATP-binding cassette,ABC)转运体超家族.CFTR功能缺陷是高加索人种中普遍存在的致死性常染色体隐性遗传疾病囊性纤维化(cystic fibrosis,CF)发生的主要原因.这种疾病患者各组织上皮细胞内Cl-转运失调.目前,与CF相关的不同突变超过1 400种.CFTR调节(regulatory,R)域负责调控,核苷酸结合域(nucleotide-binding domains,NBDs)NBD1和NBD2负责ATP结合和水解门控.近期研究发现CFTR的NBDs与其它ABC蛋白一样可以二聚化.二聚化过程中,NBD1和NBD2首-尾相连,一个NBD上的WalkerA和B模块与另一个NBD提供的标签序列(signature sequence)形成ATP结合袋(ATP-binding pockets,ABPs)ABP1和ABP2.ABPs中与ATP结合相关的氨基酸突变实验揭示,ABP1和ABP2在CFTR的ATP依赖门控中发挥不同作用.ABP2由NBD2上的Walk A和B模块与NBD1提供的标签序列形成,它与ATP结合催化通道开放,而ABP1单独与ATP结合不能促进通道开放,只能稳定通道构象.有一些CFTR突变相关疾病的特征就是门控失调,进一步深入研究CFTR的NBD1和NBD2如何通过相互作用而达到通道门控,将为药理学研究提供更多所需的机制信息,有利于为CF治疗的药物设计铺平道路.%The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1

  13. Molecular mechanism of ATP binding and ion channel activation in P2X receptors

    Energy Technology Data Exchange (ETDEWEB)

    Hattori, Motoyuki; Gouaux, Eric (Oregon HSU)

    2012-10-24

    P2X receptors are trimeric ATP-activated ion channels permeable to Na{sup +}, K{sup +} and Ca{sup 2+}. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body {beta}-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

  14. Moving out: from sterol transport to drug resistance - the ABCG subfamily of efflux pumps.

    Science.gov (United States)

    Moitra, Karobi; Silverton, Latoya; Limpert, Katy; Im, Kate; Dean, Michael

    2011-01-01

    The ATP binding cassette (ABC) proteins are typically ATP-driven transmembrane pumps that have been evolutionarily conserved from bacteria to humans. In humans these transporters are subdivided into seven subfamilies, ranging from A to G. The ABCG subfamily of transporters is the primary focus of this review. This subfamily of proteins has been conserved throughout evolution and plays a central role in several cellular processes, such as sterol homeostasis and multidrug resistance. Functional polymorphisms/mutations in some of these G-subfamily transporters have clinical consequences in humans.

  15. Enantioselective Induction of a Glutathione-S-Transferase, a Glutathione Transporter and an ABC Transporter in Maize by Metolachlor and Its (S)-Isomer

    OpenAIRE

    Sen Pang; Zhaojin Ran; Zhiqian Liu; Xiaoyu Song; Liusheng Duan; Xuefeng Li; Chengju Wang

    2012-01-01

    The metabolism of chiral herbicides in plants remains poorly understood. Glutathione conjugation reactions are one of the principal mechanisms that plants utilize to detoxify xenobiotics. The induction by rac- and S-metolachlor of the expression of three genes, ZmGST27, ZmGT1 and ZmMRP1, encoding respectively a glutathione-S-transferase, a glutathione transporter and an ATP-binding cassette (ABC) transporter was studied in maize. The results demonstrate that the inducing effect of rac- and S-...

  16. Xenobiotic, bile acid, and cholesterol transporters: function and regulation.

    Science.gov (United States)

    Klaassen, Curtis D; Aleksunes, Lauren M

    2010-03-01

    Transporters influence the disposition of chemicals within the body by participating in absorption, distribution, and elimination. Transporters of the solute carrier family (SLC) comprise a variety of proteins, including organic cation transporters (OCT) 1 to 3, organic cation/carnitine transporters (OCTN) 1 to 3, organic anion transporters (OAT) 1 to 7, various organic anion transporting polypeptide isoforms, sodium taurocholate cotransporting polypeptide, apical sodium-dependent bile acid transporter, peptide transporters (PEPT) 1 and 2, concentrative nucleoside transporters (CNT) 1 to 3, equilibrative nucleoside transporter (ENT) 1 to 3, and multidrug and toxin extrusion transporters (MATE) 1 and 2, which mediate the uptake (except MATEs) of organic anions and cations as well as peptides and nucleosides. Efflux transporters of the ATP-binding cassette superfamily, such as ATP-binding cassette transporter A1 (ABCA1), multidrug resistance proteins (MDR) 1 and 2, bile salt export pump, multidrug resistance-associated proteins (MRP) 1 to 9, breast cancer resistance protein, and ATP-binding cassette subfamily G members 5 and 8, are responsible for the unidirectional export of endogenous and exogenous substances. Other efflux transporters [ATPase copper-transporting beta polypeptide (ATP7B) and ATPase class I type 8B member 1 (ATP8B1) as well as organic solute transporters (OST) alpha and beta] also play major roles in the transport of some endogenous chemicals across biological membranes. This review article provides a comprehensive overview of these transporters (both rodent and human) with regard to tissue distribution, subcellular localization, and substrate preferences. Because uptake and efflux transporters are expressed in multiple cell types, the roles of transporters in a variety of tissues, including the liver, kidneys, intestine, brain, heart, placenta, mammary glands, immune cells, and testes are discussed. Attention is also placed upon a variety of

  17. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C; Icyuz, Mert; Chauvet, Sylvain; Tao, Binli; Hartman, John L; Kirk, Kevin L

    2016-03-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.

  18. Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

    OpenAIRE

    Iwao Ohtsu; Yusuke Kawano; Marina Suzuki; Susumu Morigasaki; Kyohei Saiki; Shunsuke Yamazaki; Gen Nonaka; Hiroshi Takagi

    2015-01-01

    Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (K m = 1.1 μM) that is ...

  19. Functional expression and characterization of plant ABC transporters in Xenopus laevis oocytes for transport engineering purposes

    DEFF Research Database (Denmark)

    Xu, Deyang; Veres, Dorottya; Belew, Zeinu Mussa

    2016-01-01

    suitable in transport engineering approaches, although their size and high number of introns make them notoriously difficult to clone. Here, we report a novel in planta “exon engineering” strategy for cloning of full-length coding sequence of ABC transporters followed by methods for biochemical......Transport engineering in bioengineering is aimed at efficient export of the final product to reduce toxicity and feedback inhibition and to increase yield. The ATP-binding cassette (ABC) transporters with their highly diverse substrate specificity and role in cellular efflux are potentially...... characterization of ABC exporters in Xenopus oocytes. Although the Xenopus oocyte expression system is particularly suitable for expression of membrane proteins and powerful in screening for novel transporter activity, only few examples of successful expression of ABC transporter has been reported. This raises...

  20. 姜黄素对脂变肝细胞胆固醇流出及ABCA1表达的影响%Effects of Curcumin on ATP Binding Cassette Transporter A1 Expression and Cholesterol Effiux in Human L-02 Hepatocyte

    Institute of Scientific and Technical Information of China (English)

    余其林; 阳学风

    2009-01-01

    目的 研究姜黄素对脂变肝细胞胆固醇的转运机制. 方法 取正常人肝L-02细胞作为研究对象.用10%(V/V)医用脂肪乳注射液和10%(V/V)胎牛血清的RPMI-1640完全培养基孵育24 h建立的脂肪变性肝细胞模型.实验分为为6个组,即正常肝细胞组、人肝L-02细胞脂肪变性模型组、姜黄素处理组(脂变细胞模型建立后,分别加入不等量姜黄素,使其终浓度分别为12.5、25、50、100 μmol/L).RT-PCR检测细胞ABCA1表达;高效液相色谱法定量检测细胞内外胆固醇含量. 结果 姜黄素呈浓度依赖性地减少细胞内脂变肝细胞内总胆固醇(TC)、胆固醇酯(CE)含量,增加培养基中游离胆固醇(FC)含量,同时增加ABCA1的表达. 结论 姜黄素能降低脂变人肝L-02细胞TC及CE含量,增加FC的外流;姜黄素增加脂变肝细胞胆固醇转运可能与ABCA1的上调有关.

  1. ω-carboxyl Like Linoleic Acid Oxides Promoting the Expression of ATP-binding Cassette Transporter A1 (ABCA1)%ω-羧基样亚油酸氧化物促进ATP结合盒转运子A1(ABCA1)表达

    Institute of Scientific and Technical Information of China (English)

    马艳华; 刘庆平; 娜琴; 苑红; 扈瑞平

    2015-01-01

    ω-羧基样亚油酸氧化物(7-KC-9-COOH)是由Cu2+氧化低密度脂蛋白(oxLDL)中得到的负性胆固醇氧化物.研究其对ATP结合盒(ABC)转运子超家族成员ABCA1表达的调控作用,并探讨其对高密度脂蛋白(HDL)介导的胆固醇流出的影响.THP-1单核细胞经佛波酯(PMA)诱导分化为巨噬细胞,化学合成的7-KC-9-COOH以时间梯度孵育细胞,随后采用RTPCR,western blot研究其对ABCA1基因和蛋白水平表达的影响.7-KC-9-COOH显著促进了ABCAl mRNA和蛋白水平表达,其中作用2h效果最为显著,基因水平增加48.6%(*P<0.05),蛋白水平增加285.3%,较空白组升高近3倍(**P<0.01).抑制其上游核转录因子PPARγ与LXRα,其蛋白表达量下降51.2%.ω-COOH亚油酸氧化物7-KC-9-COOH经由PPARγ-LXRα信号通路提高了ABCA1的表达,促进了胆固醇逆转运(RCT)和HDL介导的胆固醇流出.

  2. ATP结合盒转运子1R219K基因多态性与阿尔茨海默病相关性研究%Research on association between polymorphism of ATP-binding cassette transporter A1 and Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    王娟; 陈卫蓉; 肖志杰

    2013-01-01

    Objective To assess the effect of single-nucleotide polymorphisms (SNPs) in the cholesterol-related genes ABCA1 exon R219K variation on the risk of AD and cognitive function.Methods Using case-control study,a total of sporadic 104 AD patients and 104 individuals above 60 years without cognitive impairment from a population of Chinese Han nationality in Changsha area were studied to determine the polymorphisms of ABCA1 R219K.Adopt SPSS 13.0 for statistical analysis.Results There were significant differences in the frequencies of three genotypes (RR、RK and KK) between the AD patients and controls (x2 =8.777,P =0.012).The frequency of KK genotype in AD group is obviously lower than that of controls (x2 =5.261,P =0.022).The frequency of KK+RK genotype in AD patients was 54.8%,was significantly lower than that of controls (70.2%,P =0.022).There was significantly higher levels of HDL-C,apoA-I and cognitive scores in the carriers of KK genotype and K allele (P < 0.05).There was significantly higher levels of MMSE and WMS scores in the carriers of K K genotype and K allele (P < 0.05).Further in accordance with memory types including long-term memory,short-term memory,transient memory,compared with RR genotype,the scores of short-term memory,transient memory of RK,KK and RK + KK genotypes were higher (P < 0.05),but there was no significant difference in the score of long-term memory (P > 0.05).Multifactor logistic regression analysis showed that carrying K allele was a single protective factor for AD.Conclusions The polymorphism of ABCA1R219K is associated with AD.Carrying K allele may play a positive role in serum lipids and cognition,and produce a protective effect on risk of AD.%目的 探讨胆固醇相关基因ABCA1外显子R219K遗传变异对阿尔茨海默病(AD)发病风险及认知功能的影响.方法 采用病例对照研究方法测定湖南长沙地区汉族人群104例散发性AD患者以及104名60岁以上认知功能正常者的ABCA1R219K基因多态性.采用SPSS 13.0软件进行统计分析.结果 两组间RR、RK及KK 3种基因型分布差异有统计学意义(x2=8.777,P=0.012),AD组KK型频率显著低于对照组(x2=5.261,P=0.022).AD组(RK+KK)型频率较对照组显著降低(54.8% & 70.2%,P=0.022).KK型及K等位基因携带者血浆HDL—C、apoA-I水平增高,差异有统计学意义(P<0.05).在总体研究人群中,KK型及RK+ KK型携带者的MMSE评分以及WMS评分显著增高(P<0.05);进一步按照长时记忆、短时记忆和瞬时记忆3种记忆类型分析,与RR型相比,RK、KK型及RK+KK型携带者的短时记忆及瞬时记忆得分显著增高(P<0.05),而长时记忆得分的比较差异无统计学意义(P>0.05).Logistic回归结果表明K等位基因携带是AD发病的独立保护因素.结论 ABCA1R219K变异与AD的发病有关,K等住基因携带对血脂及认知功能可能产生有益作用,并对AD的发生可能有一定保护作用.

  3. ATP binding and aspartate protonation enhance photoinduced electron transfer in plant cryptochrome.

    Science.gov (United States)

    Cailliez, Fabien; Müller, Pavel; Gallois, Michaël; de la Lande, Aurélien

    2014-09-17

    Cryptochromes are flavoproteins encountered in most vegetal and animal species. They play a role of blue-light receptors in plants and in invertebrates. The putative resting state of the FAD cofactor in these proteins is its fully oxidized form, FADox. Upon blue-light excitation, the isoalloxazine ring (ISO) may undergo an ultrafast reduction by a nearby tryptophan residue W400. This primary reduction triggers a cascade of electron and proton transfers, ultimately leading to the formation of the FADH° radical. A recent experimental study has shown that the yield of FADH° formation in Arabidopsis cryptochrome can be strongly modulated by ATP binding and by pH, affecting the protonation state of D396 (proton donor to FAD°(-)). Here we provide a detailed molecular analysis of these effects by means of combined classical molecular dynamics simulations and time-dependent density functional theory calculations. When ATP is present and D396 protonated, FAD remains in close contact with W400, thereby enhancing electron transfer (ET) from W400 to ISO*. In contrast, deprotonation of D396 and absence of ATP introduce flexibility to the photoactive site prior to FAD excitation, with the consequence of increased ISO-W400 distance and diminished tunneling rate by almost two orders of magnitude. We show that under these conditions, ET from the adenine moiety of FAD becomes a competitive relaxation pathway. Overall, our data suggest that the observed effects of ATP and pH on the FAD photoreduction find their roots in the earliest stage of the photoreduction process; i.e., ATP binding and the protonation state of D396 determine the preferred pathway of ISO* relaxation.

  4. ABC Transporters and the Alzheimer's Disease Enigma.

    Science.gov (United States)

    Wolf, Andrea; Bauer, Björn; Hartz, Anika M S

    2012-01-01

    Alzheimer's disease (AD) is considered the "disease of the twenty-first century." With a 10-fold increase in global incidence over the past 100 years, AD is now reaching epidemic proportions and by all projections, AD patient numbers will continue to rise. Despite intense research efforts, AD remains a mystery and effective therapies are still unavailable. This represents an unmet need resulting in clinical, social, and economic problems. Over the last decade, a new AD research focus has emerged: ATP-binding cassette (ABC) transporters. In this article, we provide an overview of the ABC transporters ABCA1, ABCA2, P-glycoprotein (ABCB1), MRP1 (ABCC1), and BCRP (ABCG2), all of which are expressed in the brain and have been implicated in AD. We summarize recent findings on the role of these five transporters in AD, and discuss their potential to serve as therapeutic targets.

  5. Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae.

    Science.gov (United States)

    Müller, Axel; Severi, Emmanuele; Mulligan, Christopher; Watts, Andrew G; Kelly, David J; Wilson, Keith S; Wilkinson, Anthony J; Thomas, Gavin H

    2006-08-04

    Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an alpha-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes.

  6. Enhanced Grain Iron Levels in Rice Expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN Gene Cassette

    Science.gov (United States)

    Boonyaves, Kulaporn; Wu, Ting-Ying; Gruissem, Wilhelm; Bhullar, Navreet K.

    2017-01-01

    Micronutrient malnutrition is widespread, especially in poor populations across the globe, and iron deficiency anemia is one of the most prevalent forms of micronutrient deficiencies. Iron deficiency anemia has severe consequences for human health, working ability, and quality of life. Several interventions including iron supplementation and food fortification have been attempted and met with varied degrees of success. Rice, which is a staple food for over half of the world’s population, is an important target crop for iron biofortification. The genetic variability of iron content in the rice germplasm is very narrow, and thus, conventional breeding has not been successful in developing high iron rice varieties. Therefore, genetic engineering approaches have targeted at increasing iron uptake, translocation, and storage in the rice endosperm. We previously reported that AtIRT1, when expressed together with AtNAS1 and PvFERRITIN (PvFER) in high-iron (NFP) rice, has a synergistic effect of further increasing the iron concentration of polished rice grains. We have now engineered rice expressing AtIRT1, AtNAS1, and PvFER as a single locus gene cassette and compared the resulting lines with transgenic lines expressing AtIRT1 and PvFER gene cassettes. We also evaluated the efficacies of the MsENOD12B and native AtIRT1 promoters for the expression of AtIRT1 in rice in both types of gene cassettes, and found the native AtIRT1 promoter to be a better choice for driving the AtIRT1 expression in our biofortification strategy. All the single insertion transgenic lines have significant increases of iron concentration, both in polished and unpolished grains, but the concerted expression of AtIRT1, AtNAS1, and PvFER resulted to be a more effective strategy in achieving the highest iron increases of up to 10.46 μg/g dry weight. Furthermore, the transformed high iron lines grew better under iron deficiency growth conditions and also have significantly increased grain zinc

  7. Development and physiological regulation of intestinal lipid absorption. III. Intestinal transporters and cholesterol absorption.

    Science.gov (United States)

    Hui, David Y; Labonté, Eric D; Howles, Philip N

    2008-04-01

    Intestinal cholesterol absorption is modulated by transport proteins in enterocytes. Cholesterol uptake from intestinal lumen requires several proteins on apical brush-border membranes, including Niemann-Pick C1-like 1 (NPC1L1), scavenger receptor B-I, and CD36, whereas two ATP-binding cassette half transporters, ABCG5 and ABCG8, on apical membranes work together for cholesterol efflux back to the intestinal lumen to limit cholesterol absorption. NPC1L1 is essential for cholesterol absorption, but its function as a cell surface transporter or an intracellular cholesterol transport protein needs clarification. Another ATP transporter, ABCA1, is present in the basolateral membrane to mediate HDL secretion from enterocytes.

  8. Bacterial transport of sulfate, molybdate, and related oxyanions.

    Science.gov (United States)

    Aguilar-Barajas, Esther; Díaz-Pérez, César; Ramírez-Díaz, Martha I; Riveros-Rosas, Héctor; Cervantes, Carlos

    2011-08-01

    Sulfur is an essential element for microorganisms and it can be obtained from varied compounds, sulfate being the preferred source. The first step for sulfate assimilation, sulfate uptake, has been studied in several bacterial species. This article reviews the properties of different bacterial (and archaeal) transporters for sulfate, molybdate, and related oxyanions. Sulfate uptake is carried out by sulfate permeases that belong to the SulT (CysPTWA), SulP, CysP/(PiT), and CysZ families. The oxyanions molybdate, tungstate, selenate and chromate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC system and tungstate by the TupABC and WtpABC systems. CysPTWA, ModABC, TupABC, and WtpABC are homologous ATP-binding cassette (ABC)-type transporters with similar organization and properties. Uptake of selenate and chromate oxyanions occurs mainly through sulfate permeases.

  9. ABC Transporters and the Alzheimer’s Disease Enigma

    Directory of Open Access Journals (Sweden)

    Andrea eWolf

    2012-06-01

    Full Text Available Alzheimer’s disease (AD is considered the disease of the 21st century. With a 10-fold increase in global incidence over the past 100 years, AD is now reaching epidemic proportions and by all projections, AD patient numbers will continue to rise. Despite intense research efforts, AD remains a mystery and effective therapies are still unavailable. This represents an unmet need resulting in clinical, social, and economic problems.Over the last decade, a new AD research focus has emerged: ATP-binding cassette (ABC transporters. In this article, we provide an overview of the ABC transporters ABCA1, ABCA2, P-glycoprotein (ABCB1, Mrp1 (ABCC1 and BCRP (ABCG2, all of which are expressed in the brain and have been implicated in AD. We summarize recent findings on the role of these five transporters in AD, and discuss their potential to serve as therapeutic targets.

  10. Data for proteomic analysis of ATP-binding proteins and kinase inhibitor target proteins using an ATP probe

    Directory of Open Access Journals (Sweden)

    Jun Adachi

    2015-12-01

    Full Text Available Interactions between ATP and ATP-binding proteins (ATPome are common and are required for most cellular processes. Thus, it is clearly important to identify and quantify these interactions for understanding basic cellular mechanisms and the pathogenesis of various diseases. We used an ATP competition assay (competition between ATP and acyl-ATP probes that enabled us to distinguish specific ATP-binding proteins from non-specific proteins (Adachi et al., 2014 [1]. As a result, we identified 539 proteins, including 178 novel ATP-binding protein candidates. We also established an ATPome selectivity profiling method for kinase inhibitors using our cataloged ATPome list. Normally only kinome selectivity is profiled in selectivity profiling of kinase inhibitors. In this data, we expand the profiled targets from the kinome to the ATPome through performance of ATPome selectivity profiling and obtained target profiles of staurosporine and (S-crizotinib. The data accompanying the manuscript on this approach (Adachi et al., 2014 [1] have been deposited to the ProteomeXchange with identifier PXD001200.

  11. ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal

    Directory of Open Access Journals (Sweden)

    Choi Cheol-Hee

    2005-10-01

    Full Text Available Abstract One of the major problems related with anticancer chemotherapy is resistance against anticancer drugs. The ATP-binding cassette (ABC transporters are a family of transporter proteins that are responsible for drug resistance and a low bioavailability of drugs by pumping a variety of drugs out cells at the expense of ATP hydrolysis. One strategy for reversal of the resistance of tumor cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. In this review, the physiological functions and structures of ABC transporters, and the development of chemosensitizers are described focusing on well-known proteins including P-glycoprotein, multidrug resistance associated protein, and breast cancer resistance protein.

  12. The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

    Science.gov (United States)

    Bebes, Attila; Kis, Kornélia; Nagy, Tünde; Kurunczi, Anita; Polyánka, Hilda; Bata-Csörgo, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2012-01-01

    Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

  13. Molecular determinants for ATP-binding in proteins: a data mining and quantum chemical analysis.

    Science.gov (United States)

    Mao, Lisong; Wang, Yanli; Liu, Yuemin; Hu, Xiche

    2004-02-20

    intermolecular interactions of large biomolecular systems becomes computationally feasible. The establishment of the molecular basis for recognition of the adenine moiety of ATP in proteins will directly impact molecular design of ATP-binding site targeted enzyme inhibitors such as kinase inhibitors.

  14. ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

    Science.gov (United States)

    Finkenwirth, Friedrich; Sippach, Michael; Landmesser, Heidi; Kirsch, Franziska; Ogienko, Anastasia; Grunzel, Miriam; Kiesler, Cornelia; Steinhoff, Heinz-Jürgen; Schneider, Erwin; Eitinger, Thomas

    2015-07-03

    Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

  15. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer.

    Science.gov (United States)

    Kim, Jae-Young; Stewart, Paul A; Borne, Adam L; Fang, Bin; Welsh, Eric A; Chen, Yian Ann; Eschrich, Steven A; Koomen, John M; Haura, Eric B

    2016-06-01

    One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  16. Activity-Based Proteomics Reveals Heterogeneous Kinome and ATP-Binding Proteome Responses to MEK Inhibition in KRAS Mutant Lung Cancer

    Directory of Open Access Journals (Sweden)

    Jae-Young Kim

    2016-04-01

    Full Text Available One way cancer cells can escape from targeted agents is through their ability to evade drug effects by rapidly rewiring signaling networks. Many protein classes, such as kinases and metabolic enzymes, are regulated by ATP binding and hydrolysis. We hypothesized that a system-level profiling of drug-induced alterations in ATP-binding proteomes could offer novel insights into adaptive responses. Here, we mapped global ATP-binding proteomes perturbed by two clinical MEK inhibitors, AZD6244 and MEK162, in KRAS mutant lung cancer cells as a model system harnessing a desthiobiotin-ATP probe coupled with LC-MS/MS. We observed strikingly unique ATP-binding proteome responses to MEK inhibition, which revealed heterogeneous drug-induced pathway signatures in each cell line. We also identified diverse kinome responses, indicating each cell adapts to MEK inhibition in unique ways. Despite the heterogeneity of kinome responses, decreased probe labeling of mitotic kinases and an increase of kinases linked to autophagy were identified to be common responses. Taken together, our study revealed a diversity of adaptive ATP-binding proteome and kinome responses to MEK inhibition in KRAS mutant lung cancer cells, and our study further demonstrated the utility of our approach to identify potential candidates of targetable ATP-binding enzymes involved in adaptive resistance and to develop rational drug combinations.

  17. Functionally Relevant Residues of Cdr1p: A Multidrug ABC Transporter of Human Pathogenic Candida albicans

    Directory of Open Access Journals (Sweden)

    Rajendra Prasad

    2011-01-01

    Full Text Available Reduced intracellular accumulation of drugs (due to rapid efflux mediated by the efflux pump proteins belonging to ABC (ATP Binding Cassette and MFS (Major Facilitators superfamily is one of the most common strategies adopted by multidrug resistance (MDR pathogenic yeasts. To combat MDR, it is essential to understand the structure and function of these transporters so that inhibitors/modulators to these can be developed. The sequence alignments of the ABC transporters reveal selective divergence within much conserved domains of Nucleotide-Binding Domains (NBDs which is unique to all fungal transporters. Recently, the role of conserved but divergent residues of Candida Drug Resistance 1 (CDR1, an ABC drug transporter of human pathogenic Candida albicans, has been examined with regard to ATP binding and hydrolysis. In this paper, we focus on some of the recent advances on the relevance of divergent and conserved amino acids of CaCdr1p and also discuss as to how drug interacts with Trans Membrane Domains (TMDs residues for its extrusion from MDR cells.

  18. ATP binding and hydrolysis-driven rate-determining events in the RFC-catalyzed PCNA clamp loading reaction.

    Science.gov (United States)

    Sakato, Miho; Zhou, Yayan; Hingorani, Manju M

    2012-02-17

    The multi-subunit replication factor C (RFC) complex loads circular proliferating cell nuclear antigen (PCNA) clamps onto DNA where they serve as mobile tethers for polymerases and coordinate the functions of many other DNA metabolic proteins. The clamp loading reaction is complex, involving multiple components (RFC, PCNA, DNA, and ATP) and events (minimally: PCNA opening/closing, DNA binding/release, and ATP binding/hydrolysis) that yield a topologically linked clamp·DNA product in less than a second. Here, we report pre-steady-state measurements of several steps in the reaction catalyzed by Saccharomyces cerevisiae RFC and present a comprehensive kinetic model based on global analysis of the data. Highlights of the reaction mechanism are that ATP binding to RFC initiates slow activation of the clamp loader, enabling it to open PCNA (at ~2 s(-1)) and bind primer-template DNA (ptDNA). Rapid binding of ptDNA leads to formation of the RFC·ATP·PCNA(open)·ptDNA complex, which catalyzes a burst of ATP hydrolysis. Another slow step in the reaction follows ATP hydrolysis and is associated with PCNA closure around ptDNA (8 s(-1)). Dissociation of PCNA·ptDNA from RFC leads to catalytic turnover. We propose that these early and late rate-determining events are intramolecular conformational changes in RFC and PCNA that control clamp opening and closure, and that ATP binding and hydrolysis switch RFC between conformations with high and low affinities, respectively, for open PCNA and ptDNA, and thus bookend the clamp loading reaction.

  19. Association of three common single nucleotide polymorphisms of ATP binding cassette G8 gene with gallstone disease: a meta-analysis.

    Directory of Open Access Journals (Sweden)

    Zhao-Yan Jiang

    Full Text Available BACKGROUND: In this study, we evaluated the association between these polymorphisms and gallstone disease using meta-analysis and compared the hepatic ABCG5/G8 mRNA expression and biliary lipids composition in patients with different genotypes of T400K and Y54C. METHODS: Data were analyzed using the Stata/SE 11.0 software and a random- effects model was applied irrespective of between-study heterogeneity. Hepatic mRNA expression of ABCG5/G8 genes in 182 patients with gallstone disease and 35 gallstone-free patients who underwent cholecystectomy were determined using real-time PCR. Genotypes of Y54C and T400K in the ABCG8 gene were determined by allelic discrimination using either genomic DNA or hepatic cDNA as template by Taqman assays. Biliary compostion in gallbladder bile was assayed in these patients as well. RESULTS: Ten papers including 13 cohorts were included for the final analysis. In the genotype model, the overall association between genotype with gallstone was significant for D19H (OR = 2.43, 95%CI: 2.23-2.64, P<0.001, and for Y54C (OR = 1.36, 95%CI: 1.01-1.83, P = 0.044, or T400K (OR = 1.17, 95%CI: 0.96-1.43. P = 0.110. In allele model, minor alleles of D19H polymorphism (allele D: OR = 2.25, 95%CI: 2.10-2.42, P<0.001 and of T400K polymorphism (allele K: OR = 1.18, 95%CI: 1.06-1.31, P<0.001 were related with an increased risk of gallstone disease. However, minor allele of Y54C polymorphism (allele Y, OR = 1.08, 95%CI: 0.96-1.21, P = 0.146 was not related with gallstone disease. I(2 statistics indicated no significant between-study heterogeneity for all genetic models for any of the three polymorphisms. Funnel plot and Egger's test suggested the absence of publication bias as well. However, no association of T400K and Y54C polymorphism with hepatic ABCG8/G5 mRNA expression or biliary lipids composition was found. CONCLUSIONS: Our study showed strong association of D19H polymorphism with gallstone disease. T400K and Y54C polymorphism, though to a less extent, may also relate with gallstone disease.

  20. Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

    Directory of Open Access Journals (Sweden)

    Gracian Tejral

    2017-03-01

    Full Text Available Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation”  “semi-open conformation  “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

  1. Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

    Science.gov (United States)

    Tejral, Gracian; Sopko, Bruno; Necas, Alois; Schoner, Wilhelm

    2017-01-01

    Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation”  “semi-open conformation  “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis. PMID:28316890

  2. ABC转运蛋白与巴西橡胶树产胶代谢%ABC Transporters and the Latex Regeneration Metabolism of Hevea brasiliensis

    Institute of Scientific and Technical Information of China (English)

    聂智毅; 黎瑜; 曾日中

    2013-01-01

    ABC转运蛋白(ATP Binding Cassette transporter)是目前已知最大、功能最广泛的蛋白家族之一。大多数ABC转运蛋白都能利用水解ATP释放的能量直接转运底物。许多研究结果显示,植物ABC转运蛋白在各种代谢产物的跨膜转运中起着重要作用。橡胶的产胶代谢是一种典型的植物类异戊二烯次生代谢,是影响橡胶产量的首要因素。相关实验结果显示, ABC转运蛋白可能参与橡胶树产胶代谢。本文介绍了模式植物拟南芥中的ABC转运蛋白研究进展,并对ABC转运蛋白与橡胶树产胶代谢的关系进行讨论。%ATP Binding Cassette transporters (ABC transporters) constitute the largest protein family with the most variety of functions. Most ABC transporters can utilize the energy of ATP hydrolysis to transport substances, and many researches reveal that ABC transporters play important roles in translocation of plant metabolites across membranes. Latex regeneration metabolism of Hevea brasiliensis is a typical of plant isoprenoid secondary metabolism and is the primary factor affecting rubber production. Researches reveal that ABC-transporters may be involved in the latex regeneration metabolism of Hevea brasiliensis. Base on the new advances in ABC-transporters in A rabidopsis thaliana, the relation between ABC-transporters and latex regeneration of Hevea brasiliensis was discussed in the review.

  3. Metabolism and transport of tamoxifen in relation to its effectiveness

    DEFF Research Database (Denmark)

    Cronin-Fenton, Deirdre P; Damkier, Per; Lash, Timothy L

    2014-01-01

    Tamoxifen reduces the rate of breast cancer recurrence by approximately a half. Tamoxifen is metabolized to more active metabolites by enzymes encoded by polymorphic genes, including cytochrome P450 2D6 (CYP2D6). Tamoxifen is a substrate for ATP-binding cassette transporter proteins. We review...... tamoxifen's clinical pharmacology and use meta-analyses to evaluate the clinical epidemiology studies conducted to date on the association between CYP2D6 inhibition and tamoxifen effectiveness. Our findings indicate that the effect of both drug-induced and/or gene-induced inhibition of CYP2D6 activity...... is likely to be null or small, or at most moderate in subjects carrying two reduced function alleles. Future research should examine the effect of polymorphisms in genes encoding enzymes in tamoxifen's complete metabolic pathway, should comprehensively evaluate other biomarkers that affect tamoxifen...

  4. Coupled ATPase-adenylate kinase activity in ABC transporters

    Science.gov (United States)

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  5. A putative ABC transporter is involved in negative regulation of biofilm formation by Listeria monocytogenes

    DEFF Research Database (Denmark)

    Zhu, Xinna; Long, Fei; Chen, Yonghui

    2008-01-01

    Listeria monocytogenes may persist for long periods in food processing environments. In some instances, this may be due to aggregation or biofilm formation. To investigate the mechanism controlling biofilm formation in the food-borne pathogen L. monocytogenes, we characterized LM-49, a mutant...... with enhanced ability of biofilm-formation generated via transposon Tn917 mutagenesis of L. monocytogenes 4b G. In this mutant, a Tn917 insertion has disrupted the coding region of the gene encoding a putative ATP binding cassette (ABC) transporter permease identical to Lmof2365_1771 (a putative ABC......-transporter permease) presented in the sequenced strain L. monocytogenes str. 4b F2365. This disrupted gene, denoted lm.G_1771, encoded a protein with 10 transmembrane helixes. The revertant, LM-49RE, was obtained by replacing lm.G_1771::Tn917 with lm.G_1771 via homologous recombination. We found that LM-49RE formed...

  6. Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

    Science.gov (United States)

    Ernst, S.; Verhalen, B.; Zarrabi, N.; Wilkens, S.; Börsch, M.

    2011-03-01

    In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.

  7. A Cassette Based System for Hydrogen Storage and Delivery

    Energy Technology Data Exchange (ETDEWEB)

    Britton Wayne E.

    2006-11-29

    A hydrogen storage system is described and evaluated. This is based upon a cassette, that is a container for managing hydrogen storage materials. The container is designed to be safe, modular, adaptable to different chemistries, inexpensive, and transportable. A second module receives the cassette and provides the necessary infrastructure to deliver hydrogen from the cassette according to enduser requirements. The modular concept has a number of advantages over approaches that are all in one stand alone systems. The advantages of a cassette based system are discussed, along with results from model and laboratory testing.

  8. Mitochondrial ABC transporters.

    Science.gov (United States)

    Lill, R; Kispal, G

    2001-01-01

    In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane. The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p. In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria. The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol. Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). MTABC3 may be a candidate gene for the lethal neonatal syndrome. The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

  9. A man-made ATP-binding protein evolved independent of nature causes abnormal growth in bacterial cells.

    Directory of Open Access Journals (Sweden)

    Joshua M Stomel

    Full Text Available Recent advances in de novo protein evolution have made it possible to create synthetic proteins from unbiased libraries that fold into stable tertiary structures with predefined functions. However, it is not known whether such proteins will be functional when expressed inside living cells or how a host organism would respond to an encounter with a non-biological protein. Here, we examine the physiology and morphology of Escherichia coli cells engineered to express a synthetic ATP-binding protein evolved entirely from non-biological origins. We show that this man-made protein disrupts the normal energetic balance of the cell by altering the levels of intracellular ATP. This disruption cascades into a series of events that ultimately limit reproductive competency by inhibiting cell division. We now describe a detailed investigation into the synthetic biology of this man-made protein in a living bacterial organism, and the effect that this protein has on normal cell physiology.

  10. Mycobacterium tuberculosis universal stress protein Rv2623 regulates bacillary growth by ATP-Binding: requirement for establishing chronic persistent infection.

    Directory of Open Access Journals (Sweden)

    Joshua E Drumm

    2009-05-01

    Full Text Available Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  11. The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.

    Directory of Open Access Journals (Sweden)

    Hao Ding

    Full Text Available Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI. The Q motif, consisting of nine amino acids (GFXXPXPIQ with an invariant glutamine (Q residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11 gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.

  12. Mycobacterium tuberculosis Universal Stress Protein Rv2623 Regulates Bacillary Growth by ATP Binding: Requirement for Establishing Chronic Persistent Infection

    Energy Technology Data Exchange (ETDEWEB)

    Drumm, J.; Mi, K; Bilder, P; Sun, M; Lim, J; Bielefeldt-Ohmann, H; Basaraba, R; So, M; Zhu, G; et. al.

    2009-01-01

    Tuberculous latency and reactivation play a significant role in the pathogenesis of tuberculosis, yet the mechanisms that regulate these processes remain unclear. The Mycobacterium tuberculosisuniversal stress protein (USP) homolog, rv2623, is among the most highly induced genes when the tubercle bacillus is subjected to hypoxia and nitrosative stress, conditions thought to promote latency. Induction of rv2623 also occurs when M. tuberculosis encounters conditions associated with growth arrest, such as the intracellular milieu of macrophages and in the lungs of mice with chronic tuberculosis. Therefore, we tested the hypothesis that Rv2623 regulates tuberculosis latency. We observed that an Rv2623-deficient mutant fails to establish chronic tuberculous infection in guinea pigs and mice, exhibiting a hypervirulence phenotype associated with increased bacterial burden and mortality. Consistent with this in vivo growth-regulatory role, constitutive overexpression of rv2623 attenuates mycobacterial growth in vitro. Biochemical analysis of purified Rv2623 suggested that this mycobacterial USP binds ATP, and the 2.9-A-resolution crystal structure revealed that Rv2623 engages ATP in a novel nucleotide-binding pocket. Structure-guided mutagenesis yielded Rv2623 mutants with reduced ATP-binding capacity. Analysis of mycobacteria overexpressing these mutants revealed that the in vitro growth-inhibitory property of Rv2623 correlates with its ability to bind ATP. Together, the results indicate that i M. tuberculosis Rv2623 regulates mycobacterial growth in vitro and in vivo, and ii Rv2623 is required for the entry of the tubercle bacillus into the chronic phase of infection in the host; in addition, iii Rv2623 binds ATP; and iv the growth-regulatory attribute of this USP is dependent on its ATP-binding activity. We propose that Rv2623 may function as an ATP-dependent signaling intermediate in a pathway that promotes persistent infection.

  13. 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.

  14. Hijacking membrane transporters for arsenic phytoextraction.

    Science.gov (United States)

    LeBlanc, Melissa S; McKinney, Elizabeth C; Meagher, Richard B; Smith, Aaron P

    2013-01-10

    Arsenic is a toxic metalloid and recognized carcinogen. Arsenate and arsenite are the most common arsenic species available for uptake by plants. As an inorganic phosphate (Pi) analog, arsenate is acquired by plant roots through endogenous Pi transport systems. Inside the cell, arsenate is reduced to the thiol-reactive form arsenite. Glutathione (GSH)-conjugates of arsenite may be extruded from the cell or sequestered in vacuoles by members of the ATP-binding cassette (ABC) family of transporters. In the present study we sought to enhance both plant arsenic uptake through Pi transporter overexpression, and plant arsenic tolerance through ABC transporter overexpression. We demonstrate that Arabidopsis thaliana plants overexpressing the high-affinity Pi transporter family members, AtPht1;1 or AtPht1;7, are hypersensitive to arsenate due to increased arsenate uptake. These plants do not exhibit increased sensitivity to arsenite. Co-overexpression of the yeast ABC transporter YCF1 in combination with AtPht1;1 or AtPht1;7 suppresses the arsenate-sensitive phenotype while further enhancing arsenic uptake. Taken together, our results support an arsenic transport mechanism in which arsenate uptake is increased through Pi transporter overexpression, and arsenic tolerance is enhanced through YCF1-mediated vacuolar sequestration. This work substantiates the viability of coupling enhanced uptake and vacuolar sequestration as a means for developing a prototypical engineered arsenic hyperaccumulator.

  15. Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport.

    Science.gov (United States)

    Coleman, Jonathan A; Quazi, Faraz; Molday, Robert S

    2013-03-01

    Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P(4)-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P(4)-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P(4)-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

  16. The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact, Head-to-Tail Configuration of the Nucleotide-Binding Domains.

    Directory of Open Access Journals (Sweden)

    Peter M Jones

    Full Text Available ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs, which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration 'sandwich' dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD 'Switch' mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.

  17. 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....

  18. A Minireview: Usefulness of Transporter-Targeted Prodrugs in Enhancing Membrane Permeability.

    Science.gov (United States)

    Murakami, Teruo

    2016-09-01

    Orally administered drugs are categorized into 4 classes depending on the solubility and permeability in a Biopharmaceutics Classification System. Prodrug derivatization is one of feasible approaches in modifying the physicochemical properties such as low solubility and low permeability without changing the in vivo pharmacological action of the parent drug. In this article, prodrug-targeted solute carrier (SLC) transporters were searched randomly by PubMed. Collected SLC transporters are amino acid transporter 1, bile acid transporter, carnitine transporter 2, glucose transporter 1, peptide transporter 1, vitamin C transporter 1, and multivitamin transporter. The usefulness of transporter-targeted prodrugs was evaluated in terms of membrane permeability, stability under acidic condition, and conversion to the parent drug. Among prodrugs collected, peptide transporter-targeted prodrugs exhibited the highest number, and some prodrugs such as valaciclovir and valganciclovir are clinically available. ATP-binding cassette efflux transporter, P-glycoprotein (P-gp), reduces the intestinal absorption of lipophilic P-gp substrate drugs, and SLC transporter-targeted prodrugs of P-gp substrate drugs circumvented the P-gp-mediated efflux transport. Thus, SLC transporter-targeted prodrug derivatization seems to be feasible approach to increase the oral bioavailability by overcoming various unwanted physicochemical properties of orally administered drugs, although the effect of food on prodrug absorption should be taken into consideration.

  19. Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli.

    Science.gov (United States)

    Ohtsu, Iwao; Kawano, Yusuke; Suzuki, Marina; Morigasaki, Susumu; Saiki, Kyohei; Yamazaki, Shunsuke; Nonaka, Gen; Takagi, Hiroshi

    2015-01-01

    Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (Km = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

  20. Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Iwao Ohtsu

    Full Text Available Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (Km = 1.1 μM that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

  1. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    Bagaria, A.; Swaminathan, S.; Kumaran, D.; Burley, S. K.

    2011-04-01

    The ATP-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and non-transport related processes such as translation of RNA and DNA repair. Typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP) and Ribose binding protein (RBP). Each of these proteins consists of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations have been reported and so for MBP. The closed/active form of the protein interacts with the integral membrane component of the system in both transport and chemotaxis. Herein, we report 1.9{angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound to

  2. Transporters in human platelets: physiologic function and impact for pharmacotherapy.

    Science.gov (United States)

    Jedlitschky, Gabriele; Greinacher, Andreas; Kroemer, Heyo K

    2012-04-12

    Platelets store signaling molecules (eg, serotonin and ADP) within their granules. Transporters mediate accumulation of these molecules in platelet granules and, on platelet activation, their translocation across the plasma membrane. The balance between transporter-mediated uptake and elimination of signaling molecules and drugs in platelets determines their intracellular concentrations and effects. Several members of the 2 major transporter families, ATP-binding cassette (ABC) transporters and solute carriers (SLCs), have been identified in platelets. An example of an ABC transporter is MRP4 (ABCC4), which facilitates ADP accumulation in dense granules. MRP4 is a versatile transporter, and various additional functions have been proposed, notably lipid mediator release and a role in aspirin resistance. Several other ABC proteins have been detected in platelets with functions in glutathione and lipid homeostasis. The serotonin transporter (SERT, SLC6A4) in the platelet plasma membrane represents a well-characterized example of the SLC family. Moreover, recent experiments indicate expression of OATP2B1 (SLCO2B1), a high affinity transporter for certain statins, in platelets. Changes in transporter localization and expression can affect platelet function and drug sensitivity. This review summarizes available data on the physiologic and pharmacologic role of transporters in platelets.

  3. Cloning and Expression Analysis of a MDR-type ABC Transporter Abundant in Rubber Tree Latex%橡胶树一个胶乳高表达MDR型ABC转运蛋白的克隆与表达研究

    Institute of Scientific and Technical Information of China (English)

    聂智毅; 李波; 白瑞璞; 黎瑜

    2011-01-01

    ABC (ATP-Binding Cassette) transporters constitute the largest protein family with the most variety of functions.They are involved in the excretion, accumulation and the transmembrane transport of plant secondary metabolites.Through screening an ethylene-induced latex SSH cDNA library combined with RACE,one ABC transporter gene EST and its full-length cDNA were obtained, which was 3753 bp with an ORF that encoded a polypeptide of 1251 amino acids.The results showed that the ABC transporter gene had multiple copies in rubber tree genome and was primarily expressed in latex.Under ethylene or jasmonic acid induction,its expression was up-regulated.Base on above analysis, we speculated that the ABC transporter was closely correlated to the transport of rubber biosynthesis metabolites.%ABC转运蛋白(ATP-Binding Cassette)是目前已知最大、功能最广泛的蛋白家族之一,与植物次生代谢物的排泌、积累及跨膜运转密切相关.本研究筛选已构建的乙烯刺激橡胶树胶乳消减文库,克隆了1个ABC转运蛋白基因EST,通过RACE技术获得其全长cDNA序列,该基因开放阅读框(ORF)共3753 bp,编码1251个氨基酸残基.研究表明其在橡胶树基因组中存在多个拷贝,主要在胶乳中表达,且在乙烯或茉莉酸刺激(刺激产胶)条件下上调表达.推测其可能与橡胶生物合成过程中的物质转运密切相关.

  4. Antagonistic changes in sensitivity to antifungal drugs by mutations of an important ABC transporter gene in a fungal pathogen.

    Directory of Open Access Journals (Sweden)

    Wenjun Guan

    Full Text Available Fungal pathogens can be lethal, especially among immunocompromised populations, such as patients with AIDS and recipients of tissue transplantation or chemotherapy. Prolonged usage of antifungal reagents can lead to drug resistance and treatment failure. Understanding mechanisms that underlie drug resistance by pathogenic microorganisms is thus vital for dealing with this emerging issue. In this study, we show that dramatic sequence changes in PDR5, an ABC (ATP-binding cassette efflux transporter protein gene in an opportunistic fungal pathogen, caused the organism to become hypersensitive to azole, a widely used antifungal drug. Surprisingly, the same mutations conferred growth advantages to the organism on polyenes, which are also commonly used antimycotics. Our results indicate that Pdr5p might be important for ergosterol homeostasis. The observed remarkable sequence divergence in the PDR5 gene in yeast strain YJM789 may represent an interesting case of adaptive loss of gene function with significant clinical implications.

  5. Simulated microgravity alters the expression of cytoskeleton- and ATP-binding-related genes in MLO-Y4 osteocytes

    Science.gov (United States)

    Chen, Zhihao; Zhao, Fan; Qi, Yiduo; Hu, Lifang; Li, Dijie; Yin, Chong; Su, Peihong; Zhang, Yan; Ma, Jianhua; Qian, Jing; Zhou, Hongpo; Zou, Yiwei; Qian, Airong

    2016-12-01

    Bone undergoes dynamic modelling and remodelling processes, and it requires gravity-mediated mechanical stimulation for the maintenance of mineral content and structure. Osteocytes are the most commonly found cells in the mature bone, and they are sensitive to mechanical changes. The purpose of this study was to investigate the effects of microgravity simulated with a random position machine (RPM) on the gene expression profile of osteocytes. Genes sensitive to RPM treatment were sorted on the basis of biological processes, interactions and signalling pathways. Overall, 504 differentially expressed genes (DEGs) in osteocytes cultured under RPM conditions were found. The DEGs were further analysed using bioinformatics tools such as DAVID and iReport. A total of 15 ATP-binding and cytoskeleton-related genes were further confirmed by quantitative real-time PCR (qRT-PCR). Our findings demonstrate that the RPM affected the expression of genes involved in cytoskeleton remodelling and the energy-transfer process in osteocytes. The identification of mechanosensitive genes may enhance our understanding of the roles of osteocytes in mechanosensation and may provide some potential targets for preventing and treating bone-related diseases.

  6. A PhoPQ-Regulated ABC Transporter System Exports Tetracycline in Pseudomonas aeruginosa.

    Science.gov (United States)

    Chen, Lin; Duan, Kangmin

    2016-05-01

    Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell.

  7. The Schizosaccharomyces pombe mam1 gene encodes an ABC transporter mediating secretion of M-factor

    DEFF Research Database (Denmark)

    Christensen, P U; Davey, William John; Nielsen, O;

    1997-01-01

    In the fission yeast Schizosaccharomyces pombe, cells of opposite mating type communicate via diffusible peptide pheromones prior to mating. We have cloned the S. pombe mam1 gene, which encodes a 1336-amino acid protein belonging to the ATP-binding cassette (ABC) superfamily. The mam1 gene is onl...

  8. Cellular arsenic transport pathways in mammals.

    Science.gov (United States)

    Roggenbeck, Barbara A; Banerjee, Mayukh; Leslie, Elaine M

    2016-11-01

    Natural contamination of drinking water with arsenic results in the exposure of millions of people world-wide to unacceptable levels of this metalloid. This is a serious global health problem because arsenic is a Group 1 (proven) human carcinogen and chronic exposure is known to cause skin, lung, and bladder tumors. Furthermore, arsenic exposure can result in a myriad of other adverse health effects including diseases of the cardiovascular, respiratory, neurological, reproductive, and endocrine systems. In addition to chronic environmental exposure to arsenic, arsenic trioxide is approved for the clinical treatment of acute promyelocytic leukemia, and is in clinical trials for other hematological malignancies as well as solid tumors. Considerable inter-individual variability in susceptibility to arsenic-induced disease and toxicity exists, and the reasons for such differences are incompletely understood. Transport pathways that influence the cellular uptake and export of arsenic contribute to regulating its cellular, tissue, and ultimately body levels. In the current review, membrane proteins (including phosphate transporters, aquaglyceroporin channels, solute carrier proteins, and ATP-binding cassette transporters) shown experimentally to contribute to the passage of inorganic, methylated, and/or glutathionylated arsenic species across cellular membranes are discussed. Furthermore, what is known about arsenic transporters in organs involved in absorption, distribution, and metabolism and how transport pathways contribute to arsenic elimination are described.

  9. 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.

  10. Phase 0 and phase III transport in various organs: combined concept of phases in xenobiotic transport and metabolism.

    Science.gov (United States)

    Döring, Barbara; Petzinger, Ernst

    2014-08-01

    The historical phasing concept of drug metabolism and elimination was introduced to comprise the two phases of metabolism: phase I metabolism for oxidations, reductions and hydrolyses, and phase II metabolism for synthesis. With this concept, biological membrane barriers obstructing the accessibility of metabolism sites in the cells for drugs were not considered. The concept of two phases was extended to a concept of four phases when drug transporters were detected that guided drugs and drug metabolites in and out of the cells. In particular, water soluble or charged drugs are virtually not able to overcome the phospholipid membrane barrier. Drug transporters belong to two main clusters of transporter families: the solute carrier (SLC) families and the ATP binding cassette (ABC) carriers. The ABC transporters comprise seven families with about 20 carriers involved in drug transport. All of them operate as pumps at the expense of ATP splitting. Embedded in the former phase concept, the term "phase III" was introduced by Ishikawa in 1992 for drug export by ABC efflux pumps. SLC comprise 52 families, from which many carriers are drug uptake transporters. Later on, this uptake process was referred to as the "phase 0 transport" of drugs. Transporters for xenobiotics in man and animal are most expressed in liver, but they are also present in extra-hepatic tissues such as in the kidney, the adrenal gland and lung. This review deals with the function of drug carriers in various organs and their impact on drug metabolism and elimination.

  11. The effects of ABCG5/G8 polymorphisms on HDL-cholesterol concentrations depend on ABCA1 genetic variants in the Boston Puerto Rican health study

    Science.gov (United States)

    Background and aims: ATP-binding cassette transporters G5/G8 (ABCG5/G8) are associated with HDL-C concentrations. To assess whether the effect of ABCG5/G8 genetic variants on HDL-C concentrations is dependent on ATP-binding cassette transporters A1 (ABCA1), we studied potential interactions between ...

  12. NCBI nr-aa BLAST: CBRC-TTRU-01-1060 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1060 ref|XP_002523691.1| ATP-binding cassette transporter, putative [Ricinus... communis] gb|EEF38631.1| ATP-binding cassette transporter, putative [Ricinus communis] XP_002523691.1 0.012 25% ...

  13. NCBI nr-aa BLAST: CBRC-MLUC-01-0864 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MLUC-01-0864 ref|XP_002522797.1| ATP-binding cassette transporter, putative [Ricinus... communis] gb|EEF39648.1| ATP-binding cassette transporter, putative [Ricinus communis] XP_002522797.1 1.3 24% ...

  14. Intriguing possibilities and beneficial aspects of transporter-conscious drug design.

    Science.gov (United States)

    Tashima, Toshihiko

    2015-08-01

    It has been revealed that many types of drugs interact with transporter proteins within an organism. Transporter proteins absorb or excrete materials, including drugs and nutrients, across the cell membrane. Some hydrophobic drugs are excreted from the cell as xenobiotics by ATP-binding cassette (ABC) transporters. However, solute carrier (SLC) transporters are tissue-specifically expressed and have substrate specificities. Thus, transporter-conscious drug design is an excellent method of delivering drugs to pharmaceutical target organs and provides advantages in absorption, distribution, excretion, and toxicity of drugs (ADMET) due to transport systems. In fact, based on this strategy, the bioavailability of prodrugs designed as peptide transporter 1 (PEPT1) substrates was better than that of the corresponding parent compounds due to the transport system in the small intestine. Furthermore, in central nervous system (CNS) drug developing, drug delivery into brain across the blood-brain barrier (BBB) is a serious problem. However, this problem can be also solved by the use of the transport systems at the BBB. Therefore, transporter-consciously designed drugs not only may effectively elicit activity but also may control adverse side effects caused by off-targets and drug-drug interactions and, consequently, may show good performance in clinical trials. In this review, I introduce possibilities and advantages of transporter-conscious drug designs.

  15. Solution structure and function in trifluoroethanol of PP-50, an ATP-binding peptide from F1ATPase.

    Science.gov (United States)

    Chuang, W J; Abeygunawardana, C; Gittis, A G; Pedersen, P L; Mildvan, A S

    1995-05-10

    PP-50, a synthetic peptide, based on residues 141-190 of the beta-subunit of mitochondrial F1ATPase, containing the GX4GKT consensus sequence for nucleoside triphosphate binding, binds ATP tightly (Kd = 17.5 microM) as found by fluorescence titration at pH 4.0. CD and 2D proton NMR studies at pH 4.0 revealed two beta-turns, regions of extended secondary structure, transient tertiary structure, and flexibility in the GX4GKT region (W.J. Chuang, C. Abeygunawardana, P. L. Pedersen, and A. S. Mildvan, 1992, Biochemistry 31, 7915-7921). CD titration of PP-50 with trifluoroethanol (TFE) reveals a decrease in ellipticity at 208 and 222 nm, saturating at 25% TFE. Computer analysis indicates that 25% TFE increases the helix content from 5.8 to 28.6%, decreases the beta-structure from 30.2 to 20.2% and decreases the coil content from 64 to 51.2%. Fluorescence titrations of H2ATP2- with PP-50 in 25% TFE yields a Kd of 7.3 microM, 2.4-fold tighter than in H2O, probably due to TFE increasing the activity of H2ATP2- . PP-50 completely quenches the fluorescence of H2ATP2- in 25% TFE, while in H2O the fluorescence quenching is only 62%. In H2O the binding of H2ATP2- increases the structure of PP-50 as detected by CD, but in 25% TFE no significant change in CD is found on binding either H2ATP2- or Mg2+ HATP (Kd = 14 microM). The complete proton NMR spectrum of PP-50 in 25% TFE has been assigned. The solution structure, determined by distance geometry, molecular dynamics with simulated annealing, and energy minimization, consists of a coil (residues 1-8), a strand (residues 9-12), a loop (residues 13-22) containing the GX4GKT consensus sequence (residues 16-23), an alpha-helix (residues 23-36), a turn (residues 38-41), and a coil (residues 42-50), similar to that of the corresponding region of the X-ray structure of F1ATPase (J.P. Abrahams, A.G.W. Leslie, R. Lutter, and J. E. Walker, 1994 Nature 370, 621-628) and to the structure of a homologous peptide from the ATP-binding site of

  16. ABC multidrug transporters in schistosomes and other parasitic flatworms.

    Science.gov (United States)

    Greenberg, Robert M

    2013-12-01

    Schistosomiasis, a neglected tropical disease affecting hundreds of millions, is caused by parasitic flatworms of the genus Schistosoma. Treatment and control of schistosomiasis relies almost exclusively on a single drug, praziquantel (PZQ), a dangerous situation for a disease of this magnitude. Though PZQ is highly effective overall, it has drawbacks, and reports of worms showing PZQ resistance, either induced in the laboratory or isolated from the field, are disconcerting. Multidrug transporters underlie multidrug resistance (MDR), a phenomenon in which resistance to a single drug is accompanied by unexpected cross-resistance to several structurally unrelated compounds. Some of the best studied multidrug transporters are members of the ancient and very large ATP-binding cassette (ABC) superfamily of efflux transporters. ABC multidrug transporters such as P-glycoprotein (Pgp; ABCB1) are also associated with drug resistance in parasites, including helminths such as schistosomes. In addition to their association with drug resistance, however, ABC transporters also function in a wide variety of physiological processes in metazoans. In this review, we examine recent studies that help define the role of schistosome ABC transporters in regulating drug susceptibility, and in normal schistosome physiology, including reproduction and excretory activity. We postulate that schistosome ABC transporters could be useful targets for compounds that enhance the effectiveness of current therapeutics as well as for agents that act as antischistosomals on their own.

  17. Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site.

    Science.gov (United States)

    Chen, Hongming; Tucker, Julie; Wang, Xiaotao; Gavine, Paul R; Phillips, Chris; Augustin, Martin A; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, Derek

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented.

  18. Sphingosine-1-Phosphate Transporters as Targets for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Masayuki Nagahashi

    2014-01-01

    Full Text Available Sphingosine-1-phosphate (S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells, angiogenesis, and lymphangiogenesis, all of which are involved in cancer progression. S1P is generated inside cancer cells by sphingosine kinases then exported outside of the cell into the tumor microenvironment where it binds to any of five G protein coupled receptors and proceeds to regulate a variety of functions. We have recently reported on the mechanisms underlying the “inside-out” signaling of S1P, its export through the plasma membrane, and its interaction with cell surface receptors. Membrane lipids, including S1P, do not spontaneously exchange through lipid bilayers since the polar head groups do not readily go through the hydrophobic interior of the plasma membrane. Instead, specific transporter proteins exist on the membrane to exchange these lipids. This review summarizes what is known regarding S1P transport through the cell membrane via ATP-binding cassette transporters and the spinster 2 transporter and discusses the roles for these transporters in cancer and in the tumor microenvironment. Based on our research and the emerging understanding of the role of S1P signaling in cancer and in the tumor microenvironment, S1P transporters and S1P signaling hold promise as new therapeutic targets for cancer drug development.

  19. Effect of drug efflux transporters on placental transport of antiretroviral agent abacavir.

    Science.gov (United States)

    Neumanova, Zuzana; Cerveny, Lukas; Greenwood, Susan L; Ceckova, Martina; Staud, Frantisek

    2015-11-01

    Abacavir is as a frequent part of combination antiretroviral therapy used in pregnant women. The aim of this study was to investigate, using in vitro, in situ and ex vivo experimental approaches, whether the transplacental pharmacokinetics of abacavir is affected by ATP-binding cassette (ABC) efflux transporters functionally expressed in the placenta: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), multidrug resistance-associated protein 2 (ABCC2) and multidrug resistance-associated protein 5 (ABCC5). In vitro transport assays revealed that abacavir is a substrate of human ABCB1 and ABCG2 transporters but not of ABCC2 or ABCC5. In addition, in situ experiments using dually perfused rat term placenta confirmed interactions of abacavir with placental Abcb1/Abcg2. In contrast, uptake studies in human placental villous fragments did not reveal any interaction of abacavir with efflux transporters suggesting a large contribution of passive diffusion and/or influx mechanisms to net transplacental abacavir transfer.

  20. Transporter-Mediated Drug–Drug Interactions with Oral Antidiabetic Drugs

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    Jörg König

    2011-10-01

    Full Text Available Uptake transporters (e.g., members of the SLC superfamily of solute carriers and export proteins (e.g., members of the ABC transporter superfamily are important determinants for the pharmacokinetics of drugs. Alterations of drug transport due to concomitantly administered drugs that interfere with drug transport may alter the kinetics of drug substrates. In vitro and in vivo studies indicate that many drugs used for the treatment of metabolic disorders and cardiovascular diseases (e.g., oral antidiabetic drugs, statins are substrates for uptake transporters and export proteins expressed in the intestine, the liver and the kidney. Since most patients with type 2 diabetes receive more than one drug, transporter-mediated drug-drug interactions are important molecular mechanisms leading to alterations in oral antidiabetic drug pharmacokinetics with the risk of adverse drug reactions. This review focuses on uptake transporters of the SLCO/SLC21 (OATP and SLC22 (OCT/OAT family of solute carriers and export pumps of the ABC (ATP-binding cassette transporter superfamily (especially P-glycoprotein as well as the export proteins of the SLC47 (MATE family and their role for transporter-mediated drug-drug interactions with oral antidiabetic drugs.

  1. High-Density Lipoprotein-Mediated Transcellular Cholesterol Transport in Mouse Aortic Endothelial Cells

    Science.gov (United States)

    Miao, LiXia; Okoro, Emmanuel U.; Cao, ZhiJan; Yang, Hong; Motley-Johnson, Evangeline; Guo, Zhongmao

    2015-01-01

    Accumulation of unesterified cholesterol-rich lipid vesicles in the subendothelial space contributes to atherogenesis. Transport of cholesterol from the subendothelial intima back to the circulating blood inhibits atherosclerosis development; however, the mechanism for this process has not been fully defined. Using cultured mouse aortic endothelial cells (MAECs), we observed that unesterified cholesterol can be transported across the endothelial cell monolayer from the basolateral to the apical compartment. Administration of high-density lipoprotein (HDL) or apolipoprotein AI (apoAI) to the apical compartment enhanced transendothelial cholesterol transport in a concentration-dependent manner. Knockdown of ATP-binding cassette transporter G1 (ABCG1) or scavenger receptor class B type I (SR-B1), or inhibition of SR-B1 diminished HDL-induced transendothelial cholesterol transport; while knockdown of ABCA1 reduced apoAI-mediated cholesterol transport. HDL enhanced phosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt in MAECs. However, inhibition PI3K or Akt did not reduce HDL-induced transendothelial cholesterol transport. These results suggest that HDL enhances transendothelial cholesterol transport by activation of a mechanism involving ABCA1, ABCA1 and SR-B1 but not involving PI3K and Akt. PMID:26255968

  2. Concept design of the cassette toroidal mover

    Energy Technology Data Exchange (ETDEWEB)

    Maekinen, H., E-mail: harri.makinen@vtt.fi [VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere (Finland); Jaervenpaeae, J. [VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere (Finland); Valkama, P.; Vaeyrynen, J.; Amjad, F. [Tampere University of Technology, Korkeakoulunkatu 6, 33720 Tampere (Finland); Siuko, M. [VTT Technical Research Centre of Finland, P.O. Box 1300, FI-33101 Tampere (Finland); Mattila, J. [Tampere University of Technology, Korkeakoulunkatu 6, 33720 Tampere (Finland); Semeraro, L.; Esque, S. [Fusion for Energy, Torres Diagonal Litoral B3, Josep Pla 2, 08019 Barcelona (Spain)

    2011-10-15

    A full scale physical development and test facility, Divertor Test Platform 2 (DTP2), has been established in Finland for the purpose of demonstrating and developing the remote handling (RH) equipment designs for ITER using prototypes and virtual models. The major objective of the DTP2 environment is to verify and develop ITER divertor RH devices and operations. In practice this means various test trials and measurements of performance characteristics. This paper describes the design process of the Cassette Toroidal Mover (CTM). The main purpose of this design task was the development of the CTM concept. The goal of the design process was to achieve compatibility between CTM and the latest ITER divertor design. The design process was based on using a variety of tools, i.e. Catia V5, Delmia, Ansys, Mathcad and project management tools. Applicable European Standards were applied to the concept design. CTM is the cassette transporter, which carries divertor cassettes on the toroidal rails inside the ITER Vacuum Vessel (VV) during the divertor maintenance. The operation environment differs from a common industrial environment. Radiation level is 100 Gy/h. The temperature during RH operations can be 50 {sup o}C. Clearances are less than 20 mm and the loads carried weigh 9000 kg. These conditions require special solutions during the product development process. The design process consisted of defining and developing of the CTM operational sequence. This sequence includes the procedure of how the CTM - with it is onboard manipulator - prepares for and handles the divertor cassettes during RH operations. RH operations are essential part when defining CTM functions. High reliability is required in order to carry out RH tasks successfully. The recoverability of CTM is also an important design criteria. This paper describes the design process and the structure of the CTM concept.

  3. Structural models of zebrafish (Danio rerio NOD1 and NOD2 NACHT domains suggest differential ATP binding orientations: insights from computational modeling, docking and molecular dynamics simulations.

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    Jitendra Maharana

    Full Text Available Nucleotide-binding oligomerization domain-containing protein 1 (NOD1 and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (Danio rerio using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved 'Lysine' at Walker A formed hydrogen bonds (H-bonds and Aspartic acid (Walker B formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. 'Proline' of GxP motif (Pro386 of NOD1 and Pro464 of NOD2 interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.

  4. 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).

  5. Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.

    Science.gov (United States)

    Sarkadi, Balázs; Homolya, László; Szakács, Gergely; Váradi, András

    2006-10-01

    In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.

  6. Low levels of graphene and graphene oxide inhibit cellular xenobiotic defense system mediated by efflux transporters.

    Science.gov (United States)

    Liu, Su; Jiang, Wei; Wu, Bing; Yu, Jing; Yu, Haiyan; Zhang, Xu-Xiang; Torres-Duarte, Cristina; Cherr, Gary N

    2016-01-01

    Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 μg/mL graphene and 0.05 μg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

  7. Genetic organisation of the capsule transport gene region from Haemophilus paragallinarum

    Directory of Open Access Journals (Sweden)

    O. De Smidt

    2004-11-01

    Full Text Available The region involved in export of the capsule polysaccharides to the cell surface of Haemophilus paragallinarum was cloned and the genetic organisation determined. Degenerate primers designed from sequence alignment of the capsule transport genes of Haemophilus influenzae, Pasteurella multocida and Actinobacillus pleuropneumoniae were used to amplify a 2.6 kb fragment containing a segment of the H. paragallinarum capsule transport gene locus. This fragment was used as a digoxigenin labelled probe to isolate the complete H. paragallinarum capsule transport gene locus from genomic DNA. The sequence of the cloned DNA was determined and analysis revealed the presence of four genes, each showing high homology with known capsule transport genes. The four genes were designated hctA, B, C and D (for H. paragallinarum capsule transport genes and the predicted products of these genes likely encode an ATP-dependent export system responsible for transport of the capsule polysaccharides to the cell surface, possibly a member of a super family designated ABC (ATP-binding cassette transporters.

  8. Characteristics of cellular polyamine transport in prokaryotes and eukaryotes.

    Science.gov (United States)

    Igarashi, Kazuei; Kashiwagi, Keiko

    2010-07-01

    Polyamine content in cells is regulated by biosynthesis, degradation and transport. In Escherichia coli, there are two polyamine uptake systems, namely spermidine-preferential (PotABCD) and putrescine-specific (PotFGHI), which belong to the family of ATP binding cassette transporters. Putrescine-ornithine and cadaverine-lysine antiporters, PotE and CadB, each consisting of 12 transmembrane segments, are important for cell growth at acidic pH. Spermidine excretion protein (MdtJI) was also recently identified. When putrescine was used as energy source, PuuP functioned as a putrescine transporter. In Saccharomyces cerevisiae, there are four kinds of polyamine uptake proteins (DUR3, SAM3, GAP1 and AGP2), consisting of either 12 or 16 transmembrane segments. Among them, DUR3 and SAM3 mostly contribute to polyamine uptake. There are also five kinds of polyamine excretion proteins (TPO1-5), consisting of 12 transmembrane segments. Among them, TPO1 and TPO5 are the most active proteins. Since a polyamine metabolizing enzyme, spermidine/spermine N(1)-acetyltransferase, is not present in yeast, five kinds of excretion proteins may exist. The current status of polyamine transport in mammalian and plant cells are reviewed.

  9. Effects of curcumin on ion channels and transporters

    Directory of Open Access Journals (Sweden)

    Xuemei eZhang

    2014-03-01

    Full Text Available Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione], a polyphenolic compound isolated from the rhizomes of Curcuma longa (turmeric, has been shown to exhibit a wide range of pharmacological activities including anti-inflammatory, anti-cancer, anti-oxidant, anti-atherosclerotic, anti-microbial and wound healing effects. These activities of curcumin are based on its complex molecular structure and chemical features, as well as its ability to interact with multiple signaling molecules. The ability of curcumin to regulate ion channels and transporters was recognized a decade ago. The cystic fibrosis transmembrane conductance regulator (CFTR is a well-studied ion channel target of curcumin. During the process of studying its anti-cancer properties, curcumin was found to inhibit ATP-binding cassette (ABC family members including ABCA1, ABCB1, ABCC1 and ABCG2. Recent studies have revealed that many channels and transporters are modulated by curcumin, such as voltage-gated potassium (Kv channels, high-voltage-gated Ca2+ channels (HVGCC, volume-regulated anion channel (VRAC, Ca2+ release-activated Ca2+ channel (CRAC, aquaporin-4 (AQP-4, glucose transporters, etc. In this review, we aim to provide an overview of the interactions of curcumin with different types of ion channels and transporters and to help better understand and integrate the underlying molecular mechanisms of the multiple pharmacological activities of curcumin.

  10. The rem Mutations in the ATP-Binding Groove of the Rad3/XPD Helicase Lead to Xeroderma pigmentosum-Cockayne Syndrome-Like Phenotypes

    Science.gov (United States)

    Montelone, Beth A.; Aguilera, Andrés

    2014-01-01

    The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease. PMID:25500814

  11. Optimizing expression and purification of an ATP-binding gene gsiA from Escherichia coli k-12 by using GFP fusion

    Directory of Open Access Journals (Sweden)

    Zhongshan Wang

    2011-01-01

    Full Text Available The cloning, expression and purification of the glutathione (sulfur import system ATP-binding protein (gsiA was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3 was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3 provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving.

  12. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  13. A multidrug ABC transporter with a taste for salt.

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    Saroj Velamakanni

    Full Text Available BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1 in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. METHODOLOGY/PRINCIPAL FINDINGS: We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+-Na(+-Cl(- symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. CONCLUSIONS/SIGNIFICANCE: The observations on H(+-Na(+-Cl(- co-transport substantiate earlier suggestions of H(+-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.

  14. Transport of carbamazepine and drug interactions at blood-brain barrier

    Institute of Scientific and Technical Information of China (English)

    Jing-jing SUN; Lin XIE; Xiao-dong LIU

    2006-01-01

    Aim: To investigate the characteristics of carbamazepine (CBZ) transport and drug interactions at the blood-brain barrier. Methods: Cultured rat brain microvascular endothelial cells (rBMEC) were used as an in vitro model of the blood-brain barrier (BBB). When cells became confluent, CBZ uptake over time was recorded by incubation of the cells in a medium containing 10 mg/L CBZ at 37 ℃. The steady-state uptake of CBZ by rBMEC was tested for different CBZ concentrations at 37 ℃. The effects of various agents on the steady-state uptake of CBZ and efflux of CBZ from rBMEC were also studied. Results: The uptake of CBZ by rBMEC was time- and concentration-dependent. The steady-state uptake occurred at 30 min for incubation. The steady-state uptake was significantly increased (P<0.01) by treatment with dinitrophenol. The co-administration of cyclosporine A significantly increased the steady-state uptake of CBZ by the rBMEC, whereas co-administration of olanzapine significantly decreased the uptake in a concentration- and temperature-dependent manner. The efflux of CBZ from rBMEC was inhibited by CsA. Conclusion: The transport of CBZ at the BBB is mediated by many transporters. Some specific ABC (ATP-binding cassette,ABC ) efflux transporters may be involved in the transport of CBZ. Drugs influence the transport of CBZ at the BBB in different ways.

  15. Role of ABC transporters in fluoropyrimidine-based chemotherapy response.

    Science.gov (United States)

    Nies, Anne T; Magdy, Tarek; Schwab, Matthias; Zanger, Ulrich M

    2015-01-01

    Since over 50 years, 5-fluorouracil (5-FU) is in use as backbone of chemotherapy treatment regimens for a wide range of cancers including colon, breast, and head and neck carcinomas. However, drug resistance and severe toxicities such as mucositis, diarrhea, neutropenia, and vomiting in up to 40% of treated patients often lead to dose limitation or treatment discontinuation. Because the oral bioavailability of 5-FU is unpredictable and highly variable, 5-FU is commonly administered intravenously. To overcome medical complications and inconvenience associated with intravenous administration, the oral prodrugs capecitabine and tegafur have been developed. Both fluoropyrimidines are metabolically converted intracellularly to 5-FU, which then needs metabolic activation to exert its damaging activity on RNA and DNA. The low response rates of 10-15% of 5-FU monotherapy can be improved by combination regimens of infusional 5-FU and leucovorin together with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI), thereby increasing response rates to 30-40%. The impact of metabolizing enzymes in the development of fluoropyrimidine toxicity and resistance has been studied in great detail. In addition, membrane drug transporters, which are critical determinants of intracellular drug concentrations, may play a role in occurrence of toxicity and development of resistance against fluoropyrimidine-based therapy as well. This review therefore summarizes current knowledge on the role of drug transporters with particular focus on ATP-binding cassette transporters in fluoropyrimidine-based chemotherapy response.

  16. Two molybdate/tungstate ABC transporters that interact very differently with their substrate binding proteins.

    Science.gov (United States)

    Vigonsky, Elena; Ovcharenko, Elena; Lewinson, Oded

    2013-04-02

    In all kingdoms of life, ATP Binding Cassette (ABC) transporters participate in many physiological and pathological processes. Despite the diversity of their functions, they have been considered to operate by a largely conserved mechanism. One deviant is the vitamin B12 transporter BtuCD that has been shown to operate by a distinct mechanism. However, it is unknown if this deviation is an exotic example, perhaps arising from the nature of the transported moiety. Here we compared two ABC importers of identical substrate specificity (molybdate/tungstate), and find that their interactions with their substrate binding proteins are utterly different. One system forms a high-affinity, slow-dissociating complex that is destabilized by nucleotide and substrate binding. The other forms a low-affinity, transient complex that is stabilized by ligands. The results highlight significant mechanistic divergence among ABC transporters, even when they share the same substrate specificity. We propose that these differences are correlated with the different folds of the transmembrane domains of ABC transporters.

  17. MFS transporters of Candida species and their role in clinical drug resistance.

    Science.gov (United States)

    K Redhu, Archana; Shah, Abdul H; Prasad, Rajendra

    2016-06-01

    ABC (ATP-binding cassette) and MFS (major facilitator superfamily) exporters, belonging to two different superfamilies, are one of the most prominent contributors of multidrug resistance (MDR) in yeast. While the role of ABC efflux pump proteins in the development of MDR is well documented, the MFS transporters which are also implicated in clinical drug resistance have not received due attention. The MFS superfamily is the largest known family of secondary active membrane carriers, and MFS exporters are capable of transporting a host of substrates ranging from small molecules, including organic and inorganic ions, to complex biomolecules, such as peptide and lipid moieties. A few of the members of the drug/H(+) antiporter family of the MFS superfamily function as multidrug transporters and employ downhill transport of protons to efflux their respective substrates. This review focuses on the recent developments in MFS of Candida and highlights their role in drug transport by using the example of the relatively well characterized promiscuous Mdr1 efflux pump of the pathogenic yeast C. albicans.

  18. A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole

    Directory of Open Access Journals (Sweden)

    Bianca Zingales

    2015-05-01

    Full Text Available Benznidazole (BZ is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

  19. Glycolysis inhibition inactivates ABC transporters to restore drug sensitivity in malignant cells.

    Directory of Open Access Journals (Sweden)

    Ayako Nakano

    Full Text Available Cancer cells eventually acquire drug resistance largely via the aberrant expression of ATP-binding cassette (ABC transporters, ATP-dependent efflux pumps. Because cancer cells produce ATP mostly through glycolysis, in the present study we explored the effects of inhibiting glycolysis on the ABC transporter function and drug sensitivity of malignant cells. Inhibition of glycolysis by 3-bromopyruvate (3BrPA suppressed ATP production in malignant cells, and restored the retention of daunorubicin or mitoxantrone in ABC transporter-expressing, RPMI8226 (ABCG2, KG-1 (ABCB1 and HepG2 cells (ABCB1 and ABCG2. Interestingly, although side population (SP cells isolated from RPMI8226 cells exhibited higher levels of glycolysis with an increased expression of genes involved in the glycolytic pathway, 3BrPA abolished Hoechst 33342 exclusion in SP cells. 3BrPA also disrupted clonogenic capacity in malignant cell lines including RPMI8226, KG-1, and HepG2. Furthermore, 3BrPA restored cytotoxic effects of daunorubicin and doxorubicin on KG-1 and RPMI8226 cells, and markedly suppressed subcutaneous tumor growth in combination with doxorubicin in RPMI8226-implanted mice. These results collectively suggest that the inhibition of glycolysis is able to overcome drug resistance in ABC transporter-expressing malignant cells through the inactivation of ABC transporters and impairment of SP cells with enhanced glycolysis as well as clonogenic cells.

  20. Crystal structure of the human sterol transporter ABCG5/ABCG8.

    Science.gov (United States)

    Lee, Jyh-Yeuan; Kinch, Lisa N; Borek, Dominika M; Wang, Jin; Wang, Junmei; Urbatsch, Ina L; Xie, Xiao-Song; Grishin, Nikolai V; Cohen, Jonathan C; Otwinowski, Zbyszek; Hobbs, Helen H; Rosenbaum, Daniel M

    2016-05-26

    ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.

  1. Decipher the mechanisms of protein conformational changes induced by nucleotide binding through free-energy landscape analysis: ATP binding to Hsp70.

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    Adrien Nicolaï

    Full Text Available ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD of Hsp70 propagates a signal to its substrate-binding domain (SBD. Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in

  2. Crystal Structure of the Zinc-Binding Transport Protein ZnuA from Escherichia coli Reveals an Unexpected Variation in Metal Coordination

    Energy Technology Data Exchange (ETDEWEB)

    Li,H.; Jogl, G.

    2007-01-01

    Bacterial ATP-binding cassette transport systems for high-affinity uptake of zinc and manganese use a cluster 9 solute-binding protein. Structures of four cluster 9 transport proteins have been determined previously. However, the structural determinants for discrimination between zinc and manganese remain under discussion. To further investigate the variability of metal binding sites in bacterial transporters, we have determined the structure of the zinc-bound transport protein ZnuA from Escherichia coli to 1.75 {angstrom} resolution. The overall structure of ZnuA is similar to other solute-binding transporters. A scaffolding {alpha}-helix forms the backbone for two structurally related globular domains. The metal-binding site is located at the domain interface. The bound zinc ion is coordinated by three histidine residues (His78, His161 and His225) and one glutamate residue (Glu77). The functional role of Glu77 for metal binding is unexpected, because this residue is not conserved in previously determined structures of zinc and manganese-specific transport proteins. The observed metal coordination by four protein residues differs significantly from the zinc-binding site in the ZnuA transporter from Synechocystis 6803, which binds zinc via three histidine residues. In addition, the E. coli ZnuA structure reveals the presence of a disulfide bond in the C-terminal globular domain that is not present in previously determined cluster 9 transport protein structures.

  3. Screening for impact of popular herbs improving mental abilities on the transcriptional level of brain transporters

    Directory of Open Access Journals (Sweden)

    Mrozikiewicz Przemyslaw M.

    2014-06-01

    Full Text Available There are a number of compounds that can modify the activity of ABC (ATP-binding cassette and SLC (solute carrier transporters in the blood-brain barrier (BBB. The aim of this study was to investigate the effect of natural and synthetic substances on the expression level of genes encoding transporters present in the BBB (mdr1a, mdr1b, mrp1, mrp2, oatp1a4, oatp1a5 and oatp1c1. Our results showed that verapamil caused the greatest reduction in the mRNA level while other synthetic (piracetam, phenobarbital and natural (codeine, cyclosporine A, quercetin substances showed a selective inhibitory effect. Further, the extract from the roots of Panax ginseng C. A. Meyer exhibited a decrease of transcription against selected transporters whereas the extract from Ginkgo biloba L. leaves resulted in an increase of the expression level of tested genes, except for mrp2. Extract from the aerial parts of Hypericum perforatum L. was the only one to cause an increased mRNA level for mdr1 and oatp1c1. These findings suggest that herbs can play an important role in overcoming the BBB and multidrug resistance to pharmacotherapy of brain cancer and mental disorders, based on the activity of selected drug-metabolizing enzymes and transporters located in the BBB

  4. Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters.

    Science.gov (United States)

    Leslie, E M; Deeley, R G; Cole, S P

    2001-10-05

    The 190 kDa multidrug resistance protein 1 (MRP1/ABCC1) is a founding member of a subfamily of the ATP binding cassette (ABC) superfamily of transport proteins and was originally identified on the basis of its elevated expression in multidrug resistant lung cancer cells. In addition to its ability to confer resistance in tumour cells, MRP1 is ubiquitously expressed in normal tissues and is a primary active transporter of GSH, glucuronate and sulfate conjugated and unconjugated organic anions of toxicological relevance. Substrates include lipid peroxidation products, herbicides, tobacco specific nitrosamines, mycotoxins, heavy metals, and natural product and antifolate anti-cancer agents. MRP1 also transports unmodified xenobiotics but often requires GSH to do so. Active efflux is generally an important aspect of cellular detoxification since it prevents the accumulation of conjugated and unconjugated compounds that have the potential to be directly toxic. The related transporters MRP2 and MRP3 have overlapping substrate specificities with MRP1 but different tissue distributions, and evidence that they also have chemoprotective functions are discussed. Finally, MRP homologues have been described in other species including yeast and nematodes. Those isolated from the vascular plant Arabidopsis thaliana (AtMRPs) decrease the cytoplasmic concentration of conjugated toxins through sequestration in vacuoles and are implicated in providing herbicide resistance to plants.

  5. Cholesterol transport via ABCA1: new insights from solid-phase binding assay.

    Science.gov (United States)

    Reboul, Emmanuelle; Dyka, Frank M; Quazi, Faraz; Molday, Robert S

    2013-04-01

    It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).

  6. Toward Determining ATPase Mechanism in ABC Transporters: Development of the Reaction Path–Force Matching QM/MM Method

    Science.gov (United States)

    Zhou, Y.; Ojeda-May, P.; Nagaraju, M.; Pu, J.

    2016-01-01

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are ubiquitous ATP-dependent membrane proteins involved in translocations of a wide variety of substrates across cellular membranes. To understand the chemomechanical coupling mechanism as well as functional asymmetry in these systems, a quantitative description of how ABC transporters hydrolyze ATP is needed. Complementary to experimental approaches, computer simulations based on combined quantum mechanical and molecular mechanical (QM/MM) potentials have provided new insights into the catalytic mechanism in ABC transporters. Quantitatively reliable determination of the free energy requirement for enzymatic ATP hydrolysis, however, requires substantial statistical sampling on QM/MM potential. A case study shows that brute force sampling of ab initio QM/MM (AI/MM) potential energy surfaces is computationally impractical for enzyme simulations of ABC transporters. On the other hand, existing semiempirical QM/MM (SE/MM) methods, although affordable for free energy sampling, are unreliable for studying ATP hydrolysis. To close this gap, a multiscale QM/MM approach named reaction path–force matching (RP–FM) has been developed. In RP–FM, specific reaction parameters for a selected SE method are optimized against AI reference data along reaction paths by employing the force matching technique. The feasibility of the method is demonstrated for a proton transfer reaction in the gas phase and in solution. The RP–FM method may offer a general tool for simulating complex enzyme systems such as ABC transporters. PMID:27498639

  7. A pleiotropic drug resistance transporter in Nicotiana tabacum is involved in defense against the herbivore Manduca sexta.

    Science.gov (United States)

    Bienert, Manuela D; Siegmund, Stephanie E G; Drozak, Anna; Trombik, Tomasz; Bultreys, Alain; Baldwin, Ian T; Boutry, Marc

    2012-12-01

    Pleiotropic drug resistance (PDR) transporters are a group of membrane proteins belonging to the ABCG sub-family of ATP binding cassette (ABC) transporters. There is clear evidence for the involvement of plant ABC transporters in resistance to fungal and bacterial pathogens, but not in the biotic stress response to insect or herbivore attack. Here, we describe a PDR transporter, ABCG5/PDR5, from Nicotiana tabacum. GFP fusion and subcellular fractionation studies revealed that ABCG5/PDR5 is localized to the plasma membrane. Staining of transgenic plants expressing the GUS reporter gene under the control of the ABCG5/PDR5 transcription promoter and immunoblotting of wild-type plants showed that, under standard growth conditions, ABCG5/PDR5 is highly expressed in roots, stems and flowers, but is only expressed at marginal levels in leaves. Interestingly, ABCG5/PDR5 expression is induced in leaves by methyl jasmonate, wounding, pathogen infiltration, or herbivory by Manduca sexta. To address the physiological role of ABCG5/PDR5, N. tabacum plants silenced for the expression of ABCG5/PDR5 were obtained. No phenotypic modification was observed under standard conditions. However, a small increase in susceptibility to the fungus Fusarium oxysporum was observed. A stronger effect was observed in relation to herbivory: silenced plants allowed better growth and faster development of M. sexta larvae than wild-type plants, indicating an involvement of this PDR transporter in resistance to M. sexta herbivory.

  8. The Role of the Actin Cytoskeleton and Lipid Rafts in the Localization and Function of the ABCC1 Transporter

    Directory of Open Access Journals (Sweden)

    Jan Willem Kok

    2014-01-01

    Full Text Available ATP-binding cassette (ABC transporters are known to be important factors in multidrug resistance of tumor cells. Lipid rafts have been implicated in their localization in the plasma membrane, where they function as drug efflux pumps. This specific localization in rafts may support the activity of ABC/Abc transporters. This raises questions regarding the nature and composition of the lipid rafts that harbor ABC/Abc transporters and the dependence of ABC/Abc transporters—concerning their localization and activity—on lipid raft constituents. Here we review our work of the past 10 years aimed at evaluating whether ABC/Abc transporters are dependent on a particular membrane environment for their function. What is the nature of this membrane environment and which of the lipid raft constituents are important for this dependency? It turns out that cortical actin is of major importance for stabilizing the localization and function of the ABC/Abc transporter, provided it is localized in an actin-dependent subtype of lipid rafts, as is the case for human ABCC1/multidrug resistance-related protein 1 (MRP1 and rodent Abcc1/Mrp1 but not human ABCB1/P-glycoprotein (PGP. On the other hand, sphingolipids do not appear to be modulators of ABCC1/MRP1 (or Abcc1/Mrp1, even though they are coregulated during drug resistance development.

  9. A major facilitator superfamily transporter plays a dual role in polar auxin transport and drought stress tolerance in Arabidopsis.

    Science.gov (United States)

    Remy, Estelle; Cabrito, Tânia R; Baster, Pawel; Batista, Rita A; Teixeira, Miguel C; Friml, Jiri; Sá-Correia, Isabel; Duque, Paula

    2013-03-01

    Many key aspects of plant development are regulated by the polarized transport of the phytohormone auxin. Cellular auxin efflux, the rate-limiting step in this process, has been shown to rely on the coordinated action of PIN-formed (PIN) and B-type ATP binding cassette (ABCB) carriers. Here, we report that polar auxin transport in the Arabidopsis thaliana root also requires the action of a Major Facilitator Superfamily (MFS) transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1). Sequencing, promoter-reporter, and fluorescent protein fusion experiments indicate that the full-length ZIFL1.1 protein and a truncated splice isoform, ZIFL1.3, localize to the tonoplast of root cells and the plasma membrane of leaf stomatal guard cells, respectively. Using reverse genetics, we show that the ZIFL1.1 transporter regulates various root auxin-related processes, while the ZIFL1.3 isoform mediates drought tolerance by regulating stomatal closure. Auxin transport and immunolocalization assays demonstrate that ZIFL1.1 indirectly modulates cellular auxin efflux during shootward auxin transport at the root tip, likely by regulating plasma membrane PIN2 abundance. Finally, heterologous expression in yeast revealed that ZIFL1.1 and ZIFL1.3 share H(+)-coupled K(+) transport activity. Thus, by determining the subcellular and tissue distribution of two isoforms, alternative splicing dictates a dual function for the ZIFL1 transporter. We propose that this MFS carrier regulates stomatal movements and polar auxin transport by modulating potassium and proton fluxes in Arabidopsis cells.

  10. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model [v1; ref status: indexed, http://f1000r.es/41n

    Directory of Open Access Journals (Sweden)

    Sergi Vaquer

    2014-08-01

    Full Text Available Abstract Microgravity has been shown to influence the expression of ABC (ATP-Binding Cassette transporters in bacteria, fungi and mammals, but also to modify the activity of certain cellular components with structural and functional similarities to ABC transporters. Changes in activity of ABC transporters could lead to important metabolic disorders and undesired pharmacological effects during spaceflights. However, no current means exist to study the functionality of these transporters in microgravity. To this end, a Vesicular Transport Assay® (Solvo Biotechnology, Hungary was adapted to evaluate multi-drug resistance-associated protein 2 (MRP2 trans-membrane estradiol-17-β-glucuronide (E17βG transport activity, when activated by adenosine-tri-phosphate (ATP during parabolic flights. Simple diffusion, ATP-independent transport and benzbromarone inhibition were also evaluated. A high accuracy engineering system was designed to perform, monitor and synchronize all procedures. Samples were analysed using a validated high sensitivity drug detection protocol. Experiments were performed in microgravity during parabolic flights, and compared to 1g on ground results using identical equipment and procedures in all cases. Our results revealed that sufficient equipment accuracy and analytical sensitivity were reached to detect transport activity in both gravitational conditions. Additionally, transport activity levels of on ground samples were within commercial transport standards, proving the validity of the methods and equipment used. MRP2 net transport activity was significantly reduced in microgravity, so was signal detected in simple diffusion samples. Ultra-structural changes induced by gravitational stress upon vesicle membranes or transporters could explain the current results, although alternative explanations are possible. Further research is needed to provide a conclusive answer in this regard. Nevertheless, the present validated technology

  11. A high-affinity molybdate transporter in eukaryotes.

    Science.gov (United States)

    Tejada-Jiménez, Manuel; Llamas, Angel; Sanz-Luque, Emanuel; Galván, Aurora; Fernández, Emilio

    2007-12-11

    Molybdenum is an essential element for almost all living beings, which, in the form of a molybdopterin-cofactor, participates in the active site of enzymes involved in key reactions of carbon, nitrogen, and sulfur metabolism. This metal is taken up by cells in form of the oxyanion molybdate. Bacteria acquire molybdate by an ATP-binding-cassette (ABC) transport system in a widely studied process, but how eukaryotic cells take up molybdenum is unknown because molybdate transporters have not been identified so far. Here, we report a eukaryotic high-affinity molybdate transporter, encoded by the green alga Chlamydomonas reinhardtii gene MoT1. An antisense RNA strategy over the MoT1 gene showed that interference of the expression of this gene leads to the inhibition of molybdate transport activity and, in turn, of the Mo-containing enzyme nitrate reductase, indicating a function of MoT1 in molybdate transport. MOT1 functionality was also shown by heterologous expression in Saccharomyces cerevisiae. Molybdate uptake mediated by MOT1 showed a K(m) of approximately 6 nM, which is the range of the lowest K(m) values reported and was activated in the presence of nitrate. Analysis of deduced sequence from the putative protein coded by MoT1 showed motifs specifically conserved in similar proteins present in the databases, and defines a family of membrane proteins in both eukaryotes and prokaryotes probably involved in molybdate transport and distantly related to plant sulfate transporters SULTR. These findings represent an important step in the understanding of molybdate transport, a crucial process in eukaryotic cells.

  12. Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD

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    Moon Yuseok

    2009-01-01

    Full Text Available Abstract Background ATP binding cassette (ABC transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA, lipase ABC transporter domains (LARDs were designed for the secretion of fusion proteins. Results The LARDs included four glycine-rich repeats comprising a β-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD to three types of proteins; green fluorescent protein (GFP, epidermal growth factor (EGF and cytoplasmic transduction peptide (CTP. These fusion proteins were expressed in Escherichia coli together with ABC transporter of either P. fluorescens or Erwinia chrysanthemi. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant. Conclusion The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence.

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

    Science.gov (United States)

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

    2016-02-01

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

  14. ABCC4/MRP4: a MYCN-regulated transporter and potential therapeutic target in neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Tony eHuynh

    2012-12-01

    Full Text Available Resistance to cytotoxic drugs is thought to be a major cause of treatment failure in childhood neuroblastoma, and members of the ATP-binding cassette (ABC transporter superfamily may contribute to this phenomenon by active efflux of chemotherapeutic agents from cancer cells. As a member of the C subfamily of ABC transporters, multidrug resistance-associated protein MRP4/ABCC4 has the ability to export a variety of endogenous and exogenous substances across the plasma membrane. In light of its capacity for chemotherapeutic drug efflux, MRP4 has been studied in the context of drug resistance in a number of cancer cell types. However, MRP4 also influences cancer cell biology independently of chemotherapeutic drug exposure, which highlights the potential importance of endogenous MRP4 substrates in cancer biology. Furthermore, MRP4 is a direct transcriptional target of Myc family oncoproteins and expression of this transporter is a powerful independent predictor of clinical outcome in neuroblastoma. Together these features suggest that inhibition of MRP4 may be an attractive therapeutic approach for neuroblastoma and other cancers that rely on MRP4. In this respect, existing options for MRP4 inhibition are relatively non-selective and thus development of more specific anti-MRP4 compounds should be a major focus of future work in this area.

  15. Haemonchus contortus P-glycoprotein-2: in situ localisation and characterisation of macrocyclic lactone transport.

    Science.gov (United States)

    Godoy, Pablo; Lian, Jing; Beech, Robin N; Prichard, Roger K

    2015-01-01

    Haemonchus contortus is a veterinary nematode that infects small ruminants, causing serious decreases in animal production worldwide. Effective control through anthelmintic treatment has been compromised by the development of resistance to these drugs, including the macrocyclic lactones. The mechanisms of resistance in H. contortus have yet to be established but may involve efflux of the macrocyclic lactones by nematode ATP-binding-cassette transporters such as P-glycoproteins. Here we report the expression and functional activity of H. contortus P-glycoprotein 2 expressed in mammalian cells and characterise its interaction with the macrocyclic lactones, ivermectin, abamectin and moxidectin. The ability of H. contortus P-glycoprotein 2 to transport different fluorophore substrates was markedly inhibited by ivermectin and abamectin in a dose-dependent and saturable way. The profile of transport inhibition by moxidectin was markedly different. H. contortus P-glycoprotein 2 was expressed in the pharynx, the first portion of the worm's intestine and perhaps in adjacent nervous tissue, suggesting a role for this gene in regulating the uptake of avermectins and in protecting nematode tissues from the effects of macrocyclic lactone anthelmintic drugs. H. contortus P-glycoprotein 2 may thus contribute to resistance to these drugs in H. contortus.

  16. Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

    Indian Academy of Sciences (India)

    Avmeet Kohli; Vinita Gupta; Shankarling Krishnamurthy; Seyed E Hasnain; Rajendra Prasad

    2001-09-01

    CaMDR1 encodes a major facilitator superfamily (MFS) protein in Candida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p in Sf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance in C. albicans, were independently expressed in a common hypersensitive host JG436 of Saccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.

  17. The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy

    Science.gov (United States)

    El-Awady, Raafat; Saleh, Ekram; Hashim, Amna; Soliman, Nehal; Dallah, Alaa; Elrasheed, Azza; Elakraa, Ghada

    2017-01-01

    Over the years chemotherapy failure has been a vital research topic as researchers have been striving to discover reasons behind it. The extensive studies carried out on chemotherapeutic agents confirm that resistance to chemotherapy is a major reason for treatment failure. “Resistance to chemotherapy,” however, is a comprehensive phrase that refers to a variety of different mechanisms in which ATP-binding cassette (ABC) mediated efflux dominates. The ABC is one of the largest gene superfamily of transporters among both eukaryotes and prokaryotes; it represents a variety of genes that code for proteins, which perform countless functions, including drug efflux – a natural process that protects cells from foreign chemicals. Up to date, chemotherapy failure due to ABC drug efflux is an active research topic that continuously provides further evidence on multiple drug resistance (MDR), aiding scientists in tackling and overcoming this issue. This review focuses on drug resistance by ABC efflux transporters in human, viral, parasitic, fungal and bacterial cells and highlights the importance of the MDR permeability glycoprotein being the mutual ABC transporter among all studied organisms. Current developments and future directions to overcome this problem are also discussed. PMID:28119610

  18. Quantitative evaluation of the combination between cytotoxic drug and efflux transporter inhibitors based on a tumour growth inhibition model.

    Science.gov (United States)

    Sostelly, Alexandre; Payen, Léa; Guitton, Jérôme; Di Pietro, Attilio; Falson, Pierre; Honorat, Mylène; Boumendjel, Ahcène; Gèze, Annabelle; Freyer, Gilles; Tod, Michel

    2014-04-01

    ATP-Binding Cassette transporters such as ABCG2 confer resistance to various anticancer drugs including irinotecan and its active metabolite, SN38. Early quantitative evaluation of efflux transporter inhibitors-cytotoxic combination requires quantitative drug-disease models. A proof-of-concept study has been carried out for studying the effect of a new ABCG2 transporter inhibitor, MBLI87 combined to irinotecan in mice xenografted with cells overexpressing ABCG2. Mice were treated with irinotecan alone or combined to MBLI87, and tumour size was periodically measured. To model those data, a tumour growth inhibition model was developed. Unperturbed tumour growth was modelled using Simeoni's model. Drug effect kinetics was accounted for by a Kinetic-Pharmacodynamic approach. Effect of inhibitor was described with a pharmacodynamic interaction model where inhibitor enhances activity of cytotoxic. This model correctly predicted tumour growth dynamics from our study. MBLI87 increased irinotecan potency by 20% per μmol of MBLI87. This model retains enough complexity to simultaneously describe tumour growth and effect of this type of drug combination. It can thus be used as a template to early evaluate efflux transporter inhibitors in-vivo.

  19. Copper oxide and zinc oxide nanomaterials act as inhibitors of multidrug resistance transport in sea urchin embryos: their role as chemosensitizers.

    Science.gov (United States)

    Wu, Bing; Torres-Duarte, Cristina; Cole, Bryan J; Cherr, Gary N

    2015-05-05

    The ability of engineered nanomaterials (NMs) to act as inhibitors of ATP-binding cassette (ABC) efflux transporters in embryos of white sea urchin (Lytechinus pictus) was studied. Nanocopper oxide (nano-CuO), nanozinc oxide (nano-ZnO), and their corresponding metal ions (CuSO4 and ZnSO4) were used as target chemicals. The results showed that nano-CuO, nano-ZnO, CuSO4, and ZnSO4, even at relatively low concentrations (0.5 ppm), significantly increased calcein-AM (CAM, an indicator of ABC transporter activity) accumulation in sea urchin embryos at different stages of development. Exposure to nano-CuO, a very low solubility NM, at increasing times after fertilization (>30 min) decreased CAM accumulation, but nano-ZnO (much more soluble NM) did not, indicating that metal ions could cross the hardened fertilization envelope, but not undissolved metal oxide NMs. Moreover, nontoxic levels (0.5 ppm) of nano-CuO and nano-ZnO significantly increased developmental toxicity of vinblastine (an established ABC transporter substrate) and functioned as chemosensitizers. The multidrug resistance associated protein (MRP, one of ABC transporters) inhibitor MK571 significantly increased copper concentrations in embryos, indicating ABC transporters are important in maintaining low intracellular copper levels. We show that low concentrations of nano-CuO and nano-ZnO can make embryos more susceptible to other contaminants, representing a potent amplification of nanomaterial-related developmental toxicity.

  20. Vectorial transport of nucleoside analogs from the apical to the basolateral membrane in double-transfected cells expressing the human concentrative nucleoside transporter hCNT3 and the export pump ABCC4.

    Science.gov (United States)

    Rius, Maria; Keller, Daniela; Brom, Manuela; Hummel-Eisenbeiss, Johanna; Lyko, Frank; Keppler, Dietrich

    2010-07-01

    The identification of the transport proteins responsible for the uptake and the efflux of nucleosides and their metabolites enables the characterization of their vectorial transport and a better understanding of their absorption, distribution, and elimination. Human concentrative nucleoside transporters (hCNTs/SLC28A) are known to mediate the transport of natural nucleosides and some nucleoside analogs into cells in a sodium-dependent and unidirectional manner. On the other hand, several human multidrug resistance proteins [human ATP-binding cassette transporter, subfamily C (ABCC)] cause resistance against nucleoside analogs and mediate transport of phosphorylated nucleoside derivatives out of the cells in an ATP-dependent manner. For the integrated analysis of uptake and efflux of these compounds, we established a double-transfected Madin-Darby canine kidney (MDCK) II cell line stably expressing the human uptake transporter hCNT3 in the apical membrane and the human efflux pump ABCC4 in the basolateral membrane. The direction of transport was from the apical to the basolateral compartment, which is in line with the unidirectional transport and the localization of both recombinant proteins in the MDCKII cells. Recombinant hCNT3 mediated the transport of several known nucleoside substrates, and we identified 5-azacytidine as a new substrate for hCNT3. It is of interest that coexpression of both transporters was confirmed in pancreatic adenocarcinomas, which represent an important clinical indication for the therapeutic use of nucleoside analogs. Thus, our results establish a novel cell system for studies on the vectorial transport of nucleosides and their analogs from the apical to the basolateral compartment. The results contribute to a better understanding of the cellular transport characteristics of nucleoside drugs.

  1. ATP regulation of type-1 inositol 1,4,5-trisphosphate receptor activity does not require walker A-type ATP-binding motifs.

    Science.gov (United States)

    Betzenhauser, Matthew J; Wagner, Larry E; Park, Hyung Seo; Yule, David I

    2009-06-12

    ATP is known to increase the activity of the type-1 inositol 1,4,5-trisphosphate receptor (InsP3R1). This effect is attributed to the binding of ATP to glycine rich Walker A-type motifs present in the regulatory domain of the receptor. Only two such motifs are present in neuronal S2+ splice variant of InsP3R1 and are designated the ATPA and ATPB sites. The ATPA site is unique to InsP3R1, and the ATPB site is conserved among all three InsP3R isoforms. Despite the fact that both the ATPA and ATPB sites are known to bind ATP, the relative contribution of these two sites to the enhancing effects of ATP on InsP3R1 function is not known. We report here a mutational analysis of the ATPA and ATPB sites and conclude neither of these sites is required for ATP modulation of InsP3R1. ATP augmented InsP3-induced Ca2+ release from permeabilized cells expressing wild type and ATP-binding site-deficient InsP3R1. Similarly, ATP increased the single channel open probability of the mutated InsP3R1 to the same extent as wild type. ATP likely exerts its effects on InsP3R1 channel function via a novel and as yet unidentified mechanism.

  2. Two different point mutations in ABL gene ATP-binding domain conferring Primary Imatinib resistance in a Chronic Myeloid Leukemia (CML patient: A case report

    Directory of Open Access Journals (Sweden)

    Iqbal Zafar

    2004-01-01

    Full Text Available Imatinib (Gleevec is the effective therapy for BCR-ABL positive CML patients. Point mutations have been detected in ATP-binding domain of ABL gene which disturbs the binding of Gleevec to this target leading to resistance. Detection of mutations is helpful in clinical management of imatinib resistance. We established a very sensitive (ASO PCR to detect mutations in an imatinib-resistant CML patient. Mutations C944T and T1052C were detected which cause complete partial imatinib resistance, respectively. This is the first report of multiple point mutations conferring primary imatinib resistance in same patient at the same time. Understanding the biological reasons of primary imatinib resistance is one of the emerging issues of pharmacogenomics and will be helpful in understanding primary resistance of molecularly-targeted cancer therapies. It will also be of great utilization in clinical management of imatinib resistance. Moreover, this ASO-PCR assay is very effective in detecting mutations related to imatinib resistance.

  3. Genetic variation in ABCA1 predicts ischemic heart disease in the general population

    DEFF Research Database (Denmark)

    Frikke-Schmidt, Ruth; Nordestgaard, Børge G; Jensen, Gorm B;

    2008-01-01

    We tested the hypothesis that 6 nonsynonymous single nucleotide polymorphisms (SNPs) in ATP-Binding-Cassette transporter A1 (ABCA1) affect risk of ischemic heart disease (IHD) in the general population....

  4. NCBI nr-aa BLAST: CBRC-TTRU-01-0272 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-0272 ref|ZP_03055167.1| cyclodextrin ABC superfamily ATP binding casse...tte transporter, membrane protein [Bacillus pumilus ATCC 7061] gb|EDW21594.1| cyclodextrin ABC superfamily A

  5. Ectoine-induced proteins in Sinorhizobium meliloti include an Ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism.

    Science.gov (United States)

    Jebbar, Mohamed; Sohn-Bösser, Linda; Bremer, Erhard; Bernard, Théophile; Blanco, Carlos

    2005-02-01

    To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, ehuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

  6. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    Science.gov (United States)

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  7. H-loop histidine catalyzes ATP hydrolysis in the E. coli ABC-transporter HlyB.

    Science.gov (United States)

    Zhou, Yan; Ojeda-May, Pedro; Pu, Jingzhi

    2013-10-14

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters form a family of molecular motor proteins that couple ATP hydrolysis to substrate translocation across cell membranes. Each nucleotide binding domain of ABC-transporters contains a highly conserved H-loop histidine residue, whose precise mechanistic role in motor functions has remained elusive. By using combined quantum mechanical and molecular mechanical (QM/MM) calculations, we showed that the conserved H-loop residue H662 in E. coli HlyB, a bacterial ABC-transporter, can act first as a general acid and then as a general base to facilitate proton transfer in ATP hydrolysis. Without the assistance of H662, direct proton transfer from the lytic water to ATP results in a substantially higher barrier height. Our findings suggest that the essential function of the H-loop residue H662 is to provide a "chemical linchpin" that shuttles protons between reactants through a relay mechanism, thereby catalyzing ATP hydrolysis in HlyB.

  8. Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds.

    Science.gov (United States)

    Shi, Jinrui; Wang, Hongyu; Schellin, Kathleen; Li, Bailin; Faller, Marianna; Stoop, Johan M; Meeley, Robert B; Ertl, David S; Ranch, Jerry P; Glassman, Kimberly

    2007-08-01

    Phytic acid in cereal grains and oilseeds is poorly digested by monogastric animals and negatively affects animal nutrition and the environment. However, breeding programs involving mutants with less phytic acid and more inorganic phosphate (P(i)) have been frustrated by undesirable agronomic characteristics associated with the phytic acid-reducing mutations. We show that maize lpa1 mutants are defective in a multidrug resistance-associated protein (MRP) ATP-binding cassette (ABC) transporter that is expressed most highly in embryos, but also in immature endosperm, germinating seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner produced low-phytic-acid, high-Pi transgenic maize seeds that germinate normally and do not show any significant reduction in seed dry weight. This dominant transgenic approach obviates the need for incorporating recessive lpa1 mutations to create maize hybrids with reduced phytic acid. Suppressing the homologous soybean MRP gene also generated low-phytic-acid seed, suggesting that the strategy might be feasible for many crops.

  9. Key Role of Human ABC Transporter ABCG2 in Photodynamic Therapy and Photodynamic Diagnosis

    Directory of Open Access Journals (Sweden)

    Toshihisa Ishikawa

    2010-01-01

    Full Text Available Accumulating evidence indicates that ATP-binding cassette (ABC transporter ABCG2 plays a key role in regulating the cellular accumulation of porphyrin derivatives in cancer cells and thereby affects the efficacy of photodynamic therapy and photodynamic diagnosis. The activity of porphyrin efflux can be affected by genetic polymorphisms in the ABCG2 gene. On the other hand, Nrf2, an NF-E2-related transcription factor, has been shown to be involved in oxidative stress-mediated induction of the ABCG2 gene. Since patients have demonstrated individual differences in their response to photodynamic therapy, transcriptional activation and/or genetic polymorphisms of the ABCG2 gene in cancer cells may affect patients' responses to photodynamic therapy. Protein kinase inhibitors, including imatinib mesylate and gefitinib, are suggested to potentially enhance the efficacy of photodynamic therapy by blocking ABCG2-mediated porphyrin efflux from cancer cells. This review article provides an overview on the role of human ABC transporter ABCG2 in photodynamic therapy and photodynamic diagnosis.

  10. Metabolism and transport of tamoxifen in relation to its effectiveness: new perspectives on an ongoing controversy.

    Science.gov (United States)

    Cronin-Fenton, Deirdre P; Damkier, Per; Lash, Timothy L

    2014-01-01

    Tamoxifen reduces the rate of breast cancer recurrence by approximately a half. Tamoxifen is metabolized to more active metabolites by enzymes encoded by polymorphic genes, including cytochrome P450 2D6 (CYP2D6). Tamoxifen is a substrate for ATP-binding cassette transporter proteins. We review tamoxifen's clinical pharmacology and use meta-analyses to evaluate the clinical epidemiology studies conducted to date on the association between CYP2D6 inhibition and tamoxifen effectiveness. Our findings indicate that the effect of both drug-induced and/or gene-induced inhibition of CYP2D6 activity is likely to be null or small, or at most moderate in subjects carrying two reduced function alleles. Future research should examine the effect of polymorphisms in genes encoding enzymes in tamoxifen's complete metabolic pathway, should comprehensively evaluate other biomarkers that affect tamoxifen effectiveness, such as the transport enzymes, and focus on subgroups of patients, such as premenopausal breast cancer patients, for whom tamoxifen is the only guideline endocrine therapy.

  11. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways.

    Science.gov (United States)

    Willis, Lisa M; Whitfield, Chris

    2013-08-30

    Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.

  12. The Arabidopsis DESPERADO/AtWBC11 transporter is required for cutin and wax secretion.

    Science.gov (United States)

    Panikashvili, David; Savaldi-Goldstein, Sigal; Mandel, Tali; Yifhar, Tamar; Franke, Rochus B; Höfer, René; Schreiber, Lukas; Chory, Joanne; Aharoni, Asaph

    2007-12-01

    The cuticle fulfills multiple roles in the plant life cycle, including protection from environmental stresses and the regulation of organ fusion. It is largely composed of cutin, which consists of C(16-18) fatty acids. While cutin composition and biosynthesis have been studied, the export of cutin monomers out of the epidermis has remained elusive. Here, we show that DESPERADO (AtWBC11) (abbreviated DSO), encoding a plasma membrane-localized ATP-binding cassette transporter, is required for cutin transport to the extracellular matrix. The dso mutant exhibits an array of surface defects suggesting an abnormally functioning cuticle. This was accompanied by dramatic alterations in the levels of cutin monomers. Moreover, electron microscopy revealed unusual lipidic cytoplasmatic inclusions in epidermal cells, disappearance of the cuticle in postgenital fusion areas, and altered morphology of trichomes and pavement cells. We also found that DSO is induced by salt, abscisic acid, and wounding stresses and its loss of function results in plants that are highly susceptible to salt and display reduced root branching. Thus, DSO is not only essential for developmental plasticity but also plays a vital role in stress responses.

  13. Salinomycin overcomes ABC transporter-mediated multidrug and apoptosis resistance in human leukemia stem cell-like KG-1a cells

    Energy Technology Data Exchange (ETDEWEB)

    Fuchs, Dominik [Research Group Molecular Neuro-Oncology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg (Germany); Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany); Daniel, Volker; Sadeghi, Mahmoud; Opelz, Gerhard [Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany); Naujokat, Cord, E-mail: cord.naujokat@med.uni-heidelberg.de [Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg (Germany)

    2010-04-16

    Leukemia stem cells are known to exhibit multidrug resistance by expression of ATP-binding cassette (ABC) transporters which constitute transmembrane proteins capable of exporting a wide variety of chemotherapeutic drugs from the cytosol. We show here that human promyeloblastic leukemia KG-1a cells exposed to the histone deacetylase inhibitor phenylbutyrate resemble many characteristics of leukemia stem cells, including expression of functional ABC transporters such as P-glycoprotein, BCRP and MRP8. Consequently, KG-1a cells display resistance to the induction of apoptosis by various chemotherapeutic drugs. Resistance to apoptosis induction by chemotherapeutic drugs can be reversed by cyclosporine A, which effectively inhibits the activity of P-glycoprotein and BCRP, thus demonstrating ABC transporter-mediated drug resistance in KG-1a cells. However, KG-1a are highly sensitive to apoptosis induction by salinomycin, a polyether ionophore antibiotic that has recently been shown to kill human breast cancer stem cell-like cells and to induce apoptosis in human cancer cells displaying multiple mechanisms of drug and apoptosis resistance. Whereas KG-1a cells can be adapted to proliferate in the presence of apoptosis-inducing concentrations of bortezomib and doxorubicin, salinomycin does not permit long-term adaptation of the cells to apoptosis-inducing concentrations. Thus, salinomycin should be regarded as a novel and effective agent for the elimination of leukemia stem cells and other tumor cells exhibiting ABC transporter-mediated multidrug resistance.

  14. Variation and evolution of the ABC transporter genes ABCB1, ABCC1, ABCG2, ABCG5 and ABCG8: implication for pharmacogenetics and disease.

    Science.gov (United States)

    Silverton, Latoya; Dean, Michael; Moitra, Karobi

    2011-01-01

    The ATP-binding cassette (ABC) transporter genes are ubiquitous in the genomes of all vertebrates. Some of these transporters play a key role in xenobiotic defense and are endowed with the capacity to efflux harmful toxic substances. A major role in the evolution of the vertebrate ABC genes is played by gene duplication. Multiple gene duplication and deletion events have been identified in ABC genes, resulting in either gene birth or gene death indicating that the process of gene evolution is still ongoing in this group of transporters. Additionally, polymorphisms in these genes are linked to variations in expression, function, drug disposition and drug response. Single nucleotide polymorphisms in the ABC genes may be considered as markers of individual risk for adverse drug reactions or susceptibility to complex diseases as they can uniquely influence the quality and quantity of gene product. As the ABC genes continue to evolve, globalization will yield additional migration and racial admixtures that will have far reaching implications for the pharmacogenetics of this unique family of transporters in the context of human health.

  15. Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides.

    Science.gov (United States)

    Hayashi, Keisuke; Schoonbeek, Henk-Jan; De Waard, Maarten A

    2002-10-01

    Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

  16. Structure-function analysis of multidrug transporters in Lactococcus lactis

    NARCIS (Netherlands)

    van Veen, HW; Putman, M; Margolles, A; Sakamoto, K; Konings, WN

    1999-01-01

    The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding c

  17. High-density lipoprotein and atherosclerosis: Roles of lipid transporters

    Institute of Scientific and Technical Information of China (English)

    Yoshinari; Uehara; Keijiro; Saku

    2014-01-01

    Various previous studies have found a negative cor-relation between the risk of cardiovascular events and serum high-density lipoprotein(HDL) cholesterol levels. The reverse cholesterol transport, a pathway of choles-terol from peripheral tissue to liver which has several potent antiatherogenic properties. For instance, the particles of HDL mediate to transport cholesterol from cells in arterial tissues, particularly from atherosclerotic plaques, to the liver. Both ATP-binding cassette trans-porters(ABC) A1 and ABCG1 are membrane cholesterol transporters and have been implicated in mediating cholesterol effluxes from cells in the presence of HDL and apolipoprotein A-I, a major protein constituent of HDL. Previous studies demonstrated that ABCA1 and ABCG1 or the interaction between ABCA1 and ABCG1 exerted antiatherosclerotic effects. As a therapeutic approach for increasing HDL cholesterol levels, much focus has been placed on increasing HDL cholesterol levels as well as enhancing HDL biochemical functions. HDL therapies that use injections of reconstituted HDL, apoA-I mimetics, or full-length apoA-I have shown dramatic effectiveness. In particular, a novel apoA-I mi-metic peptide, Fukuoka University ApoA-I Mimetic Pep-tide, effectively removes cholesterol via specific ABCA1 and other transporters, such as ABCG1, and has an an-tiatherosclerotic effect by enhancing the biological func-tions of HDL without changing circulating HDL choles-terol levels. Thus, HDL-targeting therapy has significant atheroprotective potential, as it uses lipid transporter-targeting agents, and may prove to be a therapeutic tool for atherosclerotic cardiovascular diseases.

  18. Placental ABC transporters, cellular toxicity and stress in pregnancy.

    Science.gov (United States)

    Aye, Irving L M H; Keelan, Jeffrey A

    2013-04-25

    The human placenta, in addition to its roles as a nutrient transfer and endocrine organ, functions as a selective barrier to protect the fetus against the harmful effects of exogenous and endogenous toxins. Members of the ATP-binding cassette (ABC) family of transport proteins limit the entry of xenobiotics into the fetal circulation via vectorial efflux from the placenta to the maternal circulation. Several members of the ABC family, including proteins from the ABCA, ABCB, ABCC and ABCG subfamilies, have been shown to be functional in the placenta with clinically significant roles in xenobiotic efflux. However, recent findings suggest that these transporters also protect placental tissue by preventing the cellular accumulation of cytotoxic compounds such as lipids, sterols and their derivatives. Such protective functions are likely to be particularly important in pregnancies complicated by inflammatory or oxidative stress, where the generation of toxic metabolites is enhanced. For example, ABC transporters have been shown to protect against the harmful effects of hypoxia and oxidative stress through increased expression and efflux of oxysterols and glutathione conjugated xenobiotics. However, this protective capacity may be diminished in response to the same stressors. Several studies in primary human trophoblast cells and animal models have demonstrated decreased expression and activity of placental ABC transporters with inflammatory, oxidative or metabolic stress. Several clinical studies in pregnancies complicated by inflammatory conditions such as preeclampsia and gestational diabetes support these findings, although further studies are required to determine the clinical relevance of the relationships between placental ABC transporter expression and activity, and placental function in stressed pregnancies. Such studies are necessary to fully understand the consequences of pregnancy disorders on placental function and viability in order to optimise pregnancy

  19. ATP binding by the P-loop NTPase OsYchF1 (an unconventional G protein) contributes to biotic but not abiotic stress responses.

    Science.gov (United States)

    Cheung, Ming-Yan; Li, Xiaorong; Miao, Rui; Fong, Yu-Hang; Li, Kwan-Pok; Yung, Yuk-Lin; Yu, Mei-Hui; Wong, Kam-Bo; Chen, Zhongzhou; Lam, Hon-Ming

    2016-03-01

    G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life.

  20. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Byler, D.M.; Susi, H.; Brown, M.; Kuby, S.A.; Mildvan A.S.

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme, appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase, with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of ..beta..-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% ..cap alpha..-helix, 38% ..beta..-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possible due to disorder, it can be fit by using methods developed on well-characterized globular proteins. The CD spectrum is best fit by assuming the presence of at most 13% ..cap alpha..-helix in the peptide, 24 +/- 2% ..beta..-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformation changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assess by CD.

  1. Solution structure of the 45-residue ATP-binding peptide of adenylate kinase as determined by 2-D NMR, FTIR, and CD spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Byler, D.M.; Susi, H.; Brown, E.M.; Kuby, S.A.; Mildyan, A.S.

    1986-05-01

    In the X-ray structure of adenylate kinase residues 1-45 exist as 47% ..cap alpha..-helix, 29% ..beta..-structure (strands and turns) and 24% coil. The solution structure of a synthetic peptide corresponding to residues 1-45, which constitutes the MgATP binding site was studied by 3 independent spectroscopic methods. Globularity of the peptide was shown by its broad NMR resonances which narrow upon denaturation, and by its ability to bind MgATP with similar affinity and conformation as the intact enzyme does. COSY and NOESY NMR methods at 250 and 500 MHz reveal proximities among NH, C..cap alpha.., and C..beta.. protons indicative of >20% ..cap alpha..-helix, and >20% ..beta..-structure. Correlation of regions of secondary structure with the primary sequence by 2D NMR indicates at least one ..cap alpha..-helix (res. 23 to 29) and two ..beta..-strands (res. 12 to 15 and 34 to 38). The broad amide I band in the deconvoluted FTIR spectrum could be fit as the sum of 4 peaks due to specific secondary structures, yielding less than or equal to=45% ..cap alpha..-helix, less than or equal to=40% ..beta..-structure and greater than or equal to=15% coil. The CD spectrum, from 185-250 nm, interpreted with a 3-parameter basis set, yielded 20 +/- 5% ..cap alpha..=helix, and less than or equal to=20% ..beta..-structure. The solution structure of peptide 1-45 thus approximates that of residues 1-45 in the crystal.

  2. Structural Basis for a Ribofuranosyl Binding Protein: Insights into the Furanose Specific Transport

    Energy Technology Data Exchange (ETDEWEB)

    A Bagaria; D Kumaran; S Burley; S Swaminathan

    2011-12-31

    The APT-binding cassette transporters (ABC-transporters) are members of one of the largest protein superfamilies, with representatives in all extant phyla. These integral membrane proteins utilize the energy of ATP hydrolysis to carry out certain biological processes, including translocation of various substrates across membranes and nontransport related processes such as translation of RNA and DNA repair. typically, such transport systems in bacteria consist of an ATP binding component, a transmembrane permease, and a periplasmic receptor or binding protein. Soluble proteins found in the periplasm of gram-negative bacteria serve as the primary receptors for transport of many compounds, such as sugars, small peptides, and some ions. Ligand binding activates these periplasmic components, permitting recognition by the membrane spanning domain, which supports for transport, and, in some cases, chemotaxis. Transport and chemotaxis processes appear to be independent of one another, and a few mutants of bifunctional periplasmic components reveal the absence of one or the other function. Previously published high-resolution X-ray structures of various periplasmic ligand binding proteins include Arabinose binding protein (ABP), Allose binding protein (ALBP), Glucose-galactose binding protein (GBP), and Ribose binding protein (RBP). Each of these proteins consits of two structurally similar domains connected by a three-stranded hinge region, with ligand buried between the domains. Upon ligand binding and release, various conformational changes have been observed. For RBP, open (apo) and closed (ligand bound) conformations hafve been reported and so for MBP. The closed/active form of the protein interacts with the ingral membrane component of the system in both transport and chemotaxis. Herein, they report 1.9 {angstrom} resolution X-ray structure of the R{sub f}BP periplasmic component of an ABC-type sugar transport system from Hahella chejuensis (UniProt Id Q2S7D2) bound

  3. Inhibition or knockdown of ABC transporters enhances susceptibility of adult and juvenile schistosomes to Praziquantel.

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    Ravi S Kasinathan

    2014-10-01

    Full Text Available Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease that affects hundreds of millions. Treatment of schistosomiasis depends almost entirely on the drug praziquantel (PZQ. Though essential to treating and controlling schistosomiasis, a major limitation of PZQ is that it is not active against immature mammalian-stage schistosomes. Furthermore, there are reports of field isolates with heritable reductions in PZQ susceptibility, and researchers have selected for PZQ-resistant schistosomes in the laboratory. P-glycoprotein (Pgp; ABCB1 and other ATP binding cassette (ABC transporters remove a wide variety of toxins and xenobiotics from cells, and have been implicated in multidrug resistance (MDR. Changes in ABC transporter structure or expression levels are also associated with reduced drug susceptibility in parasitic helminths, including schistosomes. Here, we show that the activity of PZQ against schistosome adults and juveniles ex vivo is potentiated by co-administration of either the highly potent Pgp inhibitor tariquidar or combinations of inhibitors targeting multiple ABC multidrug transporters. Adult worms exposed to sublethal PZQ concentrations remain active, but co-administration of ABC transporter inhibitors results in complete loss of motility and disruption of the tegument. Notably, juvenile schistosomes (3-4 weeks post infection, normally refractory to 2 µM PZQ, become paralyzed when transporter inhibitors are added in combination with the PZQ. Experiments using the fluorescent PZQ derivative (R-PZQ-BODIPY are consistent with the transporter inhibitors increasing effective intraworm concentrations of PZQ. Adult worms in which expression of ABC transporters has been suppressed by RNA interference show increased responsiveness to PZQ and increased retention of (R-PZQ-BODIPY consistent with an important role for these proteins in setting levels of PZQ susceptibility. These results indicate that

  4. Transport of diclofenac by breast cancer resistance protein (ABCG2) and stimulation of multidrug resistance protein 2 (ABCC2)-mediated drug transport by diclofenac and benzbromarone.

    Science.gov (United States)

    Lagas, Jurjen S; van der Kruijssen, Cornelia M M; van de Wetering, Koen; Beijnen, Jos H; Schinkel, Alfred H

    2009-01-01

    Diclofenac is an important analgesic and anti-inflammatory drug, widely used for treatment of postoperative pain, rheumatoid arthritis, and chronic pain associated with cancer. Consequently, diclofenac is often used in combination regimens and undesirable drug-drug interactions may occur. Because many drug-drug interactions may occur at the level of drug transporting proteins, we studied interactions of diclofenac with apical ATP-binding cassette (ABC) multidrug efflux transporters. Using Madin-Darby canine kidney (MDCK)-II cells transfected with human P-glycoprotein (P-gp; MDR1/ABCB1), multidrug resistance protein 2 (MRP2/ABCC2), and breast cancer resistance protein (BCRP/ABCG2) and murine Bcrp1, we found that diclofenac was efficiently transported by murine Bcrp1 and moderately by human BCRP but not by P-gp or MRP2. Furthermore, in Sf9-BCRP membrane vesicles diclofenac inhibited transport of methotrexate in a concentration-dependent manner. We next used MDCK-II-MRP2 cells to study interactions of diclofenac with MRP2-mediated drug transport. Diclofenac stimulated paclitaxel, docetaxel, and saquinavir transport at only 50 microM. We further found that the uricosuric drug benzbromarone stimulated MRP2 at an even lower concentration, having maximal stimulatory activity at only 2 microM. Diclofenac and benzbromarone stimulated MRP2-mediated transport of amphipathic lipophilic drugs at 10- and 250-fold lower concentrations, respectively, than reported for other MRP2 stimulators. Because these concentrations are readily achieved in patients, adverse drug-drug interactions may occur, for example, during cancer therapy, in which drug concentrations are often critical and stimulation of elimination via MRP2 may result in suboptimal chemotherapeutic drug concentrations. Moreover, stimulation of MRP2 activity in tumors may lead to increased efflux of chemotherapeutic drugs and thereby drug resistance.

  5. Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

    Science.gov (United States)

    Bomberger, Jennifer M; Ely, Kenneth H; Bangia, Naveen; Ye, Siying; Green, Kathy A; Green, William R; Enelow, Richard I; Stanton, Bruce A

    2014-01-03

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

  6. Putative sugar transporters of the mustard leaf beetle Phaedon cochleariae: their phylogeny and role for nutrient supply in larval defensive glands.

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    Magdalena Stock

    Full Text Available BACKGROUND: Phytophagous insects have emerged successfully on the planet also because of the development of diverse and often astonishing defensive strategies against their enemies. The larvae of the mustard leaf beetle Phaedon cochleariae, for example, secrete deterrents from specialized defensive glands on their back. The secretion process involves ATP-binding cassette transporters. Therefore, sugar as one of the major energy sources to fuel the ATP synthesis for the cellular metabolism and transport processes, has to be present in the defensive glands. However, the role of sugar transporters for the production of defensive secretions was not addressed until now. RESULTS: To identify sugar transporters in P. cochleariae, a transcript catalogue was created by Illumina sequencing of cDNA libraries. A total of 68,667 transcripts were identified and 68 proteins were annotated as either members of the solute carrier 2 (SLC2 family or trehalose transporters. Phylogenetic analyses revealed an extension of the mammalian GLUT6/8 class in insects as well as one group of transporters exhibiting distinctive conserved motifs only present in the insect order Coleoptera. RNA-seq data of samples derived from the defensive glands revealed six transcripts encoding sugar transporters with more than 3,000 counts. Two of them are exclusively expressed in the glandular tissue. Reduction in secretions production was accomplished by silencing two of four selected transporters. RNA-seq experiments of transporter-silenced larvae showed the down-regulation of the silenced transporter but concurrently the up-regulation of other SLC2 transporters suggesting an adaptive system to maintain sugar homeostasis in the defensive glands. CONCLUSION: We provide the first comprehensive phylogenetic study of the SLC2 family in a phytophagous beetle species. RNAi and RNA-seq experiments underline the importance of SLC2 transporters in defensive glands to achieve a chemical defense

  7. Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk

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    Kiyoko Kaneko

    2013-11-01

    Full Text Available In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs, i.e., 421C>A (major and 376C>T (minor, in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

  8. Transcriptome Analysis to Identify the Putative Biosynthesis and Transport Genes Associated with the Medicinal Components of Achyranthes bidentata Bl.

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    Jinting Li

    2016-12-01

    Full Text Available Achyranthes bidentata is a popular perennial medicine herb used for thousands of years in China to treat various diseases. Although this herb has multiple pharmaceutical purposes in China, no transcriptomic information has been reported for this species. In addition, the understanding of several key pathways and enzymes involved in the biosynthesis of oleanolic acid and ecdysterone, two pharmacologically active classes of metabolites and major chemical constituents of A. bidentata root extracts, is limited. The aim of the present study was to characterize the transcriptome profile of the roots and leaves of A. bidentata to uncover the biosynthetic and transport mechanisms of the active components. In this study, we identified 100,987 transcripts, with an average length of 973.64 base pairs. A total of 31,634 (31.33% unigenes were annotated, and 12,762 unigenes were mapped to 303 pathways according to the Kyoto Encyclopedia of Genes and Genomes (KEGG pathway database. Moreover, we identified a total of 260 oleanolic acid and ecdysterone genes encoding biosynthetic enzymes. Furthermore, the key enzymes involved in the oleanolic acid and ecdysterone synthesis pathways were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR, revealing that the roots expressed these enzymes to a greater extent than the leaves. In addition, we identified 85 ATP-binding cassette (ABC transporters, some of which might be involved in the translocation of secondary metabolites.

  9. Transcriptome-based identification of ABC transporters in the western tarnished plant bug Lygus hesperus.

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    J Joe Hull

    Full Text Available ATP-binding cassette (ABC transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic clearance. While ABC transporters have been extensively studied in vertebrates, less is known concerning this superfamily in insects, particularly hemipteran pests. We used RNA-Seq transcriptome sequencing to identify 65 putative ABC transporter sequences (including 36 full-length sequences from the eight ABC subfamilies in the western tarnished plant bug (Lygus hesperus, a polyphagous agricultural pest. Phylogenetic analyses revealed clear orthologous relationships with ABC transporters linked to insecticide/xenobiotic clearance and indicated lineage specific expansion of the L. hesperus ABCG and ABCH subfamilies. The transcriptional profile of 13 LhABCs representative of the ABCA, ABCB, ABCC, ABCG, and ABCH subfamilies was examined across L. hesperus development and within sex-specific adult tissues. All of the transcripts were amplified from both reproductively immature and mature adults and all but LhABCA8 were expressed to some degree in eggs. Expression of LhABCA8 was spatially localized to the testis and temporally timed with male reproductive development, suggesting a potential role in sexual maturation and/or spermatozoa protection. Elevated expression of LhABCC5 in Malpighian tubules suggests a possible role in xenobiotic clearance. Our results provide the first transcriptome-wide analysis of ABC transporters in an agriculturally important hemipteran pest and, because ABC transporters are known to be important mediators of insecticidal resistance, will provide the basis for future biochemical and toxicological studies on the role of this protein family in insecticide resistance in Lygus species.

  10. Signal transduction pathways provide opportunities to enhance HDL and apoAI-dependent reverse cholesterol transport.

    Science.gov (United States)

    Mulay, Vishwaroop; Wood, Peta; Rentero, Carles; Enrich, Carlos; Grewal, Thomas

    2012-02-01

    Binding of High Density Lipoprotein (HDL) and its major apolipoprotein A-I (apoA-I) to cell surface receptors is believed to initiate a plethora of signaling cascades that promote atheroprotective cell behavior, including the removal of excess cholesterol from lipid-loaded macrophages. More specifically, HDL and apoA-I binding to scavenger receptor BI (SR-BI) and ATP-binding cassette (ABC) transporter A1 has been shown to activate protein kinase A and C (PKA, PKC), Rac/Rho GTPases, Janus Kinase 2 (JAK2), calmodulin as well as mitogen-activated protein kinases (MAPK). Some of these signaling events upregulate mobilization of cholesterol from cellular pools, while others promote efflux pathways through increased expression, stability, and cell surface localization of SR-BI and ABCA1. This review aims to summarize the current knowledge of HDL- and apoA-I -induced signal transduction pathways that are linked to cholesterol efflux and discusses the underlying mechanisms that could couple ligand binding to SR-BI and ABCA1 with signaling and cholesterol export. Additional focus is given on the potential of pharmacological intervention to modulate the activity of signaling cascades for the inhibition or regression of cholesterol accumulation in atherosclerotic lesions.

  11. Whatever happened to cassette-dosing pharmacokinetics?

    Science.gov (United States)

    Manitpisitkul, Prasarn; White, Ronald E

    2004-08-01

    Cassette dosing is a procedure that is used for rapidly assessing the pharmacokinetics of a series of discovery drug candidates by dosing a mixture of compounds rather than a single compound. Cassette dosing has advantages and disadvantages associated with its use, which leads to controversy about how and if it should be used. To assess the current practices of the pharmaceutical industry regarding cassette dosing, a survey of several pharmaceutical companies was conducted. Analysis of the survey revealed that opinion on this subject is divided within the pharmaceutical industry. In addition, it was determined that approximately only a half of those companies that perform in vivo pharmacokinetic screening use cassette dosing for this purpose.

  12. Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

    Science.gov (United States)

    Fry, D C; Byler, D M; Susi, H; Brown, E M; Kuby, S A; Mildvan, A S

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  13. 巴西橡胶树胶乳表达ABC转运蛋白HbGCN1的克隆与表达研究%loning and Expression Analysis of HbGCN1 Encoding an ABC Transporter in the Latex of Rubber Tree (Hevea brasiliensis Müll.Arg.)

    Institute of Scientific and Technical Information of China (English)

    聂智毅; 黎瑜; 李波; 夏可灿; 段翠芳

    2012-01-01

    橡胶生物合成是一种典型的植物类异戊二烯次生代谢,它是影响橡胶产量的首要因素.ABC转运蛋白(ATP Binding Cassette transporter,ABC transporter)是一类与植物次生代谢密切相关的蛋白大家族.筛选已构建的茉莉酸诱导橡胶树胶乳消减文库,并克隆了1个GCN(general control non-repressible)亚族ABC转运蛋白基因EST,通过RACE技术获得其全长cDNA序列,命名为HbGCN1.该基因开放阅读框共1 815 bp,编码605个氨基酸残基.表达分析表明:HbGCN1主要在橡胶树树皮及胶乳中表达,且在乙烯、茉莉酸或伤害诱导下上调表达,推测其可能与橡胶生物合成相关.原核表达该基因,为进一步的研究奠定基础.%Rubber biosynthesis, a typical isoprenoid biosynthesis pathway, has been the key important factor limiting rubber latex yield. ABC (ATP-BindingCassette)transporters are a large protein family which is closely involved in the plant secondary metabolites. Through screening a jasmonic -induced latex SSH cDNA library combined with RACE, one EST encoding ABC transporter of GCN subgroup and its full-length cDNA, named as HbGCNl were obtained. HbGCNl contains a ORF with 1 815 bp and encodes a polypeptide of 605 amino acids. The expression patterns show that HbGCNl is primarily expressed in latex or bark. Under ethylene, jasmonic acid and wounding induction, its expression is up-regulated. Based on the above analysis, it is speculated that HbGCNl may be closely correlated to the rubber biosynthesis. Moreover, the prokaryotie expression of the gene lay the foundation for further research.

  14. A specific interdomain interaction preserves the structural and binding properties of the ModA protein from the phytopathogen Xanthomonas citri domain interaction and transport in ModA.

    Science.gov (United States)

    Santacruz-Perez, Carolina; Pegos, Vanessa Rodrigues; Honorato, Rodrigo V; Verli, Hugo; Lindahl, Erik; Barbosa, João Alexandre Ribeiro Gonçalves; Balan, Andrea

    2013-11-01

    The periplasmic-binding proteins in ATP-binding cassette systems (ABC Transporters) are responsible for the capture and delivery of ligands to their specific transporters, triggering a series of ATP-driven conformational changes that leads to the transport of the ligand. Structurally consisting of two lobes, the proteins change conformation after interaction with the ligand. The structure of the molybdate-binding protein (ModA) from Xanthomonas citri, bound to molybdate, was previously solved by our group and an interdomain interaction, mediated by a salt bridge between K127 and D59, apparently supports the binding properties and keeps the domains closed. To determinate the importance of this interaction, we built two ModA mutants, K127S and D59A, and analysed their functional and structural properties. Based on a set of spectroscopic experiments, crystallisation trials, structure determination and molecular dynamics (MD) simulations, we showed that the salt bridge is essential to maintain the structure and binding properties. Additionally, the MD simulations revealed that this mutant adopted a more compact structure that packed down the ligand-binding pocket. From the closed bound to open structure, the positioning of the helices forming the dipole and the salt bridge are essential to induce an intermediate state.

  15. Transporter-Mediated Drug Interaction Strategy for 5-Aminolevulinic Acid (ALA-Based Photodynamic Diagnosis of Malignant Brain Tumor: Molecular Design of ABCG2 Inhibitors

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    Toshihisa Ishikawa

    2011-09-01

    Full Text Available Photodynamic diagnosis (PDD is a practical tool currently used in surgical operation of aggressive brain tumors, such as glioblastoma. PDD is achieved by a photon-induced physicochemical reaction which is induced by excitation of protoporphyrin IX (PpIX exposed to light. Fluorescence-guided gross-total resection has recently been developed in PDD, where 5-aminolevulinic acid (ALA or its ester is administered as the precursor of PpIX. ALA induces the accumulation of PpIX, a natural photo-sensitizer, in cancer cells. Recent studies provide evidence that adenosine triphosphate (ATP-binding cassette (ABC transporter ABCG2 plays a pivotal role in regulating the cellular accumulation of porphyrins in cancer cells and thereby affects the efficacy of PDD. Protein kinase inhibitors are suggested to potentially enhance the PDD efficacy by blocking ABCG2-mediated porphyrin efflux from cancer cells. It is of great interest to develop potent ABCG2-inhibitors that can be applied to PDD for brain tumor therapy. This review article addresses a pivotal role of human ABC transporter ABCG2 in PDD as well as a new approach of quantitative structure-activity relationship (QSAR analysis to design potent ABCG2-inhibitors.

  16. The Half-Size ABC Transporter FOLDED PETALS 2/ABCG13 Is Involved in Petal Elongation through Narrow Spaces in Arabidopsis thaliana Floral Buds

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    Seiji Takeda

    2014-08-01

    Full Text Available Flowers are vital for attracting pollinators to plants and in horticulture for humans. Petal morphogenesis is a central process of floral development. Petal development can be divided into three main processes: the establishment of organ identity in a concentric pattern, primordia initiation at fixed positions within a whorl, and morphogenesis, which includes petal elongation through the narrow spaces within the bud. Here, we show that the FOLDED PETALS 2 (FOP2 gene, encoding a member of the half-size ATP binding cassette (ABC transporter family ABCG13, is involved in straight elongation of petals in Arabidopsis thaliana. In fop2 mutants, flowers open with folded petals, instead of straight-elongated ones found in the wild type. The epicuticular nanoridge structures are absent in many abaxial epidermal cells of fop2 petals, and surgical or genetic generation of space in young fop2 buds restores the straight elongation of petals, suggesting that the physical contact of sepals and petals causes the petal folding. Similar petal folding has been reported in the fop1 mutant, and the petals of fop2 fop1 double mutants resemble those of both the fop1 and fop2 single mutants, although the epidermal structure and permeability of the petal surface is more affected in fop2. Our results suggest that synthesis and transport of cutin or wax in growing petals play an important role for their smooth elongation through the narrow spaces of floral buds.

  17. Functional genetic identification of PRP1, an ABC transporter superfamily member conferring pentamidine resistance in Leishmania major.

    Science.gov (United States)

    Coelho, Adriano C; Beverley, Stephen M; Cotrim, Paulo C

    2003-08-31

    Pentamidine (PEN) is a second-line agent in the treatment of leishmaniasis whose mode of action and resistance is not well understood. Here, we used a genetic strategy to search for loci able to mediate PEN resistance (PENr) when overexpressed in Leishmania major. A shuttle cosmid library containing genomic DNA inserts was transfected into wild-type promastigotes and screened for PEN-resistant transfectants. Two different cosmids identifying the same locus were found, which differed from other known Leishmania drug resistance genes. The PENr gene was mapped by deletion and transposon mutagenesis to an open reading frame (ORF) belonging to the P-glycoprotein (PGP)/MRP ATP-binding cassette (ABC) transporter superfamily that we named pentamidine resistance protein 1 (PRP1). The predicted PRP1 protein encodes 1,807 amino acids with the typical dimeric structure involving 10 transmembrane domains and two nucleotide-binding domains (NBDs). PRP1-mediated PENr could be reversed by verapamil and PRP1 overexpressors showed cross-resistance to trivalent antimony but not to pentavalent antimony (glucantime). Although the degree of PENr was modest (1.7- to 3.7-fold), this may be significant in clinical drug resistance given the marginal efficacy of PEN against Leishmania.

  18. Reciprocal regulation of pyoluteorin production with membrane transporter gene expression in Pseudomonas fluorescens Pf-5.

    Science.gov (United States)

    Brodhagen, Marion; Paulsen, Ian; Loper, Joyce E

    2005-11-01

    Pyoluteorin is a chlorinated polyketide antibiotic secreted by the rhizosphere bacterium Pseudomonas fluorescens Pf-5. Genes encoding enzymes and transcriptional regulators involved in pyoluteorin production are clustered in the genome of Pf-5. Sequence analysis of genes adjacent to the known pyoluteorin biosynthetic gene cluster revealed the presence of an ABC transporter system. We disrupted two putative ABC transporter genes by inserting transcriptional fusions to an ice nucleation reporter gene. Mutations in pltI and pltJ, which are predicted to encode a membrane fusion protein and an ATP-binding cassette of the ABC transporter, respectively, greatly reduced pyoluteorin production by Pf-5. During the transition from exponential growth to stationary phase, populations of a pltI mutant were lower than those of a pltI+ strain in a culture medium containing pyoluteorin, suggesting a role for the transport system in efflux and the resistance of Pf-5 to the antibiotic. Although pltI or pltJ mutant strains displayed low pyoluteorin production, they did not accumulate proportionately more of the antibiotic intracellularly, indicating that pltI and pltJ do not encode an exclusive exporter for pyoluteorin. Transcription of the putative pyoluteorin efflux genes pltI and pltJ was enhanced by exogenous pyoluteorin. These new observations parallel an earlier finding that pyoluteorin enhances the transcription of pyoluteorin biosynthesis genes and pyoluteorin production in Pf-5. This report provides evidence of a coordination of pyoluteorin production and the transcription of genes encoding a linked transport apparatus, wherein each requires the other for optimal expression.

  19. Differential regulation of transport proteins in the periinfarct region following reversible middle cerebral artery occlusion in rats.

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    Dazert, P; Suofu, Y; Grube, M; Popa-Wagner, A; Kroemer, H K; Jedlitschky, G; Kessler, C

    2006-11-03

    Members of various transport protein families including ATP-binding cassette transporters and solute carriers were shown to be expressed in brain capillaries, choroid plexus, astrocytes or neurons, controlling drug and metabolite distribution to and from the brain. However, data are currently very limited on how the expression of these transport systems is affected by damage to the brain such as stroke. Therefore we studied the expression of four selected transporters, P-glycoprotein (Mdr1a/b; Abcb1a/b), Mrp5 (Abcc5), Bcrp (Abcg2), and Oatp2 (Slc21a5) in a rat model for stroke. Transporter expression was analyzed by real-time polymerase chain reaction in the periinfarcted region and protein localization and cellular phenotyping were done by immunohistochemistry and confocal immunofluorescence microscopy. After stroke, P-glycoprotein staining was detected in endothelial cells of disintegrated capillaries and by day 14 in newly generated blood vessels. There was no significant difference, however, in the Mdr1a mRNA amount in the periinfarcted region compared with the contralateral site. For Bcrp, a significant mRNA up-regulation was observed from days 3-14. This up-regulation was followed by the protein as confirmed by quantitative immunohistochemistry. Oatp2, located in the vascular endothelium, was also up-regulated at day 14. For Mrp5, an up-regulation was observed in neurons in the periinfarcted region (day 14). In conclusion, after stroke the transport proteins were up-regulated with a maximum at day 14, a time point that coincides with behavioral recuperation. The study further suggests Bcrp as a pronounced marker for the regenerative process and a possible functional role of Mrp5 in surviving neurons.

  20. Potency of individual bile acids to regulate bile acid synthesis and transport genes in primary human hepatocyte cultures.

    Science.gov (United States)

    Liu, Jie; Lu, Hong; Lu, Yuan-Fu; Lei, Xiaohong; Cui, Julia Yue; Ellis, Ewa; Strom, Stephen C; Klaassen, Curtis D

    2014-10-01

    Bile acids (BAs) are known to regulate their own homeostasis, but the potency of individual bile acids is not known. This study examined the effects of cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA) on expression of BA synthesis and transport genes in human primary hepatocyte cultures. Hepatocytes were treated with the individual BAs at 10, 30, and 100μM for 48 h, and RNA was extracted for real-time PCR analysis. For the classic pathway of BA synthesis, BAs except for UDCA markedly suppressed CYP7A1 (70-95%), the rate-limiting enzyme of bile acid synthesis, but only moderately (35%) down-regulated CYP8B1 at a high concentration of 100μM. BAs had minimal effects on mRNA of two enzymes of the alternative pathway of BA synthesis, namely CYP27A1 and CYP7B1. BAs increased the two major target genes of the farnesoid X receptor (FXR), namely the small heterodimer partner (SHP) by fourfold, and markedly induced fibroblast growth factor 19 (FGF19) over 100-fold. The BA uptake transporter Na(+)-taurocholate co-transporting polypeptide was unaffected, whereas the efflux transporter bile salt export pump was increased 15-fold and OSTα/β were increased 10-100-fold by BAs. The expression of the organic anion transporting polypeptide 1B3 (OATP1B3; sixfold), ATP-binding cassette (ABC) transporter G5 (ABCG5; sixfold), multidrug associated protein-2 (MRP2; twofold), and MRP3 (threefold) were also increased, albeit to lesser degrees. In general, CDCA was the most potent and effective BA in regulating these genes important for BA homeostasis, whereas DCA and CA were intermediate, LCA the least, and UDCA ineffective.

  1. Effect of graded Nrf2 activation on phase-I and -II drug metabolizing enzymes and transporters in mouse liver.

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    Kai Connie Wu

    Full Text Available Nuclear factor erythroid 2-related factor 2 (Nrf2 is a transcription factor that induces a battery of cytoprotective genes in response to oxidative/electrophilic stress. Kelch-like ECH associating protein 1 (Keap1 sequesters Nrf2 in the cytosol. The purpose of this study was to investigate the role of Nrf2 in regulating the mRNA of genes encoding drug metabolizing enzymes and xenobiotic transporters. Microarray analysis was performed in livers of Nrf2-null, wild-type, Keap1-knockdown mice with increased Nrf2 activation, and Keap1-hepatocyte knockout mice with maximum Nrf2 activation. In general, Nrf2 did not have a marked effect on uptake transporters, but the mRNAs of organic anion transporting polypeptide 1a1, sodium taurocholate cotransporting polypeptide, and organic anion transporter 2 were decreased with Nrf2 activation. The effect of Nrf2 on cytochrome P450 (Cyp genes was minimal, with only Cyp2a5, Cyp2c50, Cyp2c54, and Cyp2g1 increased, and Cyp2u1 decreased with enhanced Nrf2 activation. However, Nrf2 increased mRNA of many other phase-I enzymes, such as aldo-keto reductases, carbonyl reductases, and aldehyde dehydrogenase 1. Many genes involved in phase-II drug metabolism were induced by Nrf2, including glutathione S-transferases, UDP- glucuronosyltransferases, and UDP-glucuronic acid synthesis enzymes. Efflux transporters, such as multidrug resistance-associated proteins, breast cancer resistant protein, as well as ATP-binding cassette g5 and g8 were induced by Nrf2. In conclusion, Nrf2 markedly alters hepatic mRNA of a large number of drug metabolizing enzymes and xenobiotic transporters, and thus Nrf2 plays a central role in xenobiotic metabolism and detoxification.

  2. Novel insights in transport mechanisms and kinetics of phenylacetic acid and penicillin-G in Penicillium chrysogenum.

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    Douma, Rutger D; Deshmukh, Amit T; de Jonge, Lodewijk P; de Jong, Bouke W; Seifar, Reza M; Heijnen, Joseph J; van Gulik, Walter M

    2012-01-01

    Although penicillin-G (PenG) production by the fungus Penicillium chrysogenum is a well-studied process, little is known about the mechanisms of transport of the precursor phenylacetic acid (PAA) and the product PenG over the cell membrane. To obtain more insight in the nature of these mechanisms, in vivo stimulus response experiments were performed with PAA and PenG in chemostat cultures of P. chrysogenum at time scales of seconds to minutes. The results indicated that PAA is able to enter the cell by passive diffusion of the undissociated acid at a high rate, but is at the same time actively excreted, possibly by an ATP-binding cassette transporter. This results in a futile cycle, dissipating a significant amount of metabolic energy, which was confirmed by increased rates of substrate and oxygen consumption, and carbon dioxide production. To estimate the kinetic properties of passive import and active export of PAA over the cell membrane, a dynamic mathematical model was constructed. With this model, a good description of the dynamic data could be obtained. Also, PenG was found to be rapidly taken up by the cells upon extracellular addition, indicating that PenG transport is reversible. The measured concentration gradient of PenG over the cell membrane corresponded well with facilitated transport. Also, for PenG transport, a dynamic model was constructed and validated with experimental data. The outcome of the model simulations was in agreement with the presence of a facilitated transport system for PenG.

  3. Hepatic Transporter Expression in Metabolic Syndrome: Phenotype, Serum Metabolic Hormones, and Transcription Factor Expression.

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    Donepudi, Ajay C; Cheng, Qiuqiong; Lu, Zhenqiang James; Cherrington, Nathan J; Slitt, Angela L

    2016-04-01

    Metabolic syndrome is a multifactorial disease associated with obesity, insulin resistance, diabetes, and the alteration of multiple metabolic hormones. Obesity rates have been rising worldwide, which increases our need to understand how this population will respond to drugs and exposure to other chemicals. The purpose of this study was to determine in lean and obese mice the ontogeny of clinical biomarkers such as serum hormone and blood glucose levels as well as the physiologic markers that correlate with nuclear receptor- and transporter-related pathways. Livers from male and female wild-type (WT) (C57BL/6) and ob/ob mice littermates were collected before, during, and after the onset of obesity. Serum hormone and mRNA levels were analyzed. Physiologic changes and gene expression during maturation and progression to obesity were performed and correlation analysis was performed using canonical correlations. Significant ontogenic changes in both WT and ob/ob mice were observed and these ontogenic changes differ in ob/ob mice with the development of obesity. In males and females, the ontogenic pattern of the expression of genes such as Abcc3, 4, Abcg2, Cyp2b10, and 4a14 started to differ from week 3, and became significant at weeks 4 and 8 in ob/ob mice compared with WT mice. In obese males, serum resistin, glucagon, and glucose levels correlated with the expression of most hepatic ATP-binding cassette (Abc) transporters, whereas in obese females, serum glucagon-like peptide 1 levels were correlated with most hepatic uptake transporters and P450 enzymes. Overall, the correlation between physiologic changes and gene expression indicate that metabolism-related hormones may play a role in regulating the genes involved in drug metabolism and transport.

  4. Isolation and Characterization of the Colletotrichum acutatum ABC Transporter CaABC1

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    Suyoung Kim

    2014-12-01

    Full Text Available Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.. Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1 gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5′-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum.

  5. Drug-protein hydrogen bonds govern the inhibition of the ATP hydrolysis of the multidrug transporter P-glycoprotein.

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    Chufan, Eduardo E; Kapoor, Khyati; Ambudkar, Suresh V

    2016-02-01

    P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter superfamily. This multidrug transporter utilizes energy from ATP hydrolysis for the efflux of a variety of hydrophobic and amphipathic compounds including anticancer drugs. Most of the substrates and modulators of P-gp stimulate its basal ATPase activity, although some inhibit it. The molecular mechanisms that are in play in either case are unknown. In this report, mutagenesis and molecular modeling studies of P-gp led to the identification of a pair of phenylalanine-tyrosine structural motifs in the transmembrane region that mediate the inhibition of ATP hydrolysis by certain drugs (zosuquidar, elacridar and tariquidar), with high affinity (IC50's ranging from 10 to 30nM). Upon mutation of any of these residues, drugs that inhibit the ATPase activity of P-gp switch to stimulation of the activity. Molecular modeling revealed that the phenylalanine residues F978 and F728 interact with tyrosine residues Y953 and Y310, respectively, in an edge-to-face conformation, which orients the tyrosines in such a way that they establish hydrogen-bond contacts with the inhibitor. Biochemical investigations along with transport studies in intact cells showed that the inhibitors bind at a high affinity site to produce inhibition of ATP hydrolysis and transport function. Upon mutation, they bind at lower affinity sites, stimulating ATP hydrolysis and only poorly inhibiting transport. These results also reveal that screening chemical compounds for their ability to inhibit the basal ATP hydrolysis can be a reliable tool to identify modulators with high affinity for P-gp.

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

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    Yangfang Li

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

  7. Molecular docking simulations provide insights in the substrate binding sites and possible substrates of the ABCC6 transporter.

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    Mohammad Jakir Hosen

    Full Text Available The human ATP-binding cassette family C member 6 (ABCC6 gene encodes an ABC transporter protein (ABCC6, primarily expressed in liver and kidney. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum (PXE, an autosomal recessive connective tissue disease characterized by ectopic mineralization of the elastic fibers. The pathophysiology underlying PXE is incompletely understood, which can at least partly be explained by the undetermined nature of the ABCC6 substrates as well as the unknown substrate recognition and binding sites. Several compounds, including anionic glutathione conjugates (N-ethylmaleimide; NEM-GS and leukotriene C4 (LTC4 were shown to be modestly transported in vitro; conversely, vitamin K3 (VK3 was demonstrated not to be transported by ABCC6. To predict the possible substrate binding pockets of the ABCC6 transporter, we generated a 3D homology model of ABCC6 in both open and closed conformation, qualified for molecular docking and virtual screening approaches. By docking 10 reported in vitro substrates in our ABCC6 3D homology models, we were able to predict the substrate binding residues of ABCC6. Further, virtual screening of 4651 metabolites from the Human Serum Metabolome Database against our open conformation model disclosed possible substrates for ABCC6, which are mostly lipid and biliary secretion compounds, some of which are found to be involved in mineralization. Docking of these possible substrates in the closed conformation model also showed high affinity. Virtual screening expands this possibility to explore more compounds that can interact with ABCC6, and may aid in understanding the mechanisms leading to PXE.

  8. Differential contributions of five ABC transporters to mutidrug resistance, antioxidion and virulence of Beauveria bassiana, an entomopathogenic fungus.

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    Song, Ting-Ting; Zhao, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    Multidrug resistance (MDR) confers agrochemical compatibility to fungal cells-based mycoinsecticdes but mechanisms involved in MDR remain poorly understood for entomopathogenic fungi, which have been widely applied as biocontrol agents against arthropod pests. Here we characterized the functions of five ATP-binding cassette (ABC) transporters, which were classified to the subfamilies ABC-B (Mdr1), ABC-C (Mrp1) and ABC-G (Pdr1, Pdr2 and Pdr5) and selected from 54 full-size ABC proteins of Beauveria bassiana based on their main domain architecture, membrane topology and transcriptional responses to three antifungal inducers. Disruption of each transporter gene resulted in significant reduction in resistance to four to six of eight fungicides or antifungal drugs tested due to their differences in structure and function. Compared with wild-type and complemented (control) strains, disruption mutants of all the five transporter genes became significantly less tolerant to the oxidants menadione and H₂O₂ based on 22-41% and 10-31% reductions of their effective concentrations required for the suppression of 50% colony growth at 25°C. Under a standardized spray, the killing actions of ΔPdr5 and ΔMrp1 mutants against Spodoptera litura second-instar larvae were delayed by 59% and 33% respectively. However, no significant virulence change was observed in three other delta mutants. Taken together, the examined five ABC transporters contribute differentially to not only the fungal MDR but antioxidant capability, a phenotype rarely associated with ABC efflux pumps in previous reports; at least some of them are required for the full virulence of B. bassiana, thereby affecting the fungal biocontrol potential. Our results indicate that ABC pump-dependent MDR mechanisms exist in entomopathogenic fungi as do in yeasts and human and plant pathogenic fungi.

  9. Differential contributions of five ABC transporters to mutidrug resistance, antioxidion and virulence of Beauveria bassiana, an entomopathogenic fungus.

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    Ting-Ting Song

    Full Text Available Multidrug resistance (MDR confers agrochemical compatibility to fungal cells-based mycoinsecticdes but mechanisms involved in MDR remain poorly understood for entomopathogenic fungi, which have been widely applied as biocontrol agents against arthropod pests. Here we characterized the functions of five ATP-binding cassette (ABC transporters, which were classified to the subfamilies ABC-B (Mdr1, ABC-C (Mrp1 and ABC-G (Pdr1, Pdr2 and Pdr5 and selected from 54 full-size ABC proteins of Beauveria bassiana based on their main domain architecture, membrane topology and transcriptional responses to three antifungal inducers. Disruption of each transporter gene resulted in significant reduction in resistance to four to six of eight fungicides or antifungal drugs tested due to their differences in structure and function. Compared with wild-type and complemented (control strains, disruption mutants of all the five transporter genes became significantly less tolerant to the oxidants menadione and H₂O₂ based on 22-41% and 10-31% reductions of their effective concentrations required for the suppression of 50% colony growth at 25°C. Under a standardized spray, the killing actions of ΔPdr5 and ΔMrp1 mutants against Spodoptera litura second-instar larvae were delayed by 59% and 33% respectively. However, no significant virulence change was observed in three other delta mutants. Taken together, the examined five ABC transporters contribute differentially to not only the fungal MDR but antioxidant capability, a phenotype rarely associated with ABC efflux pumps in previous reports; at least some of them are required for the full virulence of B. bassiana, thereby affecting the fungal biocontrol potential. Our results indicate that ABC pump-dependent MDR mechanisms exist in entomopathogenic fungi as do in yeasts and human and plant pathogenic fungi.

  10. 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

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    Kämmerer Ines

    2011-11-01

    Full Text Available Abstract Background Synthetic activators of peroxisome proliferator-activated receptors (PPARs stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1 and scavenger receptor class B type 1 (SR-BI. The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA, 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages. Methods 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. Results 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 Conclusion 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.

  11. Prolonged Oral Administration of a Pan-Retinoic Acid Receptor Antagonist Inhibits Spermatogenesis in Mice With a Rapid Recovery and Changes in the Expression of Influx and Efflux Transporters.

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    Chung, Sanny S W; Wang, Xiangyuan; Wolgemuth, Debra J

    2016-04-01

    We have previously shown that oral administration of a pan-retinoic acid receptor antagonist in mice daily at 2.5 mg/kg for 4 weeks reversibly inhibited spermatogenesis, with no detectable side effects. To elucidate the lowest dose and the longest dosing regimen that inhibits spermatogenesis but results in complete restoration of fertility upon cessation of administration of the drug, we examined the effects of daily doses as low as 1.0 mg/kg with dosing periods of 4, 8, and 16 weeks. We observed 100% sterility in all regimens, with restoration of fertility upon cessation of the drug treatment even for as long as 16 weeks. There was no change in testosterone levels in these males and the progeny examined from 2 of the recovered males were healthy and fertile, with normal testicular weight and testicular histology. Strikingly, a more rapid recovery, as assessed by mating studies, was observed at the lower dose and longer dosing periods. Insight into possible mechanisms underlying this rapid recovery was obtained at 2 levels. First, histological examination revealed that spermatogenesis was not as severely disrupted at the lower dose and with the longer treatment regimens. Second, gene expression analysis revealed that the more rapid recovery may involve the interplay of ATP-binding cassette efflux and solute carrier influx transporters in the testes.

  12. Inhibition of Human Drug Transporter Activities by the Pyrethroid Pesticides Allethrin and Tetramethrin

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    Chedik, Lisa; Bruyere, Arnaud; Le Vee, Marc; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Potin, Sophie; Fardel, Olivier

    2017-01-01

    Pyrethroids are widely-used chemical insecticides, to which humans are commonly exposed, and known to alter functional expression of drug metabolizing enzymes. Limited data have additionally suggested that drug transporters, that constitute key-actors of the drug detoxification system, may also be targeted by pyrethroids. The present study was therefore designed to analyze the potential regulatory effects of these pesticides towards activities of main ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, using transporter-overexpressing cells. The pyrethroids allethrin and tetramethrin were found to inhibit various ABC and SLC drug transporters, including multidrug resistance-associated protein (MRP) 2, breast cancer resistance protein (BCRP), organic anion transporter polypeptide (OATP) 1B1, organic anion transporter (OAT) 3, multidrug and toxin extrusion transporter (MATE) 1, organic cation transporter (OCT) 1 and OCT2, with IC50 values however ranging from 2.6 μM (OCT1 inhibition by allethrin) to 77.6 μM (OAT3 inhibition by tetramethrin) and thus much higher than pyrethroid concentrations (in the nM range) reached in environmentally pyrethroid-exposed humans. By contrast, allethrin and tetramethrin cis-stimulated OATP2B1 activity and failed to alter activities of OATP1B3, OAT1 and MATE2-K, whereas P-glycoprotein activity was additionally moderately inhibited. Twelve other pyrethoids used at 100 μM did not block activities of the various investigated transporters, or only moderately inhibited some of them (inhibition by less than 50%). In silico analysis of structure-activity relationships next revealed that molecular parameters, including molecular weight and lipophilicity, are associated with transporter inhibition by allethrin/tetramethrin and successfully predicted transporter inhibition by the pyrethroids imiprothrin and prallethrin. Taken together, these data fully demonstrated that two pyrethoids, i.e., allethrin and tetramethrin, can

  13. Regulation of human hepatic drug transporter activity and expression by diesel exhaust particle extract.

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    Marc Le Vee

    Full Text Available Diesel exhaust particles (DEPs are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC uptake transporters organic anion-transporting polypeptides (OATP 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP, whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP. Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a reference activator of the aryl hydrocarbon receptor (AhR pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute

  14. CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

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    Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.; Mori, Tetsuya; Qin, Ximing; Johnson, Carl H.; Egli, Martin; Stewart, Phoebe L. [Case Western; (Vanderbilt); (Vanderbilt-MED)

    2014-10-02

    The circadian control of cellular processes in cyanobacteria is regulated by a posttranslational oscillator formed by three Kai proteins. During the oscillator cycle, KaiA serves to promote autophosphorylation of KaiC while KaiB counteracts this effect. Here, we present a crystallographic structure of the wild-type Synechococcus elongatus KaiB and a cryo-electron microscopy (cryoEM) structure of a KaiBC complex. The crystal structure shows the expected dimer core structure and significant conformational variations of the KaiB C-terminal region, which is functionally important in maintaining rhythmicity. The KaiBC sample was formed with a C-terminally truncated form of KaiC, KaiC-Δ489, which is persistently phosphorylated. The KaiB–KaiC-Δ489 structure reveals that the KaiC hexamer can bind six monomers of KaiB, which form a continuous ring of density in the KaiBC complex. We performed cryoEM-guided molecular dynamics flexible fitting simulations with crystal structures of KaiB and KaiC to probe the KaiBC protein–protein interface. This analysis indicated a favorable binding mode for the KaiB monomer on the CII end of KaiC, involving two adjacent KaiC subunits and spanning an ATP binding cleft. A KaiC mutation, R468C, which has been shown to affect the affinity of KaiB for KaiC and lengthen the period in a bioluminescence rhythm assay, is found within the middle of the predicted KaiBC interface. The proposed KaiB binding mode blocks access to the ATP binding cleft in the CII ring of KaiC, which provides insight into how KaiB might influence the phosphorylation status of KaiC.

  15. Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.

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    Yilmaz, Mehtap; Gangopadhyay, Samudra S; Leavis, Paul; Grabarek, Zenon; Morgan, Kathleen G

    2013-02-07

    CaMKII (Ca²⁺/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The γ isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII γ at Ser²⁶, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser²⁶, we generated a phosphospecific Ser²⁶ antibody and demonstrated an increase in Ser²⁶ phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser²⁶ affects the kinase activity, we mutated Ser²⁶ to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr²⁸⁷ autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser²⁶ of CaMKII γ inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr²⁸⁷ most probably by blocking ATP binding. We propose that Ser²⁶ phosphorylation constitutes an important mechanism for switching off CaMKII activity.

  16. Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11 and MDR3 (ABCB4 expressed in the yeast Pichia pastoris.

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    Philipp Ellinger

    Full Text Available The human liver ATP-binding cassette (ABC transporters bile salt export pump (BSEP/ABCB11 and the multidrug resistance protein 3 (MDR3/ABCB4 fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

  17. The role of the iron transporter ABCB7 in refractory anemia with ring sideroblasts.

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    Jacqueline Boultwood

    Full Text Available Refractory Anemia with Ring Sideroblasts (RARS is an acquired myelodysplastic syndrome (MDS characterized by an excess iron accumulation in the mitochondria of erythroblasts. The pathogenesis of RARS and the cause of this unusual pattern of iron deposition remain unknown. We considered that the inherited X-linked sideroblastic anemia with ataxia (XLSA/A might be informative for the acquired disorder, RARS. XLSA/A is caused by partial inactivating mutations of the ABCB7 ATP-binding cassette transporter gene, which functions to enable transport of iron from the mitochondria to the cytoplasm. Furthermore, ABCB7 gene silencing in HeLa cells causes an accumulation of iron in the mitochondria. We have studied the role of ABCB7 in RARS by DNA sequencing, methylation studies, and gene expression studies in primary CD34(+ cells and in cultured erythroblasts. The DNA sequence of the ABCB7 gene is normal in patients with RARS. We have investigated ABCB7 gene expression levels in the CD34(+ cells of 122 MDS cases, comprising 35 patients with refractory anemia (RA, 33 patients with RARS and 54 patients with RA with excess blasts (RAEB, and in the CD34(+ cells of 16 healthy controls. We found that the expression levels of ABCB7 are significantly lower in the RARS group. RARS is thus characterized by lower levels of ABCB7 gene expression in comparison to other MDS subtypes. Moreover, we find a strong relationship between increasing percentage of bone marrow ring sideroblasts and decreasing ABCB7 gene expression levels. Erythroblast cell cultures confirm the low levels of ABCB7 gene expression levels in RARS. These data provide an important link between inherited and acquired forms of sideroblastic anemia and indicate that ABCB7 is a strong candidate gene for RARS.

  18. The role of sphingosine-1-phosphate transporter Spns2 in immune system function.

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    Nijnik, Anastasia; Clare, Simon; Hale, Christine; Chen, Jing; Raisen, Claire; Mottram, Lynda; Lucas, Mark; Estabel, Jeanne; Ryder, Edward; Adissu, Hibret; Adams, Niels C; Ramirez-Solis, Ramiro; White, Jacqueline K; Steel, Karen P; Dougan, Gordon; Hancock, Robert E W

    2012-07-01

    Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm1a(KOMP)Wtsi) allele (Spns2(tm1a)). The Spns2(tm1a/tm1a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm1a/tm1a) mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm1a/tm1a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.

  19. A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain

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    Song, Won-Yong; Yamaki, Tomohiro; Yamaji, Naoki; Ko, Donghwi; Jung, Ki-Hong; Fujii-Kashino, Miho; An, Gynheung; Martinoia, Enrico; Lee, Youngsook; Ma, Jian Feng

    2014-01-01

    Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice. PMID:25331872

  20. The Role of the Iron Transporter ABCB7 in Refractory Anemia with Ring Sideroblasts

    Science.gov (United States)

    Nikpour, Maryam; Pushkaran, Beena; Fidler, Carrie; Cattan, Helen; Littlewood, Tim J.; Malcovati, Luca; Della Porta, Matteo G.; Jädersten, Martin; Killick, Sally; Giagounidis, Aristoteles; Bowen, David; Hellström-Lindberg, Eva; Cazzola, Mario; Wainscoat, James S.

    2008-01-01

    Refractory Anemia with Ring Sideroblasts (RARS) is an acquired myelodysplastic syndrome (MDS) characterized by an excess iron accumulation in the mitochondria of erythroblasts. The pathogenesis of RARS and the cause of this unusual pattern of iron deposition remain unknown. We considered that the inherited X-linked sideroblastic anemia with ataxia (XLSA/A) might be informative for the acquired disorder, RARS. XLSA/A is caused by partial inactivating mutations of the ABCB7 ATP-binding cassette transporter gene, which functions to enable transport of iron from the mitochondria to the cytoplasm. Furthermore, ABCB7 gene silencing in HeLa cells causes an accumulation of iron in the mitochondria. We have studied the role of ABCB7 in RARS by DNA sequencing, methylation studies, and gene expression studies in primary CD34+ cells and in cultured erythroblasts. The DNA sequence of the ABCB7 gene is normal in patients with RARS. We have investigated ABCB7 gene expression levels in the CD34+ cells of 122 MDS cases, comprising 35 patients with refractory anemia (RA), 33 patients with RARS and 54 patients with RA with excess blasts (RAEB), and in the CD34+ cells of 16 healthy controls. We found that the expression levels of ABCB7 are significantly lower in the RARS group. RARS is thus characterized by lower levels of ABCB7 gene expression in comparison to other MDS subtypes. Moreover, we find a strong relationship between increasing percentage of bone marrow ring sideroblasts and decreasing ABCB7 gene expression levels. Erythroblast cell cultures confirm the low levels of ABCB7 gene expression levels in RARS. These data provide an important link between inherited and acquired forms of sideroblastic anemia and indicate that ABCB7 is a strong candidate gene for RARS. PMID:18398482

  1. Inhibition of soluble epoxide hydrolase in mice promotes reverse cholesterol transport and regression of atherosclerosis.

    Science.gov (United States)

    Shen, Li; Peng, Hongchun; Peng, Ran; Fan, Qingsong; Zhao, Shuiping; Xu, Danyan; Morisseau, Christophe; Chiamvimonvat, Nipavan; Hammock, Bruce D

    2015-04-01

    Adipose tissue is the body largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), which maintains plasma high-density lipoprotein (HDL) levels. HDLs have a protective role in atherosclerosis by mediating reverse cholesterol transport (RCT). Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition has various beneficial effects on cardiovascular disease. The sEH is highly expressed in adipocytes, and it converts epoxyeicosatrienoic acids (EETs) into less bioactive dihydroxyeicosatrienoic acids. We previously showed that increasing EETs levels with a sEH inhibitor (sEHI) (t-AUCB) resulted in elevated ABCA1 expression and promoted ABCA1-mediated cholesterol efflux from 3T3-L1 adipocytes. The present study investigates the impacts of t-AUCB in mice deficient for the low density lipoprotein (LDL) receptor (Ldlr(-/-) mice) with established atherosclerotic plaques. The sEH inhibitor delivered in vivo for 4 weeks decreased the activity of sEH in adipose tissue, enhanced ABCA1 expression and cholesterol efflux from adipose depots, and consequently increased HDL levels. Furthermore, t-AUCB enhanced RCT to the plasma, liver, bile and feces. It also showed the reduction of plasma LDL-C levels. Consistently, t-AUCB-treated mice showed reductions in the size of atherosclerotic plaques. These studies establish that raising adipose ABCA1 expression, cholesterol efflux, and plasma HDL levels with t-AUCB treatment promotes RCT, decreasing LDL-C and atherosclerosis regression, suggesting that sEH inhibition may be a promising strategy to treat atherosclerotic vascular disease.

  2. EPR spectroscopy of MolB2C2-a reveals mechanism of transport for a bacterial type II molybdate importer.

    Science.gov (United States)

    Rice, Austin J; Alvarez, Frances J D; Schultz, Kathryn M; Klug, Candice S; Davidson, Amy L; Pinkett, Heather W

    2013-07-19

    In bacteria, ATP-binding cassette (ABC) transporters are vital for the uptake of nutrients and cofactors. Based on differences in structure and activity, ABC importers are divided into two types. Type I transporters have been well studied and employ a tightly regulated alternating access mechanism. Less is known about Type II importers, but much of what we do know has been observed in studies of the vitamin B12 importer BtuC2D2. MolB2C2 (formally known as HI1470/71) is also a Type II importer, but its substrate, molybdate, is ∼10-fold smaller than vitamin B12. To understand mechanistic differences among Type II importers, we focused our studies on MolBC, for which alternative conformations may be required to transport its relatively small substrate. To investigate the mechanism of MolBC, we employed disulfide cross-linking and EPR spectroscopy. From these studies, we found that nucleotide binding is coupled to a conformational shift at the periplasmic gate. Unlike the larger conformational changes in BtuCD-F, this shift in MolBC-A is akin to unlocking a swinging door: allowing just enough space for molybdate to slip into the cell. The lower cytoplasmic gate, identified in BtuCD-F as "gate I," remains open throughout the MolBC-A mechanism, and cytoplasmic gate II closes in the presence of nucleotide. Combining our results, we propose a peristaltic mechanism for MolBC-A, which gives new insight in the transport of small substrates by a Type II importer.

  3. Effect of (mixtures of) flavonoids on the in vitro and in vivo bioavailability of 2-mino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) : a biologically based modelling approach

    NARCIS (Netherlands)

    Schutte, M.E.

    2007-01-01

    The transport of food ingredients across the intestinal epithelium is an important factor determining the absorption upon oral intake. Uptake of compounds in the intestine may be influenced by transport proteins such as the ATP binding cassette transporters (ABC transporters). ABC transporters have

  4. Mutations at the Signature Sequence of CFTR Create a Cd2+-gated Chloride Channel

    OpenAIRE

    Wang, Xiaohui; Bompadre, Silvia G.; Li, Min; Hwang, Tzyh-Chang

    2009-01-01

    The canonical sequence LSGGQ, also known as the signature sequence, defines the adenosine triphosphate (ATP)-binding cassette transporter superfamily. Crystallographic studies reveal that the signature sequence, together with the Walker A and Walker B motifs, forms the ATP-binding pocket upon dimerization of the two nucleotide-binding domains (NBDs) in a head-to-tail configuration. The importance of the signature sequence is attested by the fact that a glycine to aspartate mutation (i.e., G55...

  5. The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea.

    Science.gov (United States)

    Dubey, Mukesh; Jensen, Dan Funck; Karlsson, Magnus

    2016-04-01

    For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

  6. Role of MRP transporters in regulating antimicrobial drug inefficacy and oxidative stress-induced pathogenesis during HIV-1 and TB infections.

    Science.gov (United States)

    Roy, Upal; Barber, Paul; Tse-Dinh, Yuk-Ching; Batrakova, Elena V; Mondal, Debasis; Nair, Madhavan

    2015-01-01

    Multi-Drug Resistance Proteins (MRPs) are members of the ATP binding cassette (ABC) drug-efflux transporter superfamily. MRPs are known to regulate the efficacy of a broad range of anti-retroviral drugs (ARV) used in highly active antiretroviral therapy (HAART) and antibacterial agents used in Tuberculus Bacilli (TB) therapy. Due to their role in efflux of glutathione (GSH) conjugated drugs, MRPs can also regulate cellular oxidative stress, which may contribute to both HIV and/or TB pathogenesis. This review focuses on the characteristics, functional expression, and modulation of known members of the MRP family in HIV infected cells exposed to ARV drugs and discusses their known role in drug-inefficacy in HIV/TB-induced dysfunctions. Currently, nine members of the MRP family (MRP1-MRP9) have been identified, with MRP1 and MRP2 being the most extensively studied. Details of the other members of this family have not been known until recently, but differential expression has been documented in inflammatory tissues. Researchers have found that the distribution, function, and reactivity of members of MRP family vary in different types of lymphocytes and macrophages, and are differentially expressed at the basal and apical surfaces of both endothelial and epithelial cells. Therefore, the prime objective of this review is to delineate the role of MRP transporters in HAART and TB therapy and their potential in precipitating cellular dysfunctions manifested in these chronic infectious diseases. We also provide an overview of different available options and novel experimental strategies that are being utilized to overcome the drug resistance and disease pathogenesis mediated by these membrane transporters.

  7. Role of MRP Transporters in Regulating Antimicrobial Drug Inefficacy and Oxidative Stress-induced Pathogenesis during HIV-1 and TB Infections

    Directory of Open Access Journals (Sweden)

    Upal eRoy

    2015-09-01

    Full Text Available Multi-Drug Resistance Proteins (MRPs are members of the ATP binding cassette (ABC drug-efflux transporter superfamily. MRPs are known to regulate the efficacy of a broad range of anti-retroviral drugs (ARV used in highly active antiretroviral therapy (HAART and antibacterial agents used in Tuberculus Bacilli (TB therapy. Due to their role in efflux of glutathione (GSH conjugated drugs, MRPs can also regulate cellular oxidative stress, which may contribute to both HIV and/or TB pathogenesis. This review focuses on the characteristics, functional expression, and modulation of known members of the MRP family in HIV infected cells exposed to ARV drugs and discusses their known role in drug-inefficacy in HIV/TB-induced dysfunctions. Currently, nine members of the MRP family (MRP1-MRP9 have been identified, with MRP1 and MRP2 being the most extensively studied. Details of the other members of this family have not been known until recently, but differential expression has been documented in inflammatory tissues. Researchers have found that the distribution, function and reactivity of members of MRP family vary in different types of lymphocytes and macrophages, and are differentially expressed at the basal and apical surfaces of both endothelial and epithelial cells. Therefore, the prime objective of this review is to delineate the role of MRP transporters in HAART and TB therapy and their potential in precipitating cellular dysfunctions manifested in these chronic infectious diseases. We also provide an overview of different available options and novel experimental strategies that are being utilized to overcome the drug resistance and disease pathogenesis mediated by these membrane transporters.

  8. Cholesterol-lowering activity of sesamin is associated with down-regulation on genes of sterol transporters involved in cholesterol absorption.

    Science.gov (United States)

    Liang, Yin Tong; Chen, Jingnan; Jiao, Rui; Peng, Cheng; Zuo, Yuanyuan; Lei, Lin; Liu, Yuwei; Wang, Xiaobo; Ma, Ka Ying; Huang, Yu; Chen, Zhen-Yu

    2015-03-25

    Sesame seed is rich in sesamin. The present study was to (i) investigate the plasma cholesterol-lowering activity of dietary sesamin and (ii) examine the interaction of dietary sesamin with the gene expression of sterol transporters, enzymes, receptors, and proteins involved in cholesterol metabolism. Thirty hamsters were divided into three groups fed the control diet (CON) or one of two experimental diets containing 0.2% (SL) and 0.5% (SH) sesamin, respectively, for 6 weeks. Plasma total cholesterol (TC) levels in hamsters given the CON, SL, and SH diets were 6.62 ± 0.40, 5.32 ± 0.40, and 5.00 ± 0.44 mmol/L, respectively, indicating dietary sesamin could reduce plasma TC in a dose-dependent manner. Similarly, the excretion of total fecal neutral sterols was dose-dependently increased with the amounts of sesamin in diets (CON, 2.65 ± 0.57; SL, 4.30 ± 0.65; and SH, 5.84 ± 1.27 μmol/day). Addition of sesamin into diets was associated with down-regulation of mRNA of intestinal Niemann-Pick C1 like 1 protein (NPC1L1), acyl-CoA:cholesterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP-binding cassette transporters subfamily G members 5 and 8 (ABCG5 and ABCG8). Results also showed that dietary sesamin could up-regulate hepatic cholesterol-7α-hydroxylase (CYP7A1), whereas it down-regulated hepatic 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and liver X receptor alpha (LXRα). It was concluded that the cholesterol-lowering activity of sesamin was mediated by promoting the fecal excretion of sterols and modulating the genes involved in cholesterol absorption and metabolism.

  9. Structure and Metal Binding Properties of ZnuA, a Periplasmic Zinc Transporter from Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Yatsunyk,L.; Easton, J.; Kim, L.; Sugarbaker, S.; Bennett, B.; Breece, R.; Vorontsov, I.; Tierney, D.; Crowder, M.; Rosenzweig, A.

    2008-01-01

    ZnuA is the periplasmic Zn(2+)-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn(2+)-bound, and Co(2+)-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn(2+) with Co(2+) results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn(2+) periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn(2+) (estimated K (d) < 20 nM), Co(2+), Ni(2+), Cu(2+), Cu(+), and Cd(2+), but not Mn(2+). Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn(2+) substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer.

  10. Pitavastatin Differentially Modulates MicroRNA-Associated Cholesterol Transport Proteins in Macrophages.

    Directory of Open Access Journals (Sweden)

    Haijun Zhang

    Full Text Available There is emerging evidence identifying microRNAs (miRNAs as mediators of statin-induced cholesterol efflux, notably through the ATP-binding cassette transporter A1 (ABCA1 in macrophages. The objective of this study was to assess the impact of an HMG-CoA reductase inhibitor, pitavastatin, on macrophage miRNAs in the presence and absence of oxidized-LDL, a hallmark of a pro-atherogenic milieu. Treatment of human THP-1 cells with pitavastatin prevented the oxLDL-mediated suppression of miR-33a, -33b and -758 mRNA in these cells, an effect which was not uniquely attributable to induction of SREBP2. Induction of ABCA1 mRNA and protein by oxLDL was inhibited (30% by pitavastatin, while oxLDL or pitavastatin alone significantly induced and repressed ABCA1 expression, respectively. These findings are consistent with previous reports in macrophages. miRNA profiling was also performed using a miRNA array. We identified specific miRNAs which were up-regulated (122 and down-regulated (107 in THP-1 cells treated with oxLDL plus pitavastatin versus oxLDL alone, indicating distinct regulatory networks in these cells. Moreover, several of the differentially expressed miRNAs identified are functionally associated with cholesterol trafficking (six miRNAs in cells treated with oxLDL versus oxLDL plus pitavastatin. Our findings indicate that pitavastatin can differentially modulate miRNA in the presence of oxLDL; and, our results provide evidence that the net effect on cholesterol homeostasis is mediated by a network of miRNAs.

  11. Enterococcus faecalis uses a PTS permease and a host colonization-related ABC transporter for maltodextrin uptake.

    Science.gov (United States)

    Sauvageot, Nicolas; Mokhtari, Abdelhamid; Joyet, Philippe; Budin-Verneuil, Aurélie; Blancato, Víctor S; Repizo, Guillermo D; Henry, Céline; Pikis, Andreas; Thompson, John; Magni, Christian; Hartke, Axel; Deutscher, Josef

    2017-02-27

    Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6- linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG/MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT/mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6' -P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose and we show for the first time that in contrast to maltotriose its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG/MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, major health-care associated pathogens, are difficult to treat due to their increasing resistance to clinically relevant antibiotics and new

  12. Gene expression profiling of cytochromes P450, ABC transporters and their principal transcription factors in the amygdala and prefrontal cortex of alcoholics, smokers and drug-free controls by qRT-PCR.

    Science.gov (United States)

    Toselli, Francesca; de Waziers, Isabelle; Dutheil, Mary; Vincent, Marc; Wilce, Peter A; Dodd, Peter R; Beaune, Philippe; Loriot, Marie-Anne; Gillam, Elizabeth M J

    2015-01-01

    1. Ethanol consumption and smoking alter the expression of certain drug-metabolizing enzymes and transporters, potentially influencing the tissue-specific effects of xenobiotics. 2. Amygdala (AMG) and prefrontal cortex (PFC) are brain regions that modulate the effects of alcohol and smoking, yet little is known about the expression of cytochrome P450 enzymes (P450s) and ATP-binding cassette (ABC) transporters in these tissues. 3. Here, we describe the first study on the expression of 19 P450s, their redox partners, three ABC transporters and four related transcription factors in the AMG and PFC of smokers and alcoholics by quantitative RT-PCR. 4. CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, adrenodoxin and NADPH-P450 reductase, ABCB1, ABCG2, ABCA1, and transcription factors aryl hydrocarbon receptor AhR and proliferator-activated receptor α were quantified in both areas. CYP2A6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, adrenodoxin reductase and the nuclear receptors pregnane X receptor and constitutive androstane receptor were detected but below the limit of quantification. CYP1A2 and CYP2W1 were not detected. 5. Adrenodoxin expression was elevated in all case groups over controls, and smokers showed a trend toward higher CYP1A1 and CYP1B1 expression. 6. Our study shows that most xenobiotic-metabolizing P450s and associated redox partners, transporters and transcription factors are expressed in human AMG and PFC.

  13. Human small cell lung cancer NYH cells selected for resistance to the bisdioxopiperazine topoisomerase II catalytic inhibitor ICRF-187 demonstrate a functional R162Q mutation in the Walker A consensus ATP binding domain of the alpha isoform

    DEFF Research Database (Denmark)

    Wessel, I; Jensen, L H; Jensen, P B;

    1999-01-01

    -AMSA), which act by stabilizing enzyme-DNA-drug complexes at a stage in which the DNA gate strand is cleaved and the protein is covalently attached to DNA. Human small cell lung cancer NYH cells selected for resistance to ICRF-187 (NYH/187) showed a 25% increase in topoisomerase IIalpha level and no change...... in expression of the beta isoform. Sequencing of the entire topoisomerase IIalpha cDNA from NYH/187 cells demonstrated a homozygous G-->A point mutation at nucleotide 485, leading to a R162Q conversion in the Walker A consensus ATP binding site (residues 161-165 in the alpha isoform), this being the first drug......-selected mutation described at this site. Western blotting after incubation with ICRF-187 showed no depletion of the alpha isoform in NYH/187 cells in contrast to wild-type (wt) cells, whereas equal depletion of the beta isoform was observed in the two sublines. Alkaline elution assay demonstrated a lack...

  14. 4,6-Substituted-1,3,5-triazin-2(1H)-ones as monocyclic catalytic inhibitors of human DNA topoisomerase IIα targeting the ATP binding site.

    Science.gov (United States)

    Pogorelčnik, Barbara; Janežič, Matej; Sosič, Izidor; Gobec, Stanislav; Solmajer, Tom; Perdih, Andrej

    2015-08-01

    Human DNA topoisomerase IIα (htIIα) is a validated target for the development of novel anticancer agents. Starting from our discovered 4-amino-1,3,5-triazine inhibitors of htIIα, we investigated a library of 2,4,6-trisubstituted-1,3,5-triazines for novel inhibitors that bind to the htIIα ATP binding site using a combination of structure-based and ligand-based pharmacophore models and molecular docking. 4,6-substituted-1,3,5-triazin-2(1H)-ones 8, 9 and 14 were identified as novel inhibitors with activity comparable to the established drug etoposide (1). Compound 8 inhibits the htIIα decatenation in a superior fashion to etoposide. Cleavage assays demonstrated that selected compounds 8 and 14 do not act as poisons and antagonize the poison effect of etoposide. Microscale thermophoresis (MST) confirmed binding of compound 8 to the htIIα ATPase domain and compound 14 effectively inhibits the htIIα mediated ATP hydrolysis. The molecular dynamics simulation study provides further insight into the molecular recognition. The 4,6-disubstituted-1,3,5-triazin-2(1H)-ones represent the first validated monocyclic class of catalytic inhibitors that bind to the to the htIIα ATPase domain.

  15. Involvement of the carboxyl-terminal region of the yeast peroxisomal half ABC transporter Pxa2p in its interaction with Pxa1p and in transporter function.

    Directory of Open Access Journals (Sweden)

    Cheng-Yi Chuang

    Full Text Available BACKGROUND: The peroxisome is a single membrane-bound organelle in eukaryotic cells involved in lipid metabolism, including β-oxidation of fatty acids. The human genetic disorder X-linked adrenoleukodystrophy (X-ALD is caused by mutations in the ABCD1 gene (encoding ALDP, a peroxisomal half ATP-binding cassette [ABC] transporter. This disease is characterized by defective peroxisomal β-oxidation and a large accumulation of very long-chain fatty acids in brain white matter, adrenal cortex, and testis. ALDP forms a homodimer proposed to be the functional transporter, whereas the peroxisomal transporter in yeast is a heterodimer comprising two half ABC transporters, Pxa1p and Pxa2p, both orthologs of human ALDP. While the carboxyl-terminal domain of ALDP is engaged in dimerization, it remains unknown whether the same region is involved in the interaction between Pxa1p and Pxa2p. METHODS/PRINCIPAL FINDINGS: Using a yeast two-hybrid assay, we found that the carboxyl-terminal region (CT of Pxa2p, but not of Pxa1p, is required for their interaction. Further analysis indicated that the central part of the CT (designated CT2 of Pxa2p was indispensable for its interaction with the carboxyl terminally truncated Pxa1_NBD. An interaction between the CT of Pxa2p and Pxa1_NBD was not detected, but could be identified in the presence of Pxa2_NBD-CT1. A single mutation of two conserved residues (aligned with X-ALD-associated mutations at the same positions in ALDP in the CT2 of the Pxa2_NBD-CT protein impaired its interaction with Pxa1_NBD or Pxa1_NBD-CT, resulting in a mutant protein that exhibited a proteinase K digestion profile different from that of the wild-type protein. Functional analysis of these mutant proteins on oleate plates indicated that they were defective in transporter function. CONCLUSIONS/SIGNIFICANCE: The CT of Pxa2p is involved in its interaction with Pxa1p and in transporter function. This concept may be applied to human ALDP studies

  16. Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation

    Directory of Open Access Journals (Sweden)

    Teixeira Miguel C

    2012-07-01

    Full Text Available Abstract Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC Superfamily and Major Facilitator Superfamily (MFS in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to

  17. Effects of triclabendazole on secretion of danofloxacin and moxidectin into the milk of sheep: role of triclabendazole metabolites as inhibitors of the ruminant ABCG2 transporter.

    Science.gov (United States)

    Barrera, Borja; González-Lobato, Lucía; Otero, Jon A; Real, Rebeca; Prieto, Julio G; Álvarez, Ana I; Merino, Gracia

    2013-11-01

    ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) mediates drug-drug interactions that affect the secretion of drugs into milk. The aims of this study were: (1) to determine whether the major plasma metabolites of the flukicide triclabendazole (TCBZ), triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO2), inhibit ovine and bovine ABCG2 and its Y581S variant in vitro, and (2) to examine whether coadministration of TCBZ with the ABCG2 substrates danofloxacin (a fluoroquinolone) and moxidectin (a milbemycin) affects the secretion of these drugs into the milk of sheep. TCBZSO and TCBZSO2 inhibited ruminant ABCG2 in vitro by reversing the reduced mitoxantrone accumulation and reducing basal to apical transport of nitrofurantoin in cells transduced with bovine variants (S581 and Y581) and the ovine variant of ABCG2. Coadministration of TCBZ with moxidectin or danofloxacin to sheep resulted in significantly reduced levels of moxidectin, but not danofloxacin, in the milk of TCBZ-treated sheep compared to sheep administered moxidectin or danofloxacin alone. The milk area under concentration time curve (AUC 0-48 h) was 2.99±1.41 μg h/mL in the group treated with TCBZ and moxidectin, and 7.75±3.58 μg h/mL in the group treated with moxidectin alone. The AUC (0-48 h) milk/plasma ratio was 37% lower in the group treated with TCBZ and moxidectin (7.34±1.51) than in the group treated with moxidectin alone (11.68±3.61). TCBZ metabolites appear to inhibit ruminant ABCG2 and affect the secretion of ABCG2 substrates into milk of sheep.

  18. Inhibition of the Human ABC Efflux Transporters P-gp and BCRP by the BDE-47 Hydroxylated Metabolite 6-OH-BDE-47: Considerations for Human Exposure.

    Science.gov (United States)

    Marchitti, Satori A; Mazur, Christopher S; Dillingham, Caleb M; Rawat, Swati; Sharma, Anshika; Zastre, Jason; Kenneke, John F

    2017-01-01

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [((3)H)-paclitaxel and ((3)H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity. These findings suggest that some effects previously attributed to BDE-47 in biological systems may actually be due to 6-OH-BDE-47. Considerations for human exposure are discussed.

  19. Gene amplification, ABC transporters and cytochrome P450s: unraveling the molecular basis of pyrethroid resistance in the dengue vector, Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Vassiliki Bariami

    Full Text Available BACKGROUND: Pyrethroid insecticides are widely utilized in dengue control. However, the major vector, Aedes aegypti, is becoming increasingly resistant to these insecticides and this is impacting on the efficacy of control measures. The near complete transcriptome of two pyrethroid resistant populations from the Caribbean was examined to explore the molecular basis of this resistance. PRINCIPAL FINDINGS: Two previously described target site mutations, 1016I and 1534C were detected in pyrethroid resistant populations from Grand Cayman and Cuba. In addition between two and five per cent of the Ae. aegypti transcriptome was differentially expressed in the resistant populations compared to a laboratory susceptible population. Approximately 20 per cent of the genes over-expressed in resistant mosquitoes were up-regulated in both Caribbean populations (107 genes. Genes with putative monooxygenase activity were significantly over represented in the up-regulated subset, including five CYP9 P450 genes. Quantitative PCR was used to confirm the higher transcript levels of multiple cytochrome P450 genes from the CYP9J family and an ATP binding cassette transporter. Over expression of two genes, CYP9J26 and ABCB4, is due, at least in part, to gene amplification. SIGNIFICANCE: These results, and those from other studies, strongly suggest that increases in the amount of the CYP9J cytochrome P450s are an important mechanism of pyrethroid resistance in Ae. aegypti. The genetic redundancy resulting from the expansion of this gene family makes it unlikely that a single gene or mutation responsible for pyrethroid resistance will be identified in this mosquito species. However, the results from this study do pave the way for the development of new pyrethroid synergists and improved resistance diagnostics. The role of copy number polymorphisms in detoxification and transporter genes in providing protection against insecticide exposure requires further investigation.

  20. The E3 Ubiquitin Ligases, HUWE1 and NEDD4-1, Are Involved in the Post-translational Regulation of the ABCG1 and ABCG4 Lipid Transporters*

    Science.gov (United States)

    Aleidi, Shereen M.; Howe, Vicky; Sharpe, Laura J.; Yang, Alryel; Rao, Geetha; Brown, Andrew J.; Gelissen, Ingrid C.

    2015-01-01

    The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity. PMID:26296893

  1. Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales

    Directory of Open Access Journals (Sweden)

    Gogarten J Peter

    2008-01-01

    Full Text Available Abstract Background The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. Results We demonstrate that the two maltose ATP binding cassette (ABC transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to

  2. Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli

    OpenAIRE

    Pougach, Ksenia; Semenova, Ekaterina; Bogdanova, Ekaterina; Datsenko, Kirill A; Djordjevic, Marko; Wanner, Barry L.; Severinov, Konstantin

    2010-01-01

    CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laborato...

  3. Main: SB3NPABC1 [PLACE

    Lifescience Database Archive (English)

    Full Text Available SB3NPABC1 S000434 27-Jan-2004 (last modified) kehi Sclareol box3 (SB3) found at -21...6 of a plasma membrane ATP binding cassette (ABC) transporter gene (NpABC1) in N. plumbaginifolia; Mutation ...in SB3 completely abolished sclareolide-mediated expression; See S ; sclareol; ABC; transporter; SB3; Nicotiana plumbaginifolia (tobacco); TTATGAACAGTAATT ...

  4. Main: SB1NPABC1 [PLACE

    Lifescience Database Archive (English)

    Full Text Available SB1NPABC1 S000435 27-Jan-2004 (last modified) kehi Sclareol box1 (SB1) found at -82...7 of a plasma membrane ATP binding cassette (ABC) transporter gene (NpABC1) in N. plumbaginifolia; Mutation ...in SB3 completely abolished sclareolide-mediated expression; See S ; scalareol; diterpene; ABC; transporter; Nicotiana plumbaginifolia (tobacco); CACTAACACAAAGTAA ...

  5. PfMDR2 and PfMDR5 are dispensable for Plasmodium falciparum asexual parasite multiplication but change in vitro susceptibility to anti-malarial drugs

    NARCIS (Netherlands)

    Velden, M. van der; Rijpma, S.R.; Russel, F.G.M.; Sauerwein, R.W.; Koenderink, J.B.

    2015-01-01

    BACKGROUND: Membrane-associated ATP binding cassette (ABC) transport proteins hydrolyze ATP in order to translocate a broad spectrum of substrates, from single ions to macromolecules across membranes. In humans, members from this transport family have been linked to drug resistance phenotypes, e.g.,

  6. A structural classification of substrate-binding proteins

    NARCIS (Netherlands)

    Berntsson, Ronnie P. -A.; Smits, Sander H. J.; Schmitt, Lutz; Slotboom, Dirk-Jan; Poolman, Bert

    2010-01-01

    Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth

  7. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    NARCIS (Netherlands)

    Acuña-Alonzo, Víctor; Flores-Dorantes, Teresa; Kruit, Janine K; Villarreal-Molina, Teresa; Arellano-Campos, Olimpia; Hünemeier, Tábita; Moreno-Estrada, Andrés; Ortiz-López, Ma Guadalupe; Villamil-Ramírez, Hugo; León-Mimila, Paola; Villalobos-Comparan, Marisela; Jacobo-Albavera, Leonor; Ramírez-Jiménez, Salvador; Sikora, Martin; Zhang, Lin-Hua; Pape, Terry D; Granados-Silvestre, Ma de Angeles; Montufar-Robles, Isela; Tito-Alvarez, Ana M; Zurita-Salinas, Camilo; Bustos-Arriaga, José; Cedillo-Barrón, Leticia; Gómez-Trejo, Celta; Barquera-Lozano, Rodrigo; Vieira-Filho, Joao P; Granados, Julio; Romero-Hidalgo, Sandra; Huertas-Vázquez, Adriana; González-Martín, Antonio; Gorostiza, Amaya; Bonatto, Sandro L; Rodríguez-Cruz, Maricela; Wang, Li; Tusié-Luna, Teresa; Aguilar-Salinas, Carlos A; Lisker, Ruben; Moises, Regina S; Menjivar, Marta; Salzano, Francisco M; Knowler, William C; Bortolini, M Cátira; Hayden, Michael R; Baier, Leslie J; Canizales-Quinteros, Samuel

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was

  8. A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins.

    Science.gov (United States)

    Hung, Miao-Chiu; Humbert, María Victoria; Laver, Jay R; Phillips, Renee; Heckels, John E; Christodoulides, Myron

    2015-08-26

    The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines.

  9. Atomic modelling and systematic mutagenesis identify residues in multiple drug binding sites that are essential for drug resistance in the major Candida transporter Cdr1.

    Science.gov (United States)

    Nim, Shweta; Lobato, Lucia Gonzalez; Moreno, Alexis; Chaptal, Vincent; Rawal, Manpreet Kaur; Falson, Pierre; Prasad, Rajendra

    2016-11-01

    The ABC (ATP-Binding Cassette) transporter Cdr1 (Candida drug resistance 1) protein (Cdr1p) of Candida albicans, shows promiscuity towards the substrate it exports and plays a major role in antifungal resistance. It has two transmembrane domains (TMDs) comprising of six transmembrane helices (TMH) that envisage and confer the substrate specificity and two nucleotide binding domains (NBDs), interconnected by extracellular loops (ECLs) and intracellular loops (ICLs) Cdr1p. This study explores the diverse substrate specificity spectrum to get a deeper insight into the structural and functional features of Cdr1p. By screening with the variety of compounds towards an in-house TMH 252 mutant library of Cdr1p, we establish new substrates of Cdr1p. The localization of substrate-susceptible mutants in an ABCG5/G8 homology model highlights the common and specific binding pockets inside the membrane domain, where rhodamines and tetrazoliums mainly engage the N-moiety of Cdr1p, binding between TMH 2, 11 and surrounded by TMH 1, 5. Whereas, tin chlorides involve both N and C moieties located at the interface of TMH 2, 11, 1 and 5. Further, screening of the in house TMH mutant library of Cdr1p displays the TMH12 interaction with tetrazolium chloride, trimethyltin chloride and a Ca(2+) ionophore, A23187. In silico localization reveals a binding site at the TMH 12, 9 and 10 interface, which is widely exposed to the lipid interface. Together, for the first time, our study shows the molecular localization of Cdr1p substrates-binding sites and demonstrates the participation of TMH12 in a peripheral drug binding site.

  10. Transport of eicosapentaenoic acid-derived PGE₃, PGF(3α, and TXB₃ by ABCC4.

    Directory of Open Access Journals (Sweden)

    Nobuaki Tanaka

    Full Text Available Eicosapentaenoic acid-derived prostaglandin (PG E3, PGF(3α, and thromboxane (TX B3 are bioactive lipid mediators which have anti-cancer and anti-inflammatory effects. To exert their effects, PGE3, PGF(3α, and TXB3 must be released to the extracellular space from cells, but the release mechanism has been unclear. We therefore investigated the contribution of ATP-binding cassette transporter C4 (ABCC4, which has been known as a prostanoids efflux transporter, to the release of PGE3, PGF(3α, and TXB3.ATP-dependent transport of PGE3, PGF(3α, and TXB3 via ABCC4 was investigated by using inside-out membrane vesicles prepared from ABCC4-overexpressing HEK293 cells. To evaluate the contribution of ABCC4 to the release of PGE3, PGF(3α, and TXB3, we measured the extracellular and intracellular levels of PGE3, PGF(3α, and TXB3 in A549 cells when we used ABCC4 inhibitors (dipyridamole, MK571, and probenecid or ABCC4 siRNAs. The quantification of PGE3, PGF(3α, and TXB3 was performed by using liquid chromatography-tandem mass spectrometry.The apparent Km values for ABCC4-mediated transport were 2.9±0.1 µM for PGE3, 12.1±1.3 µM for PGF(3α, and 11.9±1.4 µM for TXB3 and the ATP-dependent accumulation of PGE3, PGF(3α, and TXB3 into vesicles was decreased by using typical substrates and inhibitors of ABCC4. ABCC4 inhibitors and ABCC4 knockdown showed the reduction of extracellular/intracellular ratio of PGE3 (40-60% of control and PGF(3α (60-80% of control in A549 cells.Our results suggest that PGE3, PGF(3α, and TXB3 are substrates of ABCC4 and ABCC4 partially contributes to the release of PGE3 and PGF(3α.

  11. Proteins involved in uptake, intracellular transport and basolateral secretion of fat-soluble vitamins and carotenoids by mammalian enterocytes.

    Science.gov (United States)

    Reboul, Emmanuelle; Borel, Patrick

    2011-10-01

    Our understanding of the molecular mechanisms responsible for fat-soluble vitamin uptake and transport at the intestinal level has advanced considerably over the past decade. On one hand, it has long been considered that vitamin D and E as well as β-carotene (the main provitamin A carotenoid in human diet) were absorbed by a passive diffusion process, although this could not explain the broad inter-individual variability in the absorption efficiency of these molecules. On the other hand, it was assumed that preformed vitamin A (retinol) and vitamin K1 (phylloquinone) absorption occurred via energy-dependent processes, but the transporters involved have not yet been identified. The recent discovery of intestinal proteins able to facilitate vitamin E and carotenoid uptake and secretion by the enterocyte has spurred renewed interest in studying the fundamental mechanisms involved in the absorption of these micronutrients. The proteins identified so far are cholesterol transporters such as SR-BI (scavenger receptor class B type I), CD36 (cluster determinant 36), NPC1L1 (Niemann-Pick C1-like 1) or ABCA1 (ATP-Binding Cassette A1) displaying a broad substrate specificity, but it is likely that other membrane proteins are also involved. After overviewing the metabolism of fat-soluble vitamins and carotenoids in the human upper gastrointestinal lumen, we will focus on the putative or identified proteins participating in the intestinal uptake, intracellular transport and basolateral secretion of these fat-soluble vitamins and carotenoids, and outline the uncertainties that need to be explored in the future. Identifying the proteins involved in intestinal uptake and transport of fat-soluble vitamins and carotenoids across the enterocyte is of great importance, especially as some of them are already targets for the development of drugs able to slow cholesterol absorption. Indeed, these drugs may also interfere with lipid vitamin uptake. A better understanding of the molecular

  12. Human immunodeficiency virus impairs reverse cholesterol transport from macrophages.

    Directory of Open Access Journals (Sweden)

    Zahedi Mujawar

    2006-10-01

    Full Text Available Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1-infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings

  13. 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.

    Science.gov (United States)

    Helms, Hans Christian; Hersom, Maria; Kuhlmann, Louise Borella; Badolo, Lasina; Nielsen, Carsten Uhd; Brodin, Birger

    2014-09-01

    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 if a bovine endothelial/rat astrocyte in vitro BBB co-culture model displayed polarized transport of known efflux transporter substrates. The co-culture model displayed low mannitol permeabilities of 0.95 ± 0.1 · 10(-6) cm·s(-1) and high transendothelial electrical resistances of 1,177 ± 101 Ω·cm(2). Bidirectional transport studies with (3)H-digoxin, (3)H-estrone-3-sulphate and (3)H-etoposide revealed polarized transport favouring the brain-to-blood direction for all substrates. Steady state efflux ratios of 2.5 ± 0.2 for digoxin, 4.4 ± 0.5 for estrone-3-sulphate and 2.4 ± 0.1 for etoposide were observed. These were reduced to 1.1 ± 0.08, 1.4 ± 0.2 and 1.5 ± 0.1, by addition of verapamil (digoxin), Ko143 (estrone-3-sulphate) or zosuquidar + reversan (etoposide), respectively. Brain-to-blood permeability of all substrates was investigated in the presence of the efflux transporter inhibitors verapamil, Ko143, 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.

  14. A sensor for intracellular ionic strength

    NARCIS (Netherlands)

    Biemans-Oldehinkel, Esther; Mahmood, Nik A.B.N.; Poolman, Bert

    2006-01-01

    Cystathionine-β-synthase (CBS) domains are found in >4,000 proteins in species from all kingdoms of life, yet their functions are largely unknown. Tandem CBS domains are associated with membrane transport proteins, most notably members of the ATP-binding cassette (ABC) superfamily; voltage-gated chl

  15. Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

    NARCIS (Netherlands)

    Hollenstein, K.; Comellas-Bigler, M.; Bevers, L.E.; Feiters, M.C.; Meyer-Klaucke, W.; Hagedoorn, P.-L.; Locher, K.P.

    2009-01-01

    Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO4 2−) and tungstate (WO4 2−). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across th

  16. The effect of ABCG5/G8 polymorphisms on plasma HDL cholesterol levels depends on the ABCA1 gene variation in the Boston Puerto Rican Health Study

    Science.gov (United States)

    Background: ATP-binding cassette transporters G5/G8 have shown an association with HDL-C. One of the most likely mechanisms to explain those associations is through ABCA1. Objective: To assess whether the effect of ABCG5/G8 polymorphisms on HDL-C is dependent on ABCA1, we studied potential interacti...

  17. AcEST: DK962825 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ly A member 1... 34 0.43 sp|Q8WWZ4|ABCAA_HUMAN ATP-binding cassette sub-family A ...ctase chain 5 OS=C... 31 3.6 sp|Q54R52|ABCAA_DICDI ABC transporter A family member 10 OS=Dict... 31 3.7 sp|P

  18. Cytotoxicity and binding profiles of activated Cry1Ac and Cry2Ab to three insect cell lines

    Science.gov (United States)

    While Cry1Ac has been known to bind with larval midgut proteins cadherin, APN (amino peptidase N), ALP (alkaline phosphatase) and ABCC2 (ATP-binding cassette transporter subfamily C2), little is known about the receptors of Cry2Ab. To provide a clue to the receptors of Cry2Ab, we tested the baselin...

  19. Multidrug resistance in oncology and beyond : from imaging of drug efflux pumps to cellular drug targets

    NARCIS (Netherlands)

    Nagengast, Wouter B; Oude Munnink, Thijs H; Dijkers, Eli; Hospers, Geesiena; Brouwers, Adrienne H; Schröder, Carolien P; Lub-de Hooge, Marjolijn; de Vries, Elisabeth G E

    2010-01-01

    Resistance of tumor cells to several structurally unrelated classes of natural products, including anthracyclines, taxanes, and epipodophyllotoxines, is often referred as multidrug resistance (MDR). This is associated with ATP-binding cassette transporters, which function as drug efflux pumps such a

  20. Genomic profiling of thousands of candidate polymorphisms predicts risk of relapse in 778 Danish and German childhood acute lymphoblastic leukemia patients

    DEFF Research Database (Denmark)

    Wesolowska, Agata; Borst, L.; Dalgaard, Marlene Danner;

    2015-01-01

    defined by end of induction residual leukemia, white blood cell count and variants in myeloperoxidase (MPO), estrogen receptor 1 (ESR1), lamin B1 (LMNB1) and matrix metalloproteinase-7 (MMP7) genes, ATP-binding cassette transporters and glucocorticosteroid transcription regulation pathways. Relapse rates...

  1. Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal multidrug resistance proteins

    NARCIS (Netherlands)

    Gajic, O; Buist, G; Kojic, M; Topisirovic, L; Kuipers, OP; Kok, J

    2003-01-01

    A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a

  2. Conformational changes and ligand recognition of Escherichia coli D-xylose binding protein revealed

    DEFF Research Database (Denmark)

    Sooriyaarachchi, Sanjeewani; Ubhayasekera, Wimal; Park, Chankyu

    2010-01-01

    ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-r...

  3. Function of MRP1/ABCC1 is not dependent on cholesterol or cholesterol-stabilized lipid rafts

    NARCIS (Netherlands)

    Meszaros, Peter; Klappe, Karin; Hummel, Ina; Hoekstra, Dick; Kok, Jan Willem

    2011-01-01

    MRP1 (multidrug-resistance-related protein 1)/ABCC1 (ATP-binding cassette transporter C1) has been localized in cholesterol-enriched lipid rafts, which suggests a role for these lipid rafts and/or cholesterol in MRP1 function. In the present study, we have shown for the first time that nearly comple

  4. Enhanced Foam Cell Formation, Atherosclerotic Lesion Development, and Inflammation by Combined Deletion of ABCA1 and SR-BI in Bone Marrow-Derived Cells in LDL Receptor Knockout Mice on Western-Type Diet

    NARCIS (Netherlands)

    Zhao, Ying; Pennings, Marieke; Hildebrand, Reeni B.; Ye, Dan; Calpe-Berdiel, Laura; Out, Ruud; Kjerrulf, Martin; Hurt-Camejo, Eva; Groen, Albert K.; Hoekstra, Menno; Jessup, Wendy; Chimini, Giovanna; Van Berkel, Theo J. C.; Van Eck, Miranda

    2010-01-01

    Rationale: Macrophages cannot limit the uptake of lipids and rely on cholesterol efflux mechanisms for maintaining cellular cholesterol homeostasis. Important mediators of macrophage cholesterol efflux are ATP-binding cassette transporter 1 (ABCA1), which mediates the efflux of cholesterol to lipid-

  5. A Partnership Training Program in Breast Cancer Research Using Molecular Imaging Techniques

    Science.gov (United States)

    2007-07-01

    NO via some unknown mechanism. NO-Fe(II)- Hb, formed by NO reaction with heme proteins, was detectable in the venous blood 1 h after dosing, and its...ATP-Binding Cassette Transporter ABCB5, In Melanoma Cells and In Melanocytes. Pigment Cell Research, 2005, 18 (2): 102-112. 7. Liang, X.J, Shen, D.W

  6. The Liver X Receptor (LXR) and its Target Gene ABCA1 are Regulated Upon Low Oxygen in Human Trophoblast Cells : A Reason for Alterations in Preeclampsia?

    NARCIS (Netherlands)

    Plosch, T.; Gellhaus, A.; van Straten, E. M. E.; Wolf, N.; Huijkman, N. C. A.; Schmidt, M.; Dunk, C. E.; Kuipers, F.; Winterhager, E.

    2010-01-01

    Objectives: The Liver X receptors (LXR) alpha and beta and their target genes such as the ATP-binding cassette (ABC) transporters have been shown to be crucially involved in the regulation of cellular cholesterol homeostasis. The aim of this study was to characterize the role of LXR alpha/beta in th

  7. Gclust Server: 76160 [Gclust Server

    Lifescience Database Archive (English)

    Full Text Available 76160 SCE_YLR188W=MDL1 Cluster Sequences Related Sequences(340) 695 Half-type ATP-b...es(340) Sequence length 695 Representative annotation Half-type ATP-binding cassette (ABC) transporter of th

  8. Sphingolipids in neuroblastoma : Their role in drug resistance mechanisms

    NARCIS (Netherlands)

    Sietsma, H; Dijkhuis, AJ; Kamps, W; Kok, JW

    2002-01-01

    Disseminated neuroblastoma usually calls for chemotherapy as the primary approach for treatment. Treatment failure is often attributable to drug resistance. This involves a variety of cellular mechanisms, including increased drug efflux through expression of ATP-binding cassette transporters (e.g.,

  9. Co-assortment in integron-associated gene cassette assemblages in environmental DNA samples

    Directory of Open Access Journals (Sweden)

    Michael Carolyn A

    2010-08-01

    Full Text Available Abstract Background It has been shown that integron-associated gene cassettes exist largely in tandem arrays of variable size, ranging from antibiotic resistance arrays of three to five cassettes up to arrays of more than 100 cassettes associated with the vibrios. Further, the ecology of the integron/gene cassette system has been investigated by showing that very many different cassettes are present in even small environmental samples. In this study, we seek to extend the ecological perspective on the integron/gene cassette system by investigating the way in which this diverse cassette metagenome is apportioned amongst prokaryote lineages in a natural environment. Results We used a combination of PCR-based techniques applied to environmental DNA samples and ecological analytical techniques to establish co-assortment within cassette populations, then establishing the relationship between this co-assortment and genomic structures. We then assessed the distribution of gene cassettes within the environment and found that the majority of gene cassettes existed in large co-assorting groups. Conclusions Our results suggested that the gene cassette diversity of a relatively pristine sampling environment was structured into co-assorting groups, predominantly containing large numbers of cassettes per group. These co-assorting groups consisted of different gene cassettes in stoichiometric relationship. Conservatively, we then attributed co-assorting cassettes to the gene cassette complements of single prokaryote lineages and by implication, to large integron-associated arrays. The prevalence of large arrays in the environment raises new questions about the assembly, maintenance and utility of large cassette arrays in prokaryote populations.

  10. 21 CFR 892.1880 - Wall-mounted radiographic cassette holder.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Wall-mounted radiographic cassette holder. 892.1880 Section 892.1880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... radiographic cassette holder. (a) Identification. A wall-mounted radiographic cassette holder is a device...

  11. Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae.

    Science.gov (United States)

    Raza, Ali; Kopp, Steven R; Bagnall, Neil H; Jabbar, Abdul; Kotze, Andrew C

    2016-08-01

    This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate.

  12. Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae

    Directory of Open Access Journals (Sweden)

    Ali Raza

    2016-08-01

    Full Text Available This study investigated the interaction of ATP binding cassette (ABC transport proteins with ivermectin (IVM and levamisole (LEV in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate.

  13. Expression of antibiotic resistance genes in the integrated cassettes of integrons.

    Science.gov (United States)

    Collis, C M; Hall, R M

    1995-01-01

    Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes. All transcripts detected commenced at the common promoter P(ant), and alterations in the sequence of P(ant) affected the level of resistance expressed by cassette genes. When both P(ant) and the secondary promoter P2 were present, transcription from both promoters was detected. When more than one cassette was present, the position of the cassette in the array influenced the level of antibiotic resistance expressed by the cassette gene. In all cases, the resistance level was highest when the gene was in the first cassette, i.e., closest to P(ant), and was reduced to different extents by the presence of individual upstream cassettes. In Northern (RNA) blots, multiple discrete transcripts originating at P(ant) were detected, and only the longer transcripts contained the distal genes. Together, these data suggest that premature transcription termination occurs within the cassettes. The most abundant transcripts appeared to contain one or more complete cassettes, and is possible that the 59-base elements found at the end of the cassettes (3' to the coding region) not only function as recombination sites but may also function as transcription terminators. PMID:7695299

  14. p21-ras effector domain mutants constructed by "cassette" mutagenesis

    DEFF Research Database (Denmark)

    Stone, J C; Vass, W C; Willumsen, B M;

    1988-01-01

    A series of mutations encoding single-amino-acid substitutions within the v-rasH effector domain were constructed, and the ability of the mutants to induce focal transformation of NIH 3T3 cells was studied. The mutations, which spanned codons 32 to 40, were made by a "cassette" mutagenesis...... technique that involved replacing this portion of the v-rasH effector domain with a linker carrying two BspMI sites in opposite orientations. Since BspMI cleaves outside its recognition sequence, BspMI digestion of the plasmid completely removed the linker, creating a double-stranded gap whose missing ras...... sequences were reconstructed as an oligonucleotide cassette. Based upon the ability of the mutants to induce focal transformation of NIH 3T3 cells, a range of phenotypes from virtually full activity to none (null mutants) was seen. Three classes of codons were present in this segment: one which could...

  15. A simplified erythromycin resistance cassette for Treponema denticola mutagenesis

    Science.gov (United States)

    Goetting-Minesky, M. Paula; Fenno, J. Christopher

    2010-01-01

    The primary selectable marker for genetic studies of Treponema denticola is a hybrid gene cassette containing both ermF and ermAM (ermB) genes. ErmB functions in Escherichia coli, while ErmF has been assumed to confer resistance in T. denticola. We demonstrate here that ErmB is sufficient for erythromycin selection in T. denticola and that the native ermB promoter drives ErmB expression. PMID:20691222

  16. Cassette less SOFC stack and method of assembly

    Science.gov (United States)

    Meinhardt, Kerry D

    2014-11-18

    A cassette less SOFC assembly and a method for creating such an assembly. The SOFC stack is characterized by an electrically isolated stack current path which allows welded interconnection between frame portions of the stack. In one embodiment electrically isolating a current path comprises the step of sealing a interconnect plate to a interconnect plate frame with an insulating seal. This enables the current path portion to be isolated from the structural frame an enables the cell frame to be welded together.

  17. Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa [version 3; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Érica L. Fonseca

    2015-11-01

    Full Text Available The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF, a short 5’ untranslated region (UTR and a 3’ recombination site (attC. Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW, had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription

  18. Multidrug resistance associated proteins in multidrug resistance

    OpenAIRE

    Sodani, Kamlesh; Patel, Atish; Kathawala, Rishil J.; Chen, Zhe-Sheng

    2012-01-01

    Multidrug resistance proteins (MRPs) are members of the C family of a group of proteins named ATP-binding cassette (ABC) transporters. These ABC transporters together form the largest branch of proteins within the human body. The MRP family comprises of 13 members, of which MRP1 to MRP9 are the major transporters indicated to cause multidrug resistance in tumor cells by extruding anticancer drugs out of the cell. They are mainly lipophilic anionic transporters and are reported to transport fr...

  19. Detoxification of multiple heavy metals by a half-molecule ABC transporter, HMT-1, and coelomocytes of Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Marc S Schwartz

    Full Text Available BACKGROUND: Developing methods for protecting organisms in metal-polluted environments is contingent upon our understanding of cellular detoxification mechanisms. In this regard, half-molecule ATP-binding cassette (ABC transporters of the HMT-1 subfamily are required for cadmium (Cd detoxification. HMTs have conserved structural architecture that distinguishes them from other ABC transporters and allows the identification of homologs in genomes of different species including humans. We recently discovered that HMT-1 from the simple, unicellular organism, Schizosaccharomyces pombe, SpHMT1, acts independently of phytochelatin synthase (PCS and detoxifies Cd, but not other heavy metals. Whether HMTs from multicellular organisms confer tolerance only to Cd or also to other heavy metals is not known. METHODOLOGY/PRINCIPAL FINDINGS: Using molecular genetics approaches and functional in vivo assays we showed that HMT-1 from a multicellular organism, Caenorhabditis elegans, functions distinctly from its S. pombe counterpart in that in addition to Cd it confers tolerance to arsenic (As and copper (Cu while acting independently of pcs-1. Further investigation of hmt-1 and pcs-1 revealed that these genes are expressed in different cell types, supporting the notion that hmt-1 and pcs-1 operate in distinct detoxification pathways. Interestingly, pcs-1 and hmt-1 are co-expressed in highly endocytic C. elegans cells with unknown function, the coelomocytes. By analyzing heavy metal and oxidative stress sensitivities of the coelomocyte-deficient C. elegans strain we discovered that coelomocytes are essential mainly for detoxification of heavy metals, but not of oxidative stress, a by-product of heavy metal toxicity. CONCLUSIONS/SIGNIFICANCE: We established that HMT-1 from the multicellular organism confers tolerance to multiple heavy metals and is expressed in liver-like cells, the coelomocytes, as well as head neurons and intestinal cells, which are cell types

  20. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

    Directory of Open Access Journals (Sweden)

    Wee Tek Tay

    2015-11-01

    Full Text Available The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the

  1. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    Science.gov (United States)

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  2. Characterization of a PDR type ABC transporter gene from wheat (Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    SHANG Yi; XIAO Jin; MA LuLin; WANG HaiYan; QI ZengJun; CHEN PeiDu; LIU DaJun; WANG XiuE

    2009-01-01

    DON,as a virulence factor,plays an important role in the infection of Fusarium graminearum in wheat.The infection ability of F. graminearum depends on its capacity of producing DON. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. In this study,GeneChip analysis indicated that an EST encoding an ATP-binding cassette (ABC) transporter was up-regulated by 45 times in a wheat landrace Wangshuibai,which is resistant to DON accumulation.A pair of EST-derived primers were designed based on the EST sequence,and a clone was then isolated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subsequently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp,consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calculated molecular weight is about 161 kD. BLAST analysis indicated that the gene may belong to pleiotropic drug resistance (PDR) sub-family,and hence designated as TaPDR1 (Triticum aestivum pleiotropic drug resistance). TaPDR1 was located on chromosome 5A of wheat using nulliaomic-tetrasomic lines of Chinese Spring. TaPDR1 was up-regulated by induction of both DON and F. graminearum. Expression patterns of TaPDR1 were different in wild-type Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1,a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON accumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress,such as JA and SA,nor by abiotic stresses,such as heat,cold,wounding and NaCI. However,TaPDR1 expression was

  3. Vinblastine and sulfinpyrazone export by the multidrug resistance protein MRP2 is associated with glutathione export

    OpenAIRE

    Evers, R.; Haas, M; Sparidans, R; Beijnen, J.; Wielinga, P R; Lankelma, J.; Borst, P

    2000-01-01

    The multidrug resistance proteins MRP1 and MRP2 are members of the same subfamily of ATP-binding cassette transporters. Besides organic molecules conjugated to negatively charged ligands, these proteins also transport cytotoxic drugs for which no negatively charged conjugates are known to exist. In polarized MDCKII cells, MRP1 routes to the lateral plasma membrane, and MRP2 to the apical plasma membrane. In these cells MRP1 transports daunorubicin, and MRP2 vinblastine; both transporters expo...

  4. Cassettes for solid-oxide fuel cell stacks and methods of making the same

    Science.gov (United States)

    Weil, K. Scott; Meinhardt, Kerry D; Sprenkle, Vincent L

    2012-10-23

    Solid-oxide fuel cell (SOFC) stack assembly designs are consistently investigated to develop an assembly that provides optimal performance, and durability, within desired cost parameters. A new design includes a repeat unit having a SOFC cassette and being characterized by a three-component construct. The three components include an oxidation-resistant, metal window frame hermetically joined to an electrolyte layer of a multi-layer, anode-supported ceramic cell and a pre-cassette including a separator plate having a plurality of vias that provide electrical contact between an anode-side collector within the pre-cassette and a cathode-side current collector of an adjacent cell. The third component is a cathode-side seal, which includes a standoff that supports a cathode channel spacing between each of the cassettes in a stack. Cassettes are formed by joining the pre-cassette and the window frame.

  5. Recombinogenic engineering of conjugative plasmids with fluorescent marker cassettes

    DEFF Research Database (Denmark)

    Reisner, A.; Molin, Søren; Zechner, E.L.

    2002-01-01

    An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage lambda recombination...... resistance genes and fluorescent markers. The choice of 5' non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E coli and is not restricted...

  6. Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.

    Science.gov (United States)

    Robinson, Andrew; Wu, Peter S-C; Harrop, Stephen J; Schaeffer, Patrick M; Dosztányi, Zsuzsanna; Gillings, Michael R; Holmes, Andrew J; Nevalainen, K M Helena; Stokes, H W; Otting, Gottfried; Dixon, Nicholas E; Curmi, Paul M G; Mabbutt, Bridget C

    2005-03-11

    The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.

  7. An AC electrokinetics facilitated biosensor cassette for rapid pathogen identification.

    Science.gov (United States)

    Ouyang, Mengxing; Mohan, Ruchika; Lu, Yi; Liu, Tingting; Mach, Kathleen E; Sin, Mandy L Y; McComb, Mason; Joshi, Janhvi; Gau, Vincent; Wong, Pak Kin; Liao, Joseph C

    2013-07-01

    To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future.

  8. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

    OpenAIRE

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R.; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine res...

  9. The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel alpha-glucosides through reverse phosphorolysis by maltose phosphorylase.

    Science.gov (United States)

    Nakai, Hiroyuki; Baumann, Martin J; Petersen, Bent O; Westphal, Yvonne; Schols, Henk; Dilokpimol, Adiphol; Hachem, Maher A; Lahtinen, Sampo J; Duus, Jens Ø; Svensson, Birte

    2009-12-01

    A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing beta-glucose 1-phosphate (beta-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and beta-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mmbeta-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be alpha-Glcp-(1-->4)-Glcp (maltose), alpha-Glcp-(1-->4)-GlcNp (maltosamine), alpha-Glcp-(1-->4)-GlcNAcp (N-acetyl maltosamine), alpha-Glcp-(1-->4)-Manp, alpha-Glcp-(1-->4)-Xylp and alpha-Glcp-(1-->4)- L-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.

  10. Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio

    Directory of Open Access Journals (Sweden)

    Gillings Michael R

    2006-01-01

    Full Text Available Abstract Background Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be. Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains. Results The complete Vibrio sp. DAT722 integron cassette array was determined through the streamlined approach described here. To place it in an evolutionary context, we compare the DAT722 array to known vibrio arrays and performed phylogenetic analyses for all of its components (integrase, 59-be sites, gene cassette encoded genes. It differs extensively in terms of genomic context as well as gene cassette content and organization. The phylogenetic tree of the 59-be sites collectively found in the Vibrio gene cassette pool suggests frequent transfer of cassettes within and between Vibrio species, with slower transfer rates between more phylogenetically distant relatives. We also identify multiple cases where non-integron chromosomal genes seem to have been assembled into gene cassettes and others where cassettes have been inserted into chromosomal locations outside integrons. Conclusion Our systematic approach greatly facilitates the isolation and annotation of large integrons gene cassette arrays. Comparative analysis of the Vibrio sp. DAT722 integron obtained through this approach to those found in other vibrios confirms the role of this genetic element in promoting lateral gene transfer and suggests a high rate of gene

  11. An investigation of infection control for x-ray cassettes in a diagnostic imaging department

    Energy Technology Data Exchange (ETDEWEB)

    Fox, Matthew [School of Allied Health Professions and Science, Faculty of Health, Wellbeing and Science, University Campus Suffolk, Rope Walk, Ipswich, Suffolk, IP4 1LT (United Kingdom); Harvey, Jane M. [School of Allied Health Professions and Science, Faculty of Health, Wellbeing and Science, University Campus Suffolk, Rope Walk, Ipswich, Suffolk, IP4 1LT (United Kingdom)], E-mail: j.harvey@ucs.ac.uk

    2008-11-15

    Introduction: This research was conducted to investigate if X-ray cassettes could be a possible source of pathogens capable of causing nosocomial infections, and if they could be a possible vector for cross infection within the hospital environment. Method: The research involved the swabbing of X-ray cassettes in a Diagnostic Imaging Department of a large hospital in the east of England. Two areas of the Diagnostic Imaging Department were included in the study. Research concentrated on X-ray cassettes used for mobile radiography, accident and emergency and inpatient use. Forty cassettes were swabbed in total specifically for general levels of bacterial contamination, also for the presence or absence of methicillin-resistant Staphylococcus aureus (MRSA). A mapping exercise was completed following the location of an X-ray cassette typically used in mobile radiography. The exercise noted the level of direct contact with patient's skin and other possible routes of infection. Results: The results demonstrated that there were large levels of growth of samples taken from cassettes and developed in the Microbiology Department. Coagulase-negative Staphylococcus, Micrococci, Diptheroids and species of Bacillus were all identified. The mapping exercise in which the journey of a 35/43 cm cassette used for mobile radiography was tracked found that contact with patient's skin and potential pathogens or routes of cross infection was a common occurrence whilst undertaking mobile radiography. Conclusion: The research has identified the presence of bacterial contamination on cassettes. The research established that X-ray cassettes/imaging plates are often exposed to pathogens and possible routes of cross infection; also that patient's skin often comes directly in contact with the X-ray cassette/imaging plate. The research also shows that as cassettes/imaging plates are a potential source of cross infection, the Diagnostic Imaging Department may be partly responsible

  12. Sertraline and Its Metabolite Desmethylsertraline, but not Bupropion or Its Three Major Metabolites, Have High Affinity for P-Glycoprotein

    OpenAIRE

    Wang, Jun-Sheng; Zhu, Hao-Jie; Gibson, Bryan Bradford; Markowitz, John Seth; Donovan, Jennifer Lyn; DeVane, Carl Lindsay

    2008-01-01

    The ATP-binding cassette (ABC) transporter protein subfamily Bl line (ABCBl) transporter P-glycoprotein (P-gp) plays an important role in the blood–brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertralin, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alchhol (EB), and hydroxy metabolite (HB) was studied using an ATPase ass...

  13. Syntheses, photophysical properties, and application of through-bond energy-transfer cassettes for biotechnology.

    Science.gov (United States)

    Jiao, Guan-Sheng; Thoresen, Lars H; Kim, Taeg Gyum; Haaland, Wade C; Gao, Feng; Topp, Michael R; Hochstrasser, Robin M; Metzker, Michael L; Burgess, Kevin

    2006-10-16

    We have designed fluorescent "through-bond energy-transfer cassettes" that can harvest energy of a relatively short wavelength (e.g., 490 nm), and emit it at appreciably longer wavelengths without significant loss of intensity. Probes of this type could be particularly useful in biotechnology for multiplexing experiments in which several different outputs are to be observed from a single excitation source. Cassettes 1-4 were designed, prepared, and studied as model systems to achieve this end. They were synthesized through convergent routes that feature coupling of specially prepared fluorescein- and rhodamine-derived fragments. The four cassettes were shown to emit strongly, with highly efficient energy transfer. Their emission maxima cover a broad range of wavelengths (broader than the four dye cassettes currently used for most high-throughput DNA sequencing), and they exhibit faster energy-transfer rates than a similar through-space energy-transfer cassette. Specifically, energy-transfer rates in these cassettes is around 6-7 ps, in contrast to a similar through-space energy-transfer system shown to have a decay time of around 35 ps. Moreover, the cassettes are considerably more stable to photobleaching than fluorescein, even though they each contain fluorescein-derived donors. This was confirmed by bulk fluorescent measurements, and in single-molecule-detection studies. Modification of a commercial automated DNA-sequencing apparatus to detect the emissions of these four energy-transfer cassettes enabled single-color dye-primer sequencing.

  14. Natural transformation with synthetic gene cassettes: new tools for integron research and biotechnology.

    Science.gov (United States)

    Gestal, Alicia M; Liew, Elissa F; Coleman, Nicholas V

    2011-12-01

    Integrons are genetic elements that can capture and express genes packaged as gene cassettes. Here we report new methods that allow integrons to be studied and manipulated in their native bacterial hosts. Synthetic gene cassettes encoding gentamicin resistance (aadB) and green fluorescence (gfp), or lactose metabolism (lacZY), were made by PCR and self-ligation, converted to large tandem arrays by multiple displacement amplification, and introduced into Escherichia coli or Pseudomonas stutzeri strains via electroporation or natural transformation. Recombinants (Gm(R) or Lac(+)) were obtained at frequencies ranging from 10(1) to 10(6) c.f.u. (µg DNA)(-1). Cassettes were integrated by site-specific recombination at the integron attI site in nearly all cases examined (370/384), including both promoterless and promoter-containing cassettes. Fluorometric analysis of gfp-containing recombinants revealed that expression levels from the integron-associated promoter P(C) were five- to 10-fold higher in the plasmid-borne integron In3 compared with the P. stutzeri chromosomal integrons. Integration of lacZY cassettes into P. stutzeri integrons allowed the bacteria to grow on lactose, and the lacZY gene cassette was stably maintained in the absence of selection. This study is believed to be the first to show natural transformation by gene cassettes, and integron-mediated capture of catabolic gene cassettes.

  15. 囊性纤维化跨膜转运调节体氯离子通道——跨上皮离子转运的多功能引擎%The cystic fibrosis transmembrane conductance regulator Cl-channel: a versatile engine for transepithelial ion transport

    Institute of Scientific and Technical Information of China (English)

    李红宇; 蔡志伟; 陈正豪; 鞠敏; 徐喆; David N.Sheppard

    2007-01-01

    囊性纤维化跨膜转运调节体(cystic fibrosis transmembrane conductance regulator,CFTR)是ATP结合转运体超家族(ATP-binding cassette transporter superfamily)的一名特殊成员,因为它是一个具有相当复杂调控机制的氯离子通道.CFTR由五个结构域(domain)组成:两个跨膜结构域(membrane-spanning domains,MSDs),两个核苷酸结合域(nucleotide-binding domains,NBDs)和一个特殊的调控域(regulatory domain,RD).MSDs构成一个低电导(6~12 pS)的阴离子选择性孔道(pore),其形状如同不对称的沙漏,胞外小胞内大,狭窄部分为离子筛.两个NBDs组成头尾相对的二聚体,在二聚体之间的接触面上有两个能和ATP结合的位点(位点1和位点2).CFTR的门控机制是:ATP分子与位点1和2相互作用促使NBD二聚体的结合与解离,从而引起MSDs的构象发生变化进而使通道孔打开和关闭.RD具有多样化的结构,它含有多个磷酸化共有位点(consensus phosphorylation sites).RD的磷酸化促进NBDs与ATP的结合,从而使CFTR得以激活.CFTR通过支架蛋白与其它膜受体以及蛋白激酶、磷酸酶形成大分子信号复合体.在复杂的细胞信号系统参与下,CFTR的功能活动在时间和空间上得到精确的调控.此外,CFTR的活动与细胞代谢有紧密联系:CFTR与代谢酶形成大分子复合体,当细胞能量需求增加时,CFTR活动会受到抑制而使细胞能量得以保存.CFTR广泛分布于机体上皮组织,它通过促进水盐转运而控制上皮细胞分泌物的量与组成.值得注意的是,在呼吸道,CFTR还对机体的防御机制起重要作用.CFTR功能失常严重影响跨上皮离子转运,进而引起或加重某些疾病.%The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of the ATP-binding cassette (ABC) transporter superfamily that forms a Cl- channel with complex regulation. CFTR is composed of five domains: two membrane-spanning domains (MSDs), two nucleotide

  16. WBC27, an Adenosine Tri-phosphate-binding Cassette Protein, Controls Pollen Wall Formation and Patterning in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ying Dou; Ke-Zhen Yang; Yi Zhang; Wei Wang; Xiao-Lei Liu; Li-Qun Chen; Xue-Qin Zhang; De Ye

    2011-01-01

    In flowering plants, the exine components are derived from tapetum. Despite its importance to sexual plant reproduction, little is known about the translocation of exine materials from tapetum to developing microspores. Here we report functional characterization of the arabidopsis WBC27 gene. WBC27 encodes an adenosine tri-phosphate binding cassette (ABC) transporter and is expressed preferentially in tapetum. Mutation of WBC27 disrupted the exine formation. The wbc27 mutant microspores began to degenerate once released from tetrads and most of the microspores collapsed at the uninucleate stage. Only a small number of wbc27-1 microspores could develop into tricellular pollen grains. These survival pollen grains lacked exine and germinated in the anther before anthesis. All of these results suggest that the ABC transporter, WBC27 plays important roles in the formation of arabidopsis exine, possibly by translocation of lipidic precursors of sporopollenin from tapetum to developing microspores.

  17. Structural characterization of two metastable ATP-bound states of P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Megan L O'Mara

    Full Text Available ATP Binding Cassette (ABC transporters couple the binding and hydrolysis of ATP to the transport of substrate molecules across the membrane. The mechanism by which ATP binding and/or hydrolysis drives the conformational changes associated with substrate transport has not yet been characterized fully. Here, changes in the conformation of the ABC export protein P-glycoprotein on ATP binding are examined in a series of molecular dynamics simulations. When one molecule of ATP is placed at the ATP binding site associated with each of the two nucleotide binding domains (NBDs, the membrane-embedded P-glycoprotein crystal structure adopts two distinct metastable conformations. In one, each ATP molecule interacts primarily with the Walker A motif of the corresponding NBD. In the other, the ATP molecules interacts with both Walker A motif of one NBD and the Signature motif of the opposite NBD inducing the partial dimerization of the NBDs. This interaction is more extensive in one of the two ATP binding site, leading to an asymmetric structure. The overall conformation of the transmembrane domains is not altered in either of these metastable states, indicating that the conformational changes associated with ATP binding observed in the simulations in the absence of substrate do not lead to the outward-facing conformation and thus would be insufficient in themselves to drive transport. Nevertheless, the metastable intermediate ATP-bound conformations observed are compatible with a wide range of experimental cross-linking data demonstrating the simulations do capture physiologically important conformations. Analysis of the interaction between ATP and its cofactor Mg(2+ with each NBD indicates that the coordination of ATP and Mg(2+ differs between the two NBDs. The role structural asymmetry may play in ATP binding and hydrolysis is discussed. Furthermore, we demonstrate that our results are not heavily influenced by the crystal structure chosen for initiation

  18. The superintegron integrase and the cassette promoters are co-regulated in Vibrio cholerae.

    Directory of Open Access Journals (Sweden)

    Evelyne Krin

    Full Text Available Chromosome 2 of Vibrio cholerae carries a chromosomal superintegron, composed of an integrase, a cassette integration site (attI and an array of mostly promoterless gene cassettes. We determined the precise location of the promoter, Pc, which drives the transcription of the first cassettes of the V. cholerae superintegron. We found that cassette mRNA starts 65 bp upstream of the attI site, so that the inversely oriented promoters Pc and Pint (integrase promoter partly overlap, allowing for their potential co-regulation. Pint was previously shown to be induced during the SOS response and is further controlled by the catabolite repression cAMP-CRP complex. We found that cassette expression from Pc was also controlled by the cAMP-CRP complex, but is not part of the SOS regulon. Pint and Pc promoters were both found to be induced in rich medium, at high temperature, high salinity and at the end of exponential growth phase, although at very different levels and independently of sigma factor RpoS. All these results show that expression from the integrase and cassette promoters can take place at the same time, thus leading to coordinated excisions and integrations within the superintegron and potentially coupling cassette shuffling to immediate selective advantage.

  19. Flipase-mediated cassette exchange in Sf9 insect cells for stable gene expression.

    Science.gov (United States)

    Fernandes, Fabiana; Vidigal, João; Dias, Mafalda M; Prather, Kristala L J; Coroadinha, Ana S; Teixeira, Ana P; Alves, Paula M

    2012-11-01

    Site-specific DNA integration allows predictable heterologous gene expression and circumvents extensive clone screening. Herein, the establishment of a Flipase (Flp)-mediated cassette exchange system in Sf9 insect cells for targeted gene integration is described. A tagging cassette harboring a reporter dsRed gene was randomly introduced into the cell genome after screening different transfection protocols. Single-copy integration clones were then co-transfected with both Flp-containing plasmid and an EGFP-containing targeting cassette. Successful cassette exchange was suggested by emergence of G418-resistant green colonies and confirmed by PCR analysis, showing the absence of the tagging cassette and single integration of the targeting cassette in the same locus. Upon cassette exchange, uniform EGFP expression between clones derived from the same integration site was obtained. Moreover, the resulting cell clones exhibited the expression properties of the parental cell line. EGFP production titers over 40 mg/L were of the same order of magnitude as those achieved through baculovirus infection. This Sf9 master cell line constitutes a versatile and re-usable platform to produce multiple recombinant proteins for fundamental and applied research.

  20. A functional ABCA1 gene variant is associated with low HDL-cholesterol levels and shows evidence of positive selection in Native Americans

    OpenAIRE

    2010-01-01

    It has been suggested that the higher susceptibility of Hispanics to metabolic disease is related to their Native American heritage. A frequent cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) gene variant (R230C, rs9282541) apparently exclusive to Native American individuals was associated with low high-density lipoprotein cholesterol (HDL-C) levels, obesity and type 2 diabetes in Mexican Mestizos. We performed a more extensive analysis of this variant in 4405 Native Ameri...

  1. The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

    OpenAIRE

    Rosenberg, Mark F.; O'Ryan, Liam P.; Hughes, Guy; Zhao, Zhefeng; Aleksandrov, Luba A.; Riordan, John R.; Ford, Robert C.

    2011-01-01

    Cystic fibrosis affects about 1 in 2500 live births and involves loss of transmembrane chloride flux due to a lack of a membrane protein channel termed the cystic fibrosis transmembrane conductance regulator (CFTR). We have studied CFTR structure by electron crystallography. The data were compared with existing structures of other ATP-binding cassette transporters. The protein was crystallized in the outward facing state and resembled the well characterized Sav1866 transporter. We identified ...

  2. Localizing a gate in CFTR

    OpenAIRE

    Gao, Xiaolong; Hwang, Tzyh-Chang

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), albeit a bona fide member of the ATP-binding cassette (ABC) transporter superfamily, is an ATP-gated chloride channel. However, phylogenetic analysis has led to a popular conjecture that CFTR evolves from a primordial ABC exporter by simply degenerating the cytoplasmic gate. This degraded transporter hypothesis predicts that CFTR’s gate is located at the external end of the substrate translocation pathway as the one documented in the...

  3. New insights into cystic fibrosis: molecular switches that regulate CFTR.

    Science.gov (United States)

    Guggino, William B; Stanton, Bruce A

    2006-06-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-)-selective ion channel, is a prototypic member of the ATP-binding cassette transporter superfamily that is expressed in several organs. In these organs, CFTR assembles into large, dynamic macromolecular complexes that contain signalling molecules, kinases, transport proteins, PDZ-domain-containing proteins, myosin motors, Rab GTPases, and SNAREs. Understanding how these complexes regulate the intracellular trafficking and activity of CFTR provides a unique insight into the aetiology of cystic fibrosis and other diseases.

  4. Model of mouth-to-mouth transfer of bacterial lipoproteins through inner membrane LolC, periplasmic LolA, and outer membrane LolB

    OpenAIRE

    Okuda, Suguru; Tokuda, Hajime

    2009-01-01

    Outer membrane-specific lipoproteins in Escherichia coli are released from the inner membrane by an ATP-binding cassette transporter, the LolCDE complex, which causes the formation of a soluble complex with a periplasmic molecular chaperone, LolA. LolA then transports lipoproteins to the outer membrane where an outer membrane receptor, LolB, incorporates lipoproteins into the outer membrane. The molecular mechanisms underlying the Lol-dependent lipoprotein sorting have been clarified in detai...

  5. Radiation exposure reduction by use of Kevlar cassettes in the neonatal nursery.

    Science.gov (United States)

    Herman, M W; Mak, H K; Lachman, R S

    1987-05-01

    A study was performed to determine whether the use of Kevlar cassettes in the neonatal intensive care nursery would reduce radiation exposure to patients. The radiation dose to the neonates was measured by using thermoluminescent dosimeters. In addition, the attenuation of the Kevlar cassettes and the sensitivity of the film-screen combination were compared with the previously used system. The greatest radiation reduction using a mobile X-ray unit was 27%; based on sensitivity measurements, the theoretical reduction averaged 38%. The reduction in radiation exposure resulted from reduced attenuation by the Kevlar cassette.

  6. The function of integron-associated genes cassettes in Vibrio species: the tip of the iceberg

    Directory of Open Access Journals (Sweden)

    Rita A Rapa

    2013-12-01

    Full Text Available The integron is a genetic element that incorporates mobile genes termed gene cassettes into a reserved genetic site via site-specific recombination. It is best known for its role in antibiotic resistance with one type of integron, the class 1 integron, a major player in the dissemination of antibiotic resistance genes across Gram negative pathogens and commensals. However, integrons are ancient structures with over 100 classes (including class 1 present in bacteria from the broader environment. While, the class 1 integron is only one example of an integron being mobilised into the clinical environment, it is by far the most successful. Unlike clinical class 1 integrons which are largely found on plasmids, other integron classes are found on the chromosomes of bacteria and carry diverse gene cassettes indicating a non-antibiotic resistance role(s. However, there is very limited knowledge on what these alternative roles are. This is particularly relevant to Vibrio species where gene cassettes make up approximately 1-3% of their entire genome. In this review, we discuss how emphasis on class 1 integron research has resulted in a limited understanding by the wider research community on the role of integrons in the broader environment. This has the capacity to be counterproductive in solving or improving the antibiotic resistance problem into the future. Furthermore, there is still a significant lack of knowledge on how gene cassettes in Vibrio species drive adaptation and evolution. From research in V. rotiferianus DAT722, new insight on how gene cassettes affect cellular physiology offers new alternative roles for the gene cassette resource. At least a subset of gene cassettes are involved in host surface polysaccharide modification suggesting that gene cassette may be important in processes such as bacteriophage resistance, adhesion/biofilm formation, protection from grazers and bacterial aggregation.

  7. Influence of cassette design on three-dimensional perfusion culture of artificial bone.

    Science.gov (United States)

    Du, Dajiang; Ushida, Takashi; Furukawa, Katsuko S

    2015-01-01

    Media perfusion is often required to maintain cell viability within topographically complex 3-dimensional scaffold cultures. Osteoblast-seeded scaffolds for bone regeneration require robust cell proliferation and survival both within the scaffold and over the exterior for optimal osteogenic capacity. Conventional press-fitting cassettes ensure internal fluid flow through the scaffold but may restrict external flow around the scaffold, resulting in a barren (cell-free) external scaffold surface. In this study, we aimed to solve this problem by modifying the cassette structure to enhance external flow in an oscillatory perfusion culture system. Mouse osteoblast-like MC 3T3-E1 cells were seeded in porous ceramic scaffolds and incubated for 3 days either under static culture conditions or in an oscillatory perfusion bioreactor. Scaffolds were held in the bioreactor with either conventional press-fitting cassettes or cassettes with rings to separate the scaffold exterior from the internal cassette wall. The external surfaces of scaffolds maintained under static conditions were well seeded, but cells failed to grow deeply into the core, reflecting poor internal chemotransport. Alternatively, scaffolds cultured by perfusion with press-fitting cassettes had poor cell viability at the cassette-external scaffold surface interface, but cells were widely distributed within the scaffold core. Scaffolds cultured using the modified cassettes with 1 or 2 rings exhibited uniformly distributed living cells throughout the internal pores and over the entire external surface, possibly because of the improved medium flow over the scaffold surface. This modified oscillatory perfusion culture system may facilitate the production of engineered bone with superior osteogenic capacity for grafting.

  8. Integron gene cassettes: a repository of novel protein folds with distinct interaction sites.

    Directory of Open Access Journals (Sweden)

    Visaahini Sureshan

    Full Text Available Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites, as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+β and α/β fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.

  9. Cassette loaded with a resilient packing to connect socket ends on the sea bed

    Energy Technology Data Exchange (ETDEWEB)

    Tuson, S.P.R.

    1983-06-08

    A cassette loaded with a resilient packing is used to connect a pipe on the sea bed to one end of a hollow shaft forming part of the crosspiece of a cardan joint at the base of an articulated columns mounted on the sea bed. The resilient packing comprises a stack of rubber rings and metallic washers disposed between end rings and capable of being derformed in torsion, the packing in use being compressed between an abutment on the end of the hollow shaft in the cardan joint and an abutment on the adjacent end of the pipe. The cassette comprises a tubular body which can be fitted in or removed from a housing for a bearing of the cardan joint, the end rings of the packing projecting through the opposite ends of the tubular body of the cassette. The cassette is fitted with jacks for compressing the packing, and wedges for retaining the packing in a compressed state until the cassette is fitted in the bearing housing, whereupon the jacks are again operated to compress further the packing and enable the wedges to be removed. Release of the jacks then allows the packing to expand within the cassette and engage the ends of the packing against the ends of the hollow shaft and pipe so as to form a connection therebetween. 2 drawings.

  10. Adenosine triphosphate-binding cassette member A3 gene mutation in children from one family from Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Gawahir Mohamed Ahmed Mukhtar

    2016-01-01

    Full Text Available Mutation in ABCA3, which is adenosine triphosphate-binding cassette member A3, a member of protein transporter family for phospholipids into the lamellar bodies during synthesis of surfactant, can cause lung disease related to surfactant dysfunction with autosomal recessive pattern. We reported three cases from same family with ABCA3 mutation, their gene, clinical course, and outcomes mentioning that one patient had successful lung transplantation, one started the process of the lung transplantation while the third one died during infancy. We concluded that the patients with ABCA3 gene mutations are increasing in numbers may be due to the availability of the genetic testing and high index of suspicion among physicians. Lung transplantation is the definitive treatment, but availability is limited in our region.

  11. Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system.

    Science.gov (United States)

    Szczepanowski, Rafael; Krahn, Irene; Linke, Burkhard; Goesmann, Alexander; Pühler, Alfred; Schlüter, Andreas

    2004-11-01

    Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-sul1-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette

  12. Identification of a novel post-hydrolytic state in CFTR gating

    OpenAIRE

    Jih, Kang-Yang; Sohma, Yoshiro; Li, Min; Hwang, Tzyh-Chang

    2012-01-01

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, ubiquitous proteins found in all kingdoms of life, catalyze substrates translocation across biological membranes using the free energy of ATP hydrolysis. Cystic fibrosis transmembrane conductance regulator (CFTR) is a unique member of this superfamily in that it functions as an ATP-gated chloride channel. Despite difference in function, recent studies suggest that the CFTR chloride channel and the exporter members of the ABC pr...

  13. mRNA expression of genes involved in lipid efflux and matrix degradation in occlusive and ectatic atherosclerotic disease

    OpenAIRE

    Soumian, S; Gibbs, R.; Davies, A.; Albrecht, C.

    2005-01-01

    Background: Atherosclerotic plaque behaviour is influenced by intraplaque inflammation, matrix turnover, and the lipid core volume. Peroxisome proliferator activated receptor γ (PPARγ) modulates atherosclerosis by its anti-inflammatory and anti-protease activity. PPARγ promotes lipid efflux through the liver X receptor α (LXRα) and the ATP binding cassette transporter A1 (ABCA1). Matrix metalloproteinase 9 (MMP-9) and cyclooxygenase 2 (COX-2) are implicated in plaque instability.

  14. Ground Testing of the EMCS Seed Cassette for Biocompatibility with the Tardigrade, Hypsibius dujardini

    Science.gov (United States)

    Reinsch, Sigrid; Myers, Zachary Alan; DeSimone, Julia Carol; Freeman, John L.; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David

    2014-01-01

    The European Modular Cultivation System, EMCS, was developed by ESA for plant experiments. We performed ground testing to determine whether ARC EMCS seed cassettes could be adapted for use with tardigrades for future spaceflight experiments. Tardigrades (water bears) are small invertebrates that enter the tun state in response to desiccation or other environmental stresses. Tardigrade tuns have suspended metabolism and have been shown to be survive exposure to space vacuum, high pressure, temperature and other stresses. For spaceflight experiments using the EMCS, the organisms ideally must be able to survive desiccation and storage in the cassette at ambient temperature for several weeks prior to the initiation of the experiment by the infusion of water to the cassette during spaceflight. The ability of tardigrades to survive extremes by entering the tun state make them ideal candidates for growth experiments in the EMCS cassettes. The growth substratum in the cassettes is a gridded polyether sulfone (PES) membrane. A blotter beneath the PES membrane contains dried growth medium. The goals of our study were to (1) determine whether tardigrades survive and reproduce on PES membranes, (2) develop a consistent method for dehydration of the tardigrades with high recovery rates upon rehydration, (3) to determine an appropriate food source for the tardigrades that can also be dehydrated/rehydrated and (4) successful mock rehydration experiment in cassettes with appropriate food source. We present results that show successful multigenerational growth of tardigrades on PES membranes with a variety of wet food sources. We have successfully performed a mock rehydration with tardigrades and at least one candidate food, protonema of the moss Polytrichum, that supports multigenerational growth and whose spores germinate quickly enough to match tardigrade feeding patterns post rehydration. Our results indicate that experiments on the ISS using the tardigrade, Hypsibius dujardini

  15. Effect of attC structure on cassette excision by integron integrases

    Directory of Open Access Journals (Sweden)

    Larouche André

    2011-02-01

    Full Text Available Abstract Background Integrons are genetic elements able to integrate and disseminate genes as cassettes by a site-specific recombination mechanism. These elements contain a gene coding for an integrase that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a gene cassette. Integron integrases (IntIs bind specifically to the bottom strand of attC sites. The extrahelical bases resulting from folding of attC bottom strands are important for the recognition by integrases. These enzymes are directly involved in the accumulation and formation of new cassette arrangements in the variable region of integrons. Thus, it is important to better understand interactions between IntIs and their substrates. Results We compared the ability of five IntIs to carry out excision of several cassettes flanked by different attC sites. The results showed that for most cassettes, IntI1 was the most active integrase. However, IntI2*179E and SonIntIA could easily excise cassettes containing the attCdfrA1 site located upstream, whereas IntI1 and IntI3 had only a weak excision activity for these cassettes. Analysis of the secondary structure adopted by the bottom strand of attCdfrA1 has shown that the identity of the extrahelical bases and the distance between them (A-N7-8-C differ from those of attCs contained in the cassettes most easily excisable by IntI1 (T-N6-G. We used the attCdfrA1 site upstream of the sat2 gene cassette as a template and varied the identity and spacing between the extrahelical bases in order to determine how these modifications influence the ability of IntI1, IntI2*179E, IntI3 and SonIntIA to excise cassettes. Our results show that IntI1 is more efficient in cassette excision using T-N6-G or T-N6-C attCs while IntI3 recognizes only a limited range of attCs. IntI2*179E and SonIntIA are more tolerant of changes to the identity and spacing of extrahelical

  16. Sampling efficiency of modified 37-mm sampling cassettes using computational fluid dynamics.

    Science.gov (United States)

    Anthony, T Renée; Sleeth, Darrah; Volckens, John

    2016-01-01

    In the U.S., most industrial hygiene practitioners continue to rely on the closed-face cassette (CFC) to assess worker exposures to hazardous dusts, primarily because ease of use, cost, and familiarity. However, mass concentrations measured with this classic sampler underestimate exposures to larger particles throughout the inhalable particulate mass (IPM) size range (up to aerodynamic diameters of 100 μm). To investigate whether the current 37-mm inlet cap can be redesigned to better meet the IPM sampling criterion, computational fluid dynamics (CFD) models were developed, and particle sampling efficiencies associated with various modifications to the CFC inlet cap were determined. Simulations of fluid flow (standard k-epsilon turbulent model) and particle transport (laminar trajectories, 1-116 μm) were conducted using sampling flow rates of 10 L min(-1) in slow moving air (0.2 m s(-1)) in the facing-the-wind orientation. Combinations of seven inlet shapes and three inlet diameters were evaluated as candidates to replace the current 37-mm inlet cap. For a given inlet geometry, differences in sampler efficiency between inlet diameters averaged less than 1% for particles through 100 μm, but the largest opening was found to increase the efficiency for the 116 μm particles by 14% for the flat inlet cap. A substantial reduction in sampler efficiency was identified for sampler inlets with side walls extending beyond the dimension of the external lip of the current 37-mm CFC. The inlet cap based on the 37-mm CFC dimensions with an expanded 15-mm entry provided the best agreement with facing-the-wind human aspiration efficiency. The sampler efficiency was increased with a flat entry or with a thin central lip adjacent to the new enlarged entry. This work provides a substantial body of sampling efficiency estimates as a function of particle size and inlet geometry for personal aerosol samplers.

  17. Structural and functional studies of conserved nucleotide-binding protein LptB in lipopolysaccharide transport

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhongshan [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom); College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Xiang, Quanju [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); Department of Microbiology, College of Resource and Environment Science, Sichuan Agriculture University, Yaan 625000 (China); Zhu, Xiaofeng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Dong, Haohao [Biomedical Sciences Research Complex, School of Chemistry, University of St Andrews, North Haugh, St Andrews KY16 9ST (United Kingdom); He, Chuan [School of Electronics and Information, Wuhan Technical College of Communications, No. 6 Huangjiahu West Road, Hongshan District, Wuhan, Hubei 430065 (China); Wang, Haiyan; Zhang, Yizheng [College of Life Sciences, Sichuan University, Chengdu 610065 (China); Wang, Wenjian, E-mail: Wenjian166@gmail.com [Laboratory of Department of Surgery, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080 (China); Dong, Changjiang, E-mail: C.Dong@uea.ac.uk [Biomedical Research Centre, Norwich Medical School, University of East Anglia, Norwich Research Park, NR4 7TJ (United Kingdom)

    2014-09-26

    Highlights: • Determination of the structure of the wild-type LptB in complex with ATP and Mg{sup 2+}. • Demonstrated that ATP binding residues are essential for LptB’s ATPase activity and LPS transport. • Dimerization is required for the LptB’s function and LPS transport. • Revealed relationship between activity of the LptB and the vitality of E. coli cells. - Abstract: Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram-negative bacteria, which plays an essential role in protecting the bacteria from harsh conditions and antibiotics. LPS molecules are transported from the inner membrane to the outer membrane by seven LPS transport proteins. LptB is vital in hydrolyzing ATP to provide energy for LPS transport, however this mechanism is not very clear. Here we report wild-type LptB crystal structure in complex with ATP and Mg{sup 2+}, which reveals that its structure is conserved with other nucleotide-binding proteins (NBD). Structural, functional and electron microscopic studies demonstrated that the ATP binding residues, including K42 and T43, are crucial for LptB’s ATPase activity, LPS transport and the vitality of Escherichia coli cells with the exceptions of H195A and Q85A; the H195A mutation does not lower its ATPase activity but impairs LPS transport, and Q85A does not alter ATPase activity but causes cell death. Our data also suggest that two protomers of LptB have to work together for ATP hydrolysis and LPS transport. These results have significant impacts in understanding the LPS transport mechanism and developing new antibiotics.

  18. Control model for compressible cake filtration of green liquor in cassette filter

    OpenAIRE

    Bornefelt, Kajsa

    2006-01-01

    In the closed chemical recovery cycle in the sulphate pulp mill it is important to remove non-process elements. This is done by clarification of the green liquor, either in clarifiers or in filters. This project focuses on a cassette filter developed by Kvaerner Pulping AB. The cassette filter is semi-continuous and the aim of the project was to model the filter in order to be able to control cycle time and feed towards optimization of the capacity. The green liquor sludge forms a compressibl...

  19. Construction of New Campylobacter Cloning Vectors and a New Mutational Cat Cassette

    Science.gov (United States)

    1993-01-01

    mutational cat cassette PE - 61102A PR - 3M161102 6. AUTHOR(S) TA - BS13AK Yao R, Aim RA, Trust TJ, Guerry P WU- 1291 7. PERFORMING ORGANIZATION NAME(S) AND...mutational cat cassette %~ccesion For (Site-specific mutagenesis; recombinant DNA; multiple cloning site; PCR; shuttle vectors) NTIS CRA&I OTIC TAB E...incompatibility plasmids can mobilize shuttle vectors containing E. coi and C. coi plasmid Systems of experimental genetics are in the early stages replicons

  20. Environmental control of microtubule-based bidirectional cargo-transport

    CERN Document Server

    Klein, Sarah; Santen, Ludger

    2014-01-01

    Inside cells, various cargos are transported by teams of molecular motors. Intriguingly, the motors involved generally have opposite pulling directions, and the resulting cargo dynamics is a biased stochastic motion. It is an open question how the cell can control this bias. Here we develop a model which takes explicitly into account the elastic coupling of the cargo with each motor. We show that bias can be simply controlled or even reversed in a counterintuitive manner via a change in the external force exerted on the cargo or a variation of the ATP binding rate to motors. Furthermore, the superdiffusive behavior found at short time scales indicates the emergence of motor cooperation induced by cargo-mediated coupling.

  1. A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

    Directory of Open Access Journals (Sweden)

    Khorchid Ahmad

    2003-07-01

    Full Text Available Abstract Background The engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all. Results Therefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence. Conclusions Finally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

  2. amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Solis-Escalante, D.; Kuijpers, N.G.A.; Bongaerts, N.; Bolat, I.; Bosman, L.; Pronk, J.T.; Daran, J.M.; Daran-Lapujade, P.A.S.

    2012-01-01

    Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, ar

  3. Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors

    DEFF Research Database (Denmark)

    Jespersen, Thomas; Duch, Mogens R.; Carrasco, M L;

    1999-01-01

    of spliced env mRNA for the SL3-3 derived vector relative to the Akv derived vectors, seemingly contributing to its low replication capacity. The EGFP expressing Akv-MLV was genetically stable for multiple rounds of infection; marker-cassette deletion revertants appeared after several replication rounds...

  4. Transgene organisation in potato after particle bombardment-mediated (co-) transformation using plasmids and gene cassettes

    NARCIS (Netherlands)

    Romano, A.; Raemakers, C.J.J.M.; Bernardi, J.; Visser, R.G.F.; Mooibroek, A.

    2003-01-01

    Protocols for efficient co-transformation of potato internodes with genes contained in separate plasmids or gene cassettes (i.e., linear PCR fragments comprising a promoter-gene-terminator) using particle bombardment were established. Twenty-eight out of 62 (45%) and 11 out of 65 (17%) plants transf

  5. Trinucleotide cassettes increase diversity of T7 phage-displayed peptide library

    Directory of Open Access Journals (Sweden)

    McMahon James B

    2007-10-01

    Full Text Available Abstract Background Amino acid sequence diversity is introduced into a phage-displayed peptide library by randomizing library oligonucleotide DNA. We recently evaluated the diversity of peptide libraries displayed on T7 lytic phage and M13 filamentous phage and showed that T7 phage can display a more diverse amino acid sequence repertoire due to differing processes of viral morphogenesis. Methods In this study, we evaluated and compared the diversity of a 12-mer T7 phage-displayed peptide library randomized using codon-corrected trinucleotide cassettes with a T7 and an M13 12-mer phage-displayed peptide library constructed using the degenerate codon randomization method. Results We herein demonstrate that the combination of trinucleotide cassette amino acid codon randomization and T7 phage display construction methods resulted in a significant enhancement to the functional diversity of a 12-mer peptide library. This novel library exhibited superior amino acid uniformity and order-of-magnitude increases in amino acid sequence diversity as compared to degenerate codon randomized peptide libraries. Comparative analyses of the biophysical characteristics of the 12-mer peptide libraries revealed the trinucleotide cassette-randomized library to be a unique resource. Conclusion The combination of T7 phage display and trinucleotide cassette randomization resulted in a novel resource for the potential isolation of binding peptides for new and previously studied molecular targets.

  6. Identification of a novel streptococcal gene cassette mediating SOS mutagenesis in Streptococcus uberis

    NARCIS (Netherlands)

    Varhimo, Emilia; Savijoki, Kirsi; Jalava, Jari; Kuipers, Oscar P.; Varmanen, Pekka

    2007-01-01

    Streptococci have been considered to lack the classical SOS response, defined by increased mutation after UV exposure and regulation by LexA. Here we report the identification of a potential self-regulated SOS mutagenesis gene cassette in the Streptococcaceae family. Exposure to UV light was found t

  7. Temperature variations around medication cassette and carry bag in routine use of epoprostenol administration in healthy volunteers.

    Directory of Open Access Journals (Sweden)

    Yuichi Tamura

    Full Text Available BACKGROUND: According to several treatment guidelines, epoprostenol is an important treatment option for pulmonary arterial hypertension. However, the pharmacokinetic characteristics and poor stability of epoprostenol at room temperature make its administration challenging. We therefore studied temperature fluctuations between the drug administration cassette and atmosphere to promote the safe use of epoprostenol. METHODS AND FINDINGS: Five healthy volunteers carried a portable intravenous infusion pump attached to a medication cassette containing saline in a bag during their ordinary activities over 16 days during which the mean atmospheric temperature was 29.6 ± 1.5°C. The temperature around the medication cassette was not less than 25°C on any occasion, and the mean period over 24 h during which the temperature around the cassette exceeded 35°C and 40°C was 96.9 ± 156.4 min and 24.4 ± 77.3 min, respectively. Significant correlations were observed between the temperatures outside the bag and around the cassette, as well as between temperatures around the cassette and of the saline solution in the cassette (r = 0.9258 and 0.8276, respectively. There were no differences in the temperatures outside the bag or around the cassette with respect to the bag material. CONCLUSIONS: Temperatures around a medication cassette and outside the bag containing the medication increase with sunlight exposure. The temperature around cassettes used for administering epoprostenol must therefore be kept low for as long as possible during hot summer conditions to maintain the drug stability.

  8. Utilizing ARC EMCS Seedling Cassettes as Highly Versatile Miniature Growth Chambers for Model Organism Experiments

    Science.gov (United States)

    Freeman, John L.; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David; Reinsch, S.; DeSimone, Julia C.; Myers, Zachary A.

    2014-01-01

    The aim of our ground testing was to demonstrate the capability of safely putting specific model organisms into dehydrated stasis, and to later rehydrate and successfully grow them inside flight proven ARC EMCS seedling cassettes. The ARC EMCS seedling cassettes were originally developed to support seedling growth during space flight. The seeds are attached to a solid substrate, launched dry, and then rehydrated in a small volume of media on orbit to initiate the experiment. We hypothesized that the same seedling cassettes should be capable of acting as culture chambers for a wide range of organisms with minimal or no modification. The ability to safely preserve live organisms in a dehydrated state allows for on orbit experiments to be conducted at the best time for crew operations and more importantly provides a tightly controlled physiologically relevant growth experiment with specific environmental parameters. Thus, we performed a series of ground tests that involved growing the organisms, preparing them for dehydration on gridded Polyether Sulfone (PES) membranes, dry storage at ambient temperatures for varying periods of time, followed by rehydration. Inside the culture cassettes, the PES membranes were mounted above blotters containing dehydrated growth media. These were mounted on stainless steel bases and sealed with plastic covers that have permeable membrane covered ports for gas exchange. The results showed we were able to demonstrate acceptable normal growth of C.elegans (nematodes), E.coli (bacteria), S.cerevisiae (yeast), Polytrichum (moss) spores and protonemata, C.thalictroides (fern), D.discoideum (amoeba), and H.dujardini (tardigrades). All organisms showed acceptable growth and rehydration in both petri dishes and culture cassettes initially, and after various time lengths of dehydration. At the end of on orbit ISS European Modular Cultivation System experiments the cassettes could be frozen at ultra-low temperatures, refrigerated, or chemically

  9. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Science.gov (United States)

    Burmølle, Mette; Norman, Anders; Sørensen, Søren J; Hansen, Lars Hestbjerg

    2012-01-01

    Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  10. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Directory of Open Access Journals (Sweden)

    Mette Burmølle

    Full Text Available Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids into a laboratory strain (Escherichia coli Genehogs® for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54 and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33 were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  11. Ground Testing of the EMCS Seed Cassette for Biocompatibility with the Cellular Slime Mold, Dictyostelium Discoideum

    Science.gov (United States)

    Hanely, Julia C.; Reinsch, Sigrid; Myers, Zachary A.; Freeman, John; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David G.

    2014-01-01

    The European Modular Cultivation System, EMCS, was developed by ESA for plant experiments. To expand the use of flight verified hardware for various model organisms, we performed ground experiments to determine whether ARC EMCS Seed Cassettes could be adapted for use with cellular slime mold for future space flight experiments. Dictyostelium is a cellular slime mold that can exist both as a single-celled independent organism and as a part of a multicellular colony which functions as a unit (pseudoplasmodium). Under certain stress conditions, individual amoebae will aggregate to form multicellular structures. Developmental pathways are very similar to those found in Eukaryotic organisms, making this a uniquely interesting organism for use in genetic studies. Dictyostelium has been used as a genetic model organism for prior space flight experiments. Due to the formation of spores that are resistant to unfavorable conditions such as desiccation, Dictyostelium is also a good candidate for use in the EMCS Seed Cassettes. The growth substratum in the cassettes is a gridded polyether sulfone (PES) membrane. A blotter beneath the PES membranes contains dried growth medium. The goals of this study were to (1) verify that Dictyostelium are capable of normal growth and development on PES membranes, (2) develop a method for dehydration of Dictyostelium spores with successful recovery and development after rehydration, and (3) successful mock rehydration experiments in cassettes. Our results show normal developmental progression in two strains of Dictyostelium discoideum on PES membranes with a bacterial food source. We have successfully performed a mock rehydration of spores with developmental progression from aggregation to slug formation, and production of morphologically normal spores within 9 days of rehydration. Our results indicate that experiments on the ISS using the slime mold, Dictyostelium discoideum could potentially be performed in the flight verified hardware of

  12. Reference: 698 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available r ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transpor...ter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of t...hese seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemical... one ABC transporter can be involved in the secretion of a given phytochemical an...ype. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters. Al

  13. Nanoparticle-delivered VEGF-silencing cassette and suicide gene expression cassettes inhibit colon carcinoma growth in vitro and in vivo.

    Science.gov (United States)

    Leng, Aimin; Yang, Jing; Liu, Ting; Cui, Jianfang; Li, Xiu-Hua; Zhu, Yanan; Xiong, Ting; Chen, Yuxiang

    2011-12-01

    The strategies for tumor-specific expression of suicide genes and target tumor angiogenesis have been tested in tumors. However, the anti-tumor efficacy of the combination of these two strategies, particularly, delivering suicide gene and anti-angiogenesis agent by nanoparticles, has not yet been evaluated in colon carcinoma. We constructed a cassette to silence VEGF-A expression and express a fused yCDglyTK gene driven by tumor-specific promoter (shVEGF-CDTK). The DNA carrying shVEGF-CDTK was delivered into colon carcinoma cells by calcium phosphate nanoparticles (CPNPs). Cell proliferation was measured by MTT assay, and apoptosis was detected by flow cytometry. The anti-tumor effect of the combined cassette was tested in xenograft animal model. With 5-fluorocytosine (5-FC), CPNP-delivered shVEGF-CDTK DNA (CPNP-shVEGF-CDTK) showed high expression of fused yCDglyTK gene and effectively silenced VEGF-A expression in vitro and in vivo, which significantly inhibited colon carcinoma cell proliferation and induced apoptosis in vitro. With 5-FC, the systemic delivery of CPNP-shVEGF-CDTK significantly inhibited tumor growth in the colon carcinoma xenograft animal model. The combined cassette is obviously effective in inhibiting tumor cell proliferation and inducing apoptosis in vitro and tumor growth in vivo than the CPNP-shVEGF or CPNP-CDTK alone. The combination of VEGF-A-silencing and tumor-specific expression of suicide gene is an effective strategy for colon carcinoma treatment.

  14. Light without substrate amendment: the bacterial luciferase gene cassette as a mammalian bioreporter

    Science.gov (United States)

    Close, Dan M.; Xu, Tingting; Smartt, Abby E.; Jegier, Pat; Ripp, Steven A.; Sayler, Gary S.

    2011-06-01

    Bioluminescent production represents a facile method for bioreporter detection in mammalian tissues. The lack of endogenous bioluminescent reactions in these tissues allows for high signal to noise ratios even at low signal strength compared to fluorescent signal detection. While the luciferase enzymes commonly employed for bioluminescent detection are those from class Insecta (firefly and click beetle luciferases), these are handicapped in that they require concurrent administration of a luciferin compound to elicit a bioluminescent signal. The bacterial luciferase (lux) gene cassette offers the advantages common to other bioluminescent proteins, but is simultaneously capable of synthesizing its own luciferin substrates using endogenously available cellular compounds. The longstanding shortcoming of the lux cassette has been its recalcitrance to function in the mammalian cellular environment. This paper will present an overview of the work completed to date to overcome this limitation and provide examples of mammalian lux-based bioreporter technologies that could provide the framework for advanced, biomedically relevant real-time sensor development.

  15. Methicillin-resistant Staphylococcus saprophyticus in Sweden carries various types of staphylococcal cassette chromosome mec (SCCmec).

    Science.gov (United States)

    Söderquist, B; Berglund, C

    2009-12-01

    Staphylococcus saprophyticus is a common cause of uncomplicated urinary tract infections and is usually susceptible to the antimicrobial agents used for their treatment. However, S. saprophyticus resistant to beta-lactam antibiotics and carrying mecA has been reported. Eight Swedish isolates of mecA-positive S. saprophyticus with diverse origin carrying at least three different types of staphylococcal cassette chromosome mec (SCCmec) are described here.

  16. Integration of multiple expression cassettes into mammalian genomes in a single step

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Andrijana Kriz, Katharina Schmid, Kurt Ballmer & Philipp Berger ### Abstract The modification of mammalian cells by the expression of multiple genes is a crucial technology in modern biological research. MultiLabel allows the modular assembly of independent expression units in a single plasmid which can be used for transient and stable modification of cells. In contrast to other methods, the assembly of the expression cassettes does not require restriction enzymes since i...

  17. Conservation of gene cassettes among diverse viruses of the human gut.

    Directory of Open Access Journals (Sweden)

    Samuel Minot

    Full Text Available Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.

  18. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    Directory of Open Access Journals (Sweden)

    Ming Yan

    Full Text Available Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture.

  19. Linear friction weld process monitoring of fixture cassette deformations using empirical mode decomposition

    Science.gov (United States)

    Bakker, O. J.; Gibson, C.; Wilson, P.; Lohse, N.; Popov, A. A.

    2015-10-01

    Due to its inherent advantages, linear friction welding is a solid-state joining process of increasing importance to the aerospace, automotive, medical and power generation equipment industries. Tangential oscillations and forge stroke during the burn-off phase of the joining process introduce essential dynamic forces, which can also be detrimental to the welding process. Since burn-off is a critical phase in the manufacturing stage, process monitoring is fundamental for quality and stability control purposes. This study aims to improve workholding stability through the analysis of fixture cassette deformations. Methods and procedures for process monitoring are developed and implemented in a fail-or-pass assessment system for fixture cassette deformations during the burn-off phase. Additionally, the de-noised signals are compared to results from previous production runs. The observed deformations as a consequence of the forces acting on the fixture cassette are measured directly during the welding process. Data on the linear friction-welding machine are acquired and de-noised using empirical mode decomposition, before the burn-off phase is extracted. This approach enables a direct, objective comparison of the signal features with trends from previous successful welds. The capacity of the whole process monitoring system is validated and demonstrated through the analysis of a large number of signals obtained from welding experiments.

  20. Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.

    Science.gov (United States)

    Das, Sabyasachi; Li, Jianxu; Holland, Stephen J; Iyer, Lakshminarayan M; Hirano, Masayuki; Schorpp, Michael; Aravind, L; Cooper, Max D; Boehm, Thomas

    2014-10-14

    Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA(+) and VLRC(+) lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the αβ and γδ T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes.

  1. Circulating cathodic antigen cassette test versus haematuria strip test in diagnosis of urinary schistosomiasis.

    Science.gov (United States)

    El-Ghareeb, Azza S; Abd El Motaleb, Ghada S; Waked, Nevien Maher; Osman Hany Kamel, Nancy; Aly, Nagwa Shaban

    2016-12-01

    Urinary schistosomiasis caused by Schistosoma haematobium constitutes a major public health problem in many tropical and sub-tropical countries. This study was conducted to evaluate circulating cathodic antigen cassette test and haematuria strip test for detection of S. haematobium in urine samples and to evaluate their screening performance among the study population. Microscopy was used as a gold standard. A total of 600 urine samples were examined by microscopy for detection of S. haematobium eggs, screened for microhaematuria using Self-Stik reagent strips and screened for circulating cathodic antigen (CCA) using the urine-CCA cassette test. The specificity of CCA, microhaematuria and macrohaematuria was 96.4, 40.6 and 31.2 % respectively while the sensitivity was 88.2, 99.3 and 100 % respectively which was statistically significant (P < 0.001). These findings suggest that using of urine-CCA cassette test in diagnosis of urinary schistosomiasis is highly specific (96.4 %) compared with the highly sensitive haematuria strip test (100 %). The degree of agreement between microscopic examination and CCA detection was 99.3 % with highly statistically significant difference (P < 0.001). The combination of two techniques could potentially use for screening and mapping of S. haematobium infection.

  2. Extending the Glucosyl Ceramide Cassette Approach: Application in the Total Synthesis of Ganglioside GalNAc-GM1b

    Directory of Open Access Journals (Sweden)

    Miku Konishi

    2013-12-01

    Full Text Available The development of a novel cyclic glucosyl ceramide cassette acceptor for efficient glycolipid syntheses was investigated. p-Methoxybenzyl (PMB groups were selected as protecting groups at C2 and C3 of the glucose residue with the aim of improving the functionality of the cassette acceptor. The choice of the PMB group resulted in a loss of β-selectivity, which was corrected by using an appropriate tether to control the spatial arrangement and the nitrile solvent effect. To investigate the effect of linker structure on the β-selectivity of intramolecular glycosylation, several linkers for tethering the glucose and ceramide moiety were designed and prepared, namely, succinyl, glutaryl, dimethylmalonyl, and phthaloyl esters. The succinyl ester linker was the best for accessing the cassette form. The newly designed glucosyl ceramide cassette acceptor was then applied in the total synthesis of ganglioside GalNAc-GM1b.

  3. Enhanced performance of methamphetamine lateral flow cassettes using an electronic lateral flow reader.

    Science.gov (United States)

    Smith, Jerome P; Sammons, Deborah L; Robertson, Shirley A; Snawder, John E

    2015-01-01

    Surface contamination from methamphetamine in meth labs continues to be a problem. We had previously developed a lateral flow assay cassette for field detection of methamphetamine contamination that is commercially available and has been used by a number of groups to assess contamination. This cassette uses the complete disappearance of the test line as an end point for detection of 50 ng/100 cm2 of methamphetamine contamination for surface sampling with cotton swabs. In the present study, we further evaluate the response of the cassettes using an electronic lateral flow reader to measure the intensities of the test and control lines. The cassettes were capable of detecting 0.25 ng/ml for calibration solutions. For 100 cm2 ceramic tiles that were spiked with methamphetamine and wiped with cotton-tipped wooden swabs wetted in assay/sampling buffer, 1 ng/tile was detected using the reader. Semi-quantitative results can be produced over the range 0-10 ng/ml for calibration solutions and 0-25 ng/tile for spiked tiles using either a 4-parameter logistic fit of test line intensity versus concentration or spiked mass or the ratio of the control line to the test line intensity fit to concentration or spiked mass. Recovery from the tiles was determined to be about 30% using the fitted curves. Comparison of the control line to the test line was also examined as a possible visual detection end point and it was found that the control line became more intense than the test line at 0.5 to 1 ng/ml for calibration solutions or 1 to 2 ng/tile for spiked tiles. Thus the lateral flow cassettes for methamphetamine have the potential to produce more sensitive semi-quantitative results if an electronic lateral flow reader is used and can be more sensitive for detection if the comparison of the control line to the test line is used as the visual end point.

  4. THE ROLE OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN (MRP) IN THE BLOOD-BRAIN BARRIER AND OPIOID ANALGESIA

    OpenAIRE

    Su, Wendy; Pasternak, Gavril W.

    2013-01-01

    The blood brain barrier protects the brain from circulating compounds and drugs. The ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) is involved with the barrier, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood, raising the possibility of a similar role for other transporters. Multidrug resistance associated protein (MRP), a 190 kDa protein similar to Pgp is also ABC transport that has been im...

  5. Staphylococcal Cassette Chromosome mec Types Among Methicillin-Resistant Staphylococcus aureus in Northern Iran

    Science.gov (United States)

    Taherirad, Akram; Jahanbakhsh, Roghayeh; Shakeri, Fatemeh; Anvary, Shaghayegh; Ghaemi, Ezzat Allah

    2016-01-01

    Background Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of nosocomial and community-acquired infections around the world. Staphylococcal cassette chromosome mec (SCCmec) typing methods are often used to study MRSA molecular epidemiology. Objectives The current study was designed to explore the distribution profiles of different SCCmec types among methicillin-resistant S. aureus strains isolated from hospitals in Gorgan, in northern Iran, and to correlate the types into observed bacterial virulence factors. Materials and Methods Staphylococcal cassette chromosome mec typing of 62 MRSA strains isolated from patients and health-care workers in Gorgan was performed using multiplex polymerase chain reaction (PCR) assay. The prevalence of the strains was then compared according to isolation source, antibiotic susceptibility profiles, biofilm production, and the presence of the Panton-Valentine gene in isolates. Results The most common SCCmec type was type III, with a frequency rate of 76%, followed by types IV, I, and V, with frequency rates of 11.2%, 4.8%, and 3.2%, respectively; three isolates (4.8%) were not typeable by this method. SCCmec type I was only isolated from blood culture, and types IV and V were mainly isolated from wounds and urine samples; SCCmec type III was isolated from all of the clinically samples. All of the MRSA strains that were isolated from healthy carriers were type III. Multidrug resistance in the type III strains was higher compared to the other types. The frequencies of Panton-Valentine and biofilm production were significantly lower in the type III strains compared to the other SCCmec types (P < 0.05). Conclusions Similarly to other geographical regions of Iran, the SCCmec type III MRSA strain was the most frequently isolated strain from patients in Gorgan. Staphylococcal cassette chromosome mec type III showed fewer virulence factors compared to other SCCmec types. PMID:27800133

  6. Hygromycin B and apramycin antibiotic resistance cassettes for use in Campylobacter jejuni.

    Directory of Open Access Journals (Sweden)

    Andrew Cameron

    Full Text Available Campylobacter jejuni genetic manipulation is restricted by the limited number of antibiotic resistance cassettes available for use in this diarrheal pathogen. In this study, two antibiotic resistance cassettes were developed, encoding for hygromycin B and apramycin resistance, for use in mutagenesis or for selection of gene expression and complementation constructs in C. jejuni. First, the marker genes were successfully modified to allow for insertional mutagenesis or deletion of a gene-of-interest, and were bracketed with restriction sites for the facilitation of site-specific cloning. These hygromycin B and apramycin markers are encoded by plasmids pAC1H and pAC1A, respectively. We also modified an insertional gene-delivery vector to create pRRH and pRRA, containing the hygromycin B and apramycin resistance genes, and 3 unique restriction sites for the directional introduction of genes into the conserved multi-copy rRNA gene clusters of the C. jejuni chromosome. We determined the effective antibiotic concentrations required for selection, and established that no harmful effects or fitness costs were associated with carrying hygromycin B or apramycin resistance under standard C. jejuni laboratory conditions. Using these markers, the arylsulfatase reporter gene astA was deleted, and the ability to genetically complement the astA deletion using pRRH and pRRA for astA gene insertion was demonstrated. Furthermore, the relative levels of expression from the endogenous astA promoter were compared to that of polycistronic mRNA expression from the constitutive promoter upstream of the resistance gene. The development of additional antibiotic resistance cassettes for use in Campylobacter will enable multiple gene deletion and expression combinations as well as more in-depth study of multi-gene systems important for the survival and pathogenesis of this important bacterium.

  7. A new tocograph with cassette recording system and separate servo graphic recorder.

    Science.gov (United States)

    Zahn, V; Seitz, P

    1979-01-01

    In order to register contractional activity, especially in the case of high-risk pregnancies, a tocograph was develop by means of which the contractions are registered by a small cassette recorder, which the patient can carry by about with her. A separate graphic recorder is responsible for the playback and this recorder remains at the doctor's practice. The patient is able to register her contractions herself as the unit is so simple to use. The recording section weighs only 500 grams, including the specially developed pressure transducer with optical distance-meter. The tocograph is produced in series.

  8. THE REAL ISSUE WITH WALL DEPOSITS IN CLOSED FILTER CASSETTES - WHAT'S THE SAMPLE?

    Energy Technology Data Exchange (ETDEWEB)

    Brisson, M.

    2009-09-12

    The measurement of aerosol dusts has long been utilized to assess the exposure of workers to metals. Tools used to sample and measure aerosol dusts have gone through many transitions over the past century. In particular, there have been several different techniques used to sample for beryllium, not all of which might be expected to produce the same result. Today, beryllium samples are generally collected using filters housed in holders of several different designs, some of which are expected to produce a sample that mimics the human capacity for dust inhalation. The presence of dust on the interior walls of cassettes used to hold filters during metals sampling has been discussed in the literature for a number of metals, including beryllium, with widely varying data. It appears that even in the best designs, particulates can enter the sampling cassette and deposit on the interior walls rather than on the sampling medium. The causes are not well understood but are believed to include particle bounce, electrostatic forces, particle size, particle density, and airflow turbulence. Historically, the filter catch has been considered to be the sample, but the presence of wall deposits, and the potential that the filter catch is not representative of the exposure to the worker, puts that historical position into question. This leads to a fundamental question: What is the sample? This article reviews the background behind the issue, poses the above-mentioned question, and discusses options and a possible path forward for addressing that question.

  9. Fully Automated Data Collection Using PAM and the Development of PAM/SPACE Reversible Cassettes

    Science.gov (United States)

    Hiraki, Masahiko; Watanabe, Shokei; Chavas, Leonard M. G.; Yamada, Yusuke; Matsugaki, Naohiro; Igarashi, Noriyuki; Wakatsuki, Soichi; Fujihashi, Masahiro; Miki, Kunio; Baba, Seiki; Ueno, Go; Yamamoto, Masaki; Suzuki, Mamoru; Nakagawa, Atsushi; Watanabe, Nobuhisa; Tanaka, Isao

    2010-06-01

    To remotely control and automatically collect data in high-throughput X-ray data collection experiments, the Structural Biology Research Center at the Photon Factory (PF) developed and installed sample exchange robots PAM (PF Automated Mounting system) at PF macromolecular crystallography beamlines; BL-5A, BL-17A, AR-NW12A and AR-NE3A. We developed and installed software that manages the flow of the automated X-ray experiments; sample exchanges, loop-centering and X-ray diffraction data collection. The fully automated data collection function has been available since February 2009. To identify sample cassettes, PAM employs a two-dimensional bar code reader. New beamlines, BL-1A at the Photon Factory and BL32XU at SPring-8, are currently under construction as part of Targeted Proteins Research Program (TPRP) by the Ministry of Education, Culture, Sports, Science and Technology of Japan. However, different robots, PAM and SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), will be installed at BL-1A and BL32XU, respectively. For the convenience of the users of both facilities, pins and cassettes for PAM and SPACE are developed as part of the TPRP.

  10. Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

    Science.gov (United States)

    Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

    2016-02-01

    Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence.

  11. Recent advances on host plants and expression cassettes' structure and function in plant molecular pharming.

    Science.gov (United States)

    Makhzoum, Abdullah; Benyammi, Roukia; Moustafa, Khaled; Trémouillaux-Guiller, Jocelyne

    2014-04-01

    Plant molecular pharming is a promising system to produce important recombinant proteins such as therapeutic antibodies, pharmaceuticals, enzymes, growth factors, and vaccines. The system provides an interesting alternative method to the direct extraction of proteins from inappropriate source material while offering the possibility to overcome problems related to product safety and source availability. Multiple factors including plant hosts, genes of interest, expression vector cassettes, and extraction and purification techniques play important roles in the plant molecular pharming. Plant species, as a biosynthesis platform, are a crucial factor in achieving high yields of recombinant protein in plant. The choice of recombinant gene and its expression strategy is also of great importance in ensuring a high amount of the recombinant proteins. Many studies have been conducted to improve expression, accumulation, and purification of the recombinant protein from molecular pharming systems. Re-engineered vectors and expression cassettes are also pivotal tools in enhancing gene expression at the transcription and translation level, and increasing protein accumulation, stability, retention and targeting of specific organelles. In this review, we report recent advances and strategies of plant molecular pharming while focusing on the choice of plant hosts and the role of some molecular pharming elements and approaches: promoters, codon optimization, signal sequences, and peptides used for upstream design, purification and downstream processing.

  12. Site-specific integration and tailoring of cassette design for sustainable gene transfer.

    Science.gov (United States)

    Lombardo, Angelo; Cesana, Daniela; Genovese, Pietro; Di Stefano, Bruno; Provasi, Elena; Colombo, Daniele F; Neri, Margherita; Magnani, Zulma; Cantore, Alessio; Lo Riso, Pietro; Damo, Martina; Pello, Oscar M; Holmes, Michael C; Gregory, Philip D; Gritti, Angela; Broccoli, Vania; Bonini, Chiara; Naldini, Luigi

    2011-08-21

    Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

  13. Electricity generation from cattle manure slurry by cassette-electrode microbial fuel cells.

    Science.gov (United States)

    Inoue, Kengo; Ito, Toshihiro; Kawano, Yoshihiro; Iguchi, Atsushi; Miyahara, Morio; Suzuki, Yoshihiro; Watanabe, Kazuya

    2013-11-01

    Cassette-electrode microbial fuel cells (CE-MFCs) are efficient and scalable devices for electricity production from organic waste. Previous studies have demonstrated that CE-MFCs are capable of generating electricity from artificial wastewater at relatively high efficiencies. In this study, a single-cassette CE-MFC was constructed, and its capacity for electricity generation from cattle manure suspended in water (solid to water ratio of 1:50) was examined. The CE-MFC reactor was operated in batch mode for 49 days; electricity generation became stable 2 weeks after initiating the operation. The maximum power density was measured at 16.3 W m⁻³ on day 26. Sequencing analysis of PCR-amplified 16S rRNA gene fragments obtained from the original manure and from anode biofilms suggested that Chloroflexi and Geobacteraceae were abundant in the anode biofilm (29% and 18%, respectively), whereas no Geobacteraceae sequences were detected in the original manure sample. The results of this study suggest that CE-MFCs can be used to generate electricity from water-suspended cattle manure in a scalable MFC system.

  14. Sub classification and targeted characterization of prophage-encoded two-component cell lysis cassette

    Indian Academy of Sciences (India)

    K V Srividhya; S Krishnaswamy

    2007-08-01

    Bacteriophage induced lysis of host bacterial cell is mediated by a two component cell lysis cassette comprised of holin and lysozyme. Prophages are integrated forms of bacteriophages in bacterial genomes providing a repertoire for bacterial evolution. Analysis using the prophage database (http://bicmku.in:8082) constructed by us showed 47 prophages were associated with putative two component cell lysis genes. These proteins cluster into four different subgroups. In this process, a putative holin (essd) and endolysin (ybcS), encoded by the defective lambdoid prophage DLP12 was found to be similar to two component cell lysis genes in functional bacteriophages like p21 and P1. The holin essd was found to have a characteristic dual start motif with two transmembrane regions and C-terminal charged residues as in class II holins. Expression of a fusion construct of essd in Escherichia coli showed slow growth. However, under appropriate conditions, this protein could be over expressed and purified for structure function studies. The second component of the cell lysis cassette, ybcS, was found to have an N-terminal SAR (Signal Arrest Release) transmembrane domain. The construct of ybcS has been over expressed in E. coli and the purified protein was functional, exhibiting lytic activity against E. coli and Salmonella typhi cell wall substrate. Such targeted sequence-structure-function characterization of proteins encoded by cryptic prophages will help understand the contribution of prophage proteins to bacterial evolution.

  15. Conserved allosteric hot spots in the transmembrane domains of cystic fibrosis transmembrane conductance regulator (CFTR) channels and multidrug resistance protein (MRP) pumps.

    Science.gov (United States)

    Wei, Shipeng; Roessler, Bryan C