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Sample records for atp-binding cassette transporter

  1. Human ATP-binding cassette (ABC transporter family

    Directory of Open Access Journals (Sweden)

    Vasiliou Vasilis

    2009-04-01

    Full Text Available Abstract There exist four fundamentally different classes of membrane-bound transport proteins: ion channels; transporters; aquaporins; and ATP-powered pumps. ATP-binding cassette (ABC transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx or out (efflux of cells. In mammals, ABC transporters are expressed predominantly in the liver, intestine, blood-brain barrier, blood-testis barrier, placenta and kidney. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. That ABC genes are important is underscored by the fact that mutations in at least I I of these genes are already known to cause severe inherited diseases (eg cystic fibrosis and X-linked adrenoleukodystrophy [X-ALD]. ABC transporters also participate in the movement of most drugs and their metabolites across cell surface and cellular organelle membranes; thus, defects in these genes can be important in terms of cancer therapy, pharmacokinetics and innumerable pharmacogenetic disorders.

  2. Pharmacogenetics of ATP-binding cassette transporters and clinical implications.

    Science.gov (United States)

    Cascorbi, Ingolf; Haenisch, Sierk

    2010-01-01

    Drug resistance is a severe limitation of chemotherapy of various malignancies. In particular efflux transporters of the ATP-binding cassette family such as ABCB1 (P-glycoprotein), the ABCC (multidrug resistance-associated protein) family, and ABCG2 (breast cancer resistance protein) have been identified as major determinants of chemoresistance in tumor cells. Bioavailability depends not only on the activity of drug metabolizing enzymes but also to a major extent on the activity of drug transport across biomembranes. They are expressed in the apical membranes of many barrier tissues such as the intestine, liver, blood-brain barrier, kidney, placenta, testis, and in lymphocytes, thus contributing to plasma, liquor, but also intracellular drug disposition. Since expression and function exhibit a broad variability, it was hypothesized that hereditary variances in the genes of membrane transporters could explain at least in part interindividual differences of pharmacokinetics of a variety of anticancer drugs and many others contributing to the clinical outcome of certain leukemias and further malignancies.

  3. ATP-binding cassette transporters in reproduction: a new frontier

    Science.gov (United States)

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

    2016-01-01

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

  4. Identification and Characterization of an ATP Binding Cassette l-Carnitine Transporter in Listeria monocytogenes

    OpenAIRE

    Fraser, Katy R.; Harvie, Duncan; Coote, Peter J.; O'Byrne, Conor P.

    2000-01-01

    We identified an operon in Listeria monocytogenes EGD with high levels of sequence similarity to the operons encoding the OpuC and OpuB compatible solute transporters from Bacillus subtilis, which are members of the ATP binding cassette (ABC) substrate binding protein-dependent transporter superfamily. The operon, designated opuC, consists of four genes which are predicted to encode an ATP binding protein (OpuCA), an extracellular substrate binding protein (OpuCC), and two membrane-associated...

  5. 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....... 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. Serum albumin can serve as sterol...... donor in ATP-binding cassette-transporter-dependent sterol uptake, a process potentially important for growth of Candida glabrata inside infected humans....

  6. ATP-binding cassette (ABC) transporters in normal and pathological lung

    NARCIS (Netherlands)

    van der Deen, M; de Vries, EGE; Timens, W; Scheper, RJ; Timmer-Bosscha, H; Postma, DS

    2005-01-01

    ATP-binding cassette ( ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein ( P-gp), multidrug resistance-associated protein 1 ( MRP1) and

  7. Metabolism of ATP-binding cassette drug transporter inhibitors: complicating factor for multidrug resistance

    NARCIS (Netherlands)

    Cnubben, N.H.P.; Wortelboer, H.M.; Zanden, van J.J.; Rietjens, I.M.C.M.; Bladeren, van P.J.

    2005-01-01

    Membrane transport proteins belonging to the ATP-binding cassette (ABC) family of transport proteins play a central role in the defence of organisms against toxic compounds, including anticancer drugs. However, for compounds that are designed to display a toxic effect, this defence system diminishes

  8. Metabolism of ATP-binding cassette drug transporter inhibitors: complicating factor for multidrug resistance.

    NARCIS (Netherlands)

    Cnubben, N.H.; Wortelboer, H.M.; Zanden, J.J. van; Rietjens, I.M.; Bladeren, P.J. van

    2005-01-01

    Membrane transport proteins belonging to the ATP-binding cassette (ABC) family of transport proteins play a central role in the defence of organisms against toxic compounds, including anticancer drugs. However, for compounds that are designed to display a toxic effect, this defence system diminishes

  9. Association of ATP-binding cassette transporter-A1 polymorphism ...

    Indian Academy of Sciences (India)

    environment might be modified in a more personalized fash- ion to help prevent or delay ... Journal of Genetics, Vol. 90, No. 1, April 2011 ..... aged children. Metabolism 53, 182–186. Yin K., Liao D. F. and Tang C. K. 2010 ATP-binding mem- brane cassette transporter A1 (ABCA1): a possible link between inflammation and ...

  10. Role of family D ATP-binding cassette transporters (ABCD) in cancer.

    Science.gov (United States)

    Hlaváč, Viktor; Souček, Pavel

    2015-10-01

    ATP-binding cassette (ABC) transporters, belonging to the family D, are expressed in peroxisomes, endoplasmic reticulum or lysosomes. ABCD transporters play a role in transport of lipids, bile acids and vitamin B12 and associate with peroxisomal disorders. ABCD1 performs transport of coenzyme A esters of very-long-chain fatty acids (VLCFA) in peroxisomes and a number of mutations in ABCD1 gene were linked to an X-linked adrenoleucodystrophy (X-ALD). The role of ABCD transporters in tumour growth has not been studied in detail, but there is some evidence that ABCDs levels differ between undifferentiated stem or tumour cells and differentiated cells suggesting a possible link to tumorigenesis. In this mini-review, we discuss the available information about the role of ABCD transporters in cancer. © 2015 Authors; published by Portland Press Limited.

  11. Molecular basis of multidrug transport by ATP-binding cassette transporters : A proposed two-cylinder engine model

    NARCIS (Netherlands)

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

    ATP-binding cassette multidrug transporters are probably present in all living cells, and are able to export a variety of structurally unrelated compounds at the expense of ATP hydrolysis. The elevated expression of these proteins in multidrug resistant cells interferes with the drug-based control

  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. Phylogenetic analysis of the ATP-binding cassette transporter family in three mosquito species.

    Science.gov (United States)

    Lu, Hong; Xu, Yongyu; Cui, Feng

    2016-09-01

    The ATP-binding cassette (ABC) transporter family functions in the ATP-dependent transportation of various substrates across biological membranes. ABC proteins participate in various biological processes and insecticide resistance in insects, and are divided into eight subfamilies (A-H). Mosquitoes are important vectors of human diseases, but the mechanism by which the ABC transporter family evolves in mosquitoes is unknown. In this study, we classified and compared the ABC transporter families of three mosquitoes, namely, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus. The three mosquitoes have 55, 69, and 70 ABC genes, respectively. The C. p. quinquefasciatus had approximately 40% and 65% expansion in the ABCG subfamily, mainly in ABCG1/G4, compared with the two other mosquito species. The ABCB, ABCD, ABCE, and ABCF subfamilies were conserved in the three mosquito species. The C. p. quinquefasciatus transcriptomes during development showed that the ABCG and ABCC genes were mainly highly expressed at the egg and pupal stages. The pigment-transport relative brown, white, and scarlet, as well as the ABCF subfamily, were highly expressed at the egg stage. The highly expressed genes in larvae included three ABCA3 genes. The majority of the highly expressed genes in adults were ABCG1/4 genes. These results provided insights into the evolution of the ABC transporter family in mosquitoes. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Expression of ATP-binding cassette membrane transporters in a HIV-1 transgenic rat model.

    Science.gov (United States)

    Robillard, Kevin R; Hoque, Md Tozammel; Bendayan, Reina

    2014-02-21

    P-glycoprotein (P-gp, product of Mdr1a and Mdr1b genes), multidrug resistance associated proteins (Mrps), and breast cancer resistance protein (Bcrp), all members of the ATP-binding cassette (ABC) membrane-associated drug transporters superfamily, can significantly restrict the entry of antiretroviral drugs (ARVs) into organs which exhibit a barrier function such as the central nervous system (CNS) and the male genital tract (MGT). In vitro, HIV-1 viral proteins such as glycoprotein-120 (gp120) and transcriptional transactivator (tat) have been shown to alter the expression of these transporters and ARVs permeability. The objective of this study was to compare mRNA expression of these transporters, in vivo, in several tissues obtained from HIV-1 transgenic rats (Tg-rat) (8 and 24 weeks) with those of age-matched wild-type rats. At 24 weeks, significant changes in several drug transporter mRNA expressions were observed, in particular, in brain, kidney, liver and testes. These findings suggest that HIV-1 viral proteins can alter the expression of ABC drug transporters, in vivo, in the context of HIV-1 and further regulate ARVs permeability in several organs including the CNS and MGT, two sites which have been reported to display very low ARVs permeability in the clinic. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC Transporters

    Directory of Open Access Journals (Sweden)

    Pierre Andreoletti

    2017-07-01

    Full Text Available The peroxisomal ATP-binding Cassette (ABC transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD. Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85  Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues.

  16. 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

    2017-08-01

    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. Copyright © 2017. Published by Elsevier B.V.

  17. Role of pharmacogenetics of ATP-binding cassette transporters in the pharmacokinetics of drugs.

    Science.gov (United States)

    Cascorbi, Ingolf

    2006-11-01

    Interindividual differences of drug response are an important cause of treatment failures and adverse drug reactions. The identification of polymorphisms explaining distinct phenotypes of drug metabolizing enzymes contributed in part to the understanding of individual variations of drug plasma levels. However, bioavailability also depends on a major extent from the expression and activity of drug transport across biomembranes. In particular efflux transporters of the ATP-binding cassette (ABC) family such as ABCB1 (P-glycoprotein, P-gp), the ABCC (multidrug resistance-related protein, MRP) family and ABCG2 (breast cancer resistance protein, BCRP) have been identified as major determinants of chemoresistance in tumor cells. They are expressed in the apical membranes of many barrier tissue such as the intestine, liver, blood-brain barrier, kidney, placenta, testis and in lymphocytes, thus contributing to plasma, liquor, but also intracellular drug disposition. Since expression and function exhibit a broad variability, it was hypothesized that hereditary variances in the genes of membrane transporters could explain at least in part interindividual differences of pharmacokinetics and clinical outcome of a variety of drugs. This review focuses on the functional significance of single nucleotide polymorphisms (SNP) of ABCB1, ABCC1, ABCC2, and ABCG2 in in vitro systems, in vivo tissues and drug disposition, as well as on the clinical outcome of major indications.

  18. ATP-binding cassette (ABC transporters in normal and pathological lung

    Directory of Open Access Journals (Sweden)

    Timmer-Bosscha Hetty

    2005-06-01

    Full Text Available Abstract ATP-binding cassette (ABC transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp, multidrug resistance-associated protein 1 (MRP1 and breast cancer resistance protein (BCRP are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (malfunction in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases.

  19. 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

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

    Science.gov (United States)

    Lara, Flavio Alves; Pohl, Paula C.; Gandara, Ana Caroline; Ferreira, Jessica da Silva; Nascimento-Silva, Maria Clara; Bechara, Gervásio Henrique; Sorgine, Marcos H. F.; Almeida, Igor C.; Vaz, Itabajara da Silva; Oliveira, Pedro L.

    2015-01-01

    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 represent a new

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

    OpenAIRE

    Aberuyi, N; S. Rahgozar; 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...

  2. A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6.

    Directory of Open Access Journals (Sweden)

    Kishore Polireddy

    Full Text Available ABCB6 is a member of the adenosine triphosphate (ATP-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS, can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.

  3. 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...

  4. Association of ATP-binding cassette transporter-A1 polymorphism ...

    Indian Academy of Sciences (India)

    binding cassette transporter-A1 polymorphism with apolipoprotein AI level in Tehranian population. Sohrab Halalkhor Seyed Alireza Mesbah-Namin Maryam Sadat Daneshpour Mehdi Hedayati Fereidoun Azizi. Research Note Volume 90 Issue 1 ...

  5. 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

  6. 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 (...

  7. ATP-binding membrane cassette transporter A1 (ABCA1): a possible link between inflammation and reverse cholesterol transport.

    Science.gov (United States)

    Yin, Kai; Liao, Duan-fang; Tang, Chao-ke

    2010-01-01

    Atherosclerosis is characterized by a chronic inflammatory condition that involves numerous cellular and molecular inflammatory components. A wide array of inflammatory mediators, such as cytokines and proteins produced by macrophages and other cells, play a critical role in the development and progression of the disease. ATP-binding membrane cassette transporter A1 (ABCA1) is crucial for cellular cholesterol efflux and reverse cholesterol transport (RCT) and is also identified as an important target in antiatherosclerosis treatment. Evidence from several recent studies indicates that inflammation, along with other atherogenic-related mediators, plays distinct regulating roles in ABCA1 expression. Proatherogenic cytokines such as interferon (IFN)-γ and interleukin (IL)-1β have been shown to inhibit the expression of ABCA1, while antiatherogenic cytokines, including IL-10 and transforming growth factor (TGF)-β1, have been shown to promote the expression of ABCA1. Moreover, some cytokines such as tumor necrosis factor (TNF)-α seem to regulate ABCA1 expression in species-specific and dose-dependent manners. Inflammatory proteins such as C-reactive protein (CRP) and cyclooxygenase (COX)-2 are likely to inhibit ABCA1 expression during inflammation, and inflammation induced by lipopolysaccharide (LPS) was also found to block the expression of ABCA1. Interestingly, recent experiments revealed ABCA1 can function as an antiinflammatory receptor to suppress the expression of inflammatory factors, suggesting that ABCA1 may be the molecular basis for the interaction between inflammation and RCT. This review aims to summarize recent findings on the role of inflammatory cytokines, inflammatory proteins, inflammatory lipids, and the endotoxin-mediated inflammatory process in expression of ABCA1. Also covered is the current understanding of the function of ABCA1 in modulating the immune response and inflammation through its direct and indirect antiinflammatory mechanisms

  8. Identification and characterization of novel loss of function mutations in ATP-binding cassette transporter A1 in patients with low plasma high-density lipoprotein cholesterol

    NARCIS (Netherlands)

    Candini, C.; Schimmel, A. W.; Peter, J.; Bochem, A. E.; Holleboom, A. G.; Vergeer, M.; Dullaart, R. P. F.; Dallinga-Thie, G. M.; Hovingh, G. K.; Khoo, K. L.; Fasano, T.; Bocchi, L.; Calandra, S.; Kuivenhoven, J. A.; Motazacker, M. M.

    2010-01-01

    Objectives: The current literature provides little information on the frequency of mutations in the ATP-binding cassette transporter A1 (ABCA1) in patients with low high-density lipoprotein cholesterol (HDL) levels that are referred to the clinic. In 78 patients with low plasma levels of HDL

  9. Endocrine Disruptors Differentially Target ATP-Binding Cassette Transporters in the Blood-Testis Barrier and Affect Leydig Cell Testosterone Secretion In Vitro

    NARCIS (Netherlands)

    Dankers, A.C.A.; Roelofs, M.J.; Piersma, A.H.; Sweep, F.C.; Russel, F.G.M.; Berg, M. van den; Duursen, M.B. van; Masereeuw, R.

    2013-01-01

    Endocrine-disrupting chemicals (EDCs) are considered to cause testicular toxicity primarily via interference with steroid hormone function. Alternatively, EDCs could possibly exert their effects by interaction with ATP-binding cassette (ABC) transporters that are expressed in the blood-testis

  10. Regulation of ATP-binding cassette transporters and cholesterol efflux by glucose in primary human monocytes and murine bone marrow-derived macrophages

    Science.gov (United States)

    Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. Two models were used...

  11. ATP-binding-cassette transporters in hematopoietic stem cells and their utility as therapeutical targets in acute and chronic myeloid leukemia.

    NARCIS (Netherlands)

    Raaijmakers, M.

    2007-01-01

    ATP-binding-cassette (ABC) transporters are evolutionary extremely well-conserved transmembrane proteins that are highly expressed in hematopoietic stem cells (HSCs). The physiological function in human stem cells is believed to be protection against genetic damage caused by both environmental and

  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. Relation between hepatic expression of ATP-binding cassette transporters G5 and G8 and biliary cholesterol secretion in mice

    NARCIS (Netherlands)

    Kosters, A; Frijters, RJJM; Schaap, FG; Vink, E; Plosch, T; Ottenhoff, R; Jirsa, M; De Cuyper, IM; Kuipers, F; Groen, AK

    Background/Aims: Mutations in genes encoding the ATP-binding cassette (ABC)-transporters ABCG5 and ABCG8 underlie sitosterolemia, which is characterized by elevated plasma levels of phytosterols due to increased intestinal absorption and impaired biliary secretion of sterols. The aim of our study

  14. Oct-3/4 modulates the drug-resistant phenotype of glioblastoma cells through expression of ATP binding cassette transporter G2.

    Science.gov (United States)

    Hosokawa, Yuki; Takahashi, Hisaaki; Inoue, Akihiro; Kawabe, Yuya; Funahashi, Yu; Kameda, Kenji; Sugimoto, Kana; Yano, Hajime; Harada, Hironobu; Kohno, Shohei; Ohue, Shiro; Ohnishi, Takanori; Tanaka, Junya

    2015-06-01

    Drug resistance is a major obstacle for the efficacy of chemotherapeutic treatment of tumors. Oct-3/4, a self-renewal regulator in stem cells, is expressed in various kinds of solid tumors including glioblastoma. Although Oct-3/4 expression has been implicated in the malignancy and prognosis of glioblastomas, little is known of its involvement in drug resistances of glioblastoma. The involvement of Oct-3/4 in drug resistance of glioblastoma cells was assessed by lactate dehydrogenase assay, efflux assay of an anticancer drug, poly ADP-ribose polymerase cleavage, and in vivo xenograft experiments. Involvement of a drug efflux pump ATP binding cassette transporter G2 in Oct-3/4-induced drug resistance was evaluated by quantitative PCR analysis and knockdown by shRNA. Oct-3/4 decreased the susceptibility to chemotherapeutic drugs by enhancing excretion of drugs through a drug efflux pump gene, ATP binding cassette transporter G2. Moreover, the expression of Oct-3/4 was well correlated to ATP binding cassette transporter G2 expression in clinical GB tissues. Oct-3/4 elevated the ATP binding cassette transporter G2 expression, leading to acquisition of a drug-resistant phenotype by glioblastoma cells. If the drug-resistance of glioblastoma cells could be suppressed, it should be a highly ameliorative treatment for glioblastoma patients. Therefore, signaling pathways from Oct-3/4 to ATP binding cassette transporter G2 should be intensively elucidated to develop new therapeutic interventions for better efficacy of anti-cancer drugs. Copyright © 2015 Elsevier B.V. All rights reserved.

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

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    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. Association of ATP binding cassette transporter G8 rs4148217 SNP and serum lipid levels in Mulao and Han nationalities

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    Li Qing

    2012-05-01

    Full Text Available Abstract Background The association of ATP binding cassette transporter G8 gene (ABCG8 rs4148217 single nucleotide polymorphism (SNP and serum lipid profiles is still controversial in diverse racial/ethnic groups. Mulao nationality is an isolated minority in China. The aim of this study was to evaluate the association of ABCG8 rs4148217 SNP and several environmental factors with serum lipid levels in the Guangxi Mulao and Han populations. Methods A total of 634 subjects of Mulao nationality and 717 participants of Han nationality were randomly selected from our previous samples. Genotyping of the ABCG8 rs4148217 SNP was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. Results The genotypic and allelic frequencies of ABCG8 rs4148217 SNP were different between the two nationalities (P P P P P P P  Conclusions The ABCG8 rs4148217 SNP is associated with serum TG, HDL-C and ApoA1 levels in our study populations, but this association is different between the Mulao and Han populations. There is a sex (female-specific association in both ethnic groups.

  17. ATP-binding cassette transporter G5 and G8 polymorphisms and several environmental factors with serum lipid levels.

    Science.gov (United States)

    Li, Qing; Yin, Rui-Xing; Wei, Xian-Liang; Yan, Ting-Ting; Aung, Lynn Htet Htet; Wu, Dong-Feng; Wu, Jin-Zhen; Lin, Wei-Xiong; Liu, Cheng-Wu; Pan, Shang-Ling

    2012-01-01

    The association of ATP-binding cassette (ABC) transporter single nucleotide polymorphisms (SNPs) and serum lipid profiles is inconsistent. The present study was undertaken to detect the association of ABCG5/G8 SNPs and several environmental factors with serum lipid levels. Genotyping of the ABCG5 (rs4131229 and rs6720173) and ABCG8 (rs3806471 and rs4148211) SNPs was performed in 719 unrelated subjects of Mulao nationality and 782 participants of Han nationality. There were no differences in the genotypic and allelic frequencies of four SNPs between the two ethnic groups besides the genotypic frequencies of rs4131229 SNP in Han. The levels of triglyceride (TG), apolipoprotein (Apo) A1, and ApoA1/ApoB ratio (rs4131229); low-density lipoprotein cholesterol (LDL-C) and ApoB (rs6720173); high-density lipoprotein cholesterol (HDL-C), ApoA1, ApoB, and ApoA1/ApoB ratio (rs3806471); and HDL-C, ApoA1, and ApoA1/ApoB ratio (rs4148211) in Han were different among their genotypes (PG8 SNPs and serum lipid levels are different between the Mulao and Han populations, or between males and females, suggesting that there may be a racial/ethnic- and/or sex-specific association between ABCG5/G8 SNPs and some serum lipid parameters.

  18. Genome-wide identification, characterization and phylogenetic analysis of 50 catfish ATP-binding cassette (ABC transporter genes.

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    Shikai Liu

    Full Text Available BACKGROUND: Although a large set of full-length transcripts was recently assembled in catfish, annotation of large gene families, especially those with duplications, is still a great challenge. Most often, complexities in annotation cause mis-identification and thereby much confusion in the scientific literature. As such, detailed phylogenetic analysis and/or orthology analysis are required for annotation of genes involved in gene families. The ATP-binding cassette (ABC transporter gene superfamily is a large gene family that encodes membrane proteins that transport a diverse set of substrates across membranes, playing important roles in protecting organisms from diverse environment. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we identified a set of 50 ABC transporters in catfish genome. Phylogenetic analysis allowed their identification and annotation into seven subfamilies, including 9 ABCA genes, 12 ABCB genes, 12 ABCC genes, 5 ABCD genes, 2 ABCE genes, 4 ABCF genes and 6 ABCG genes. Most ABC transporters are conserved among vertebrates, though cases of recent gene duplications and gene losses do exist. Gene duplications in catfish were found for ABCA1, ABCB3, ABCB6, ABCC5, ABCD3, ABCE1, ABCF2 and ABCG2. CONCLUSION/SIGNIFICANCE: The whole set of catfish ABC transporters provide the essential genomic resources for future biochemical, toxicological and physiological studies of ABC drug efflux transporters. The establishment of orthologies should allow functional inferences with the information from model species, though the function of lineage-specific genes can be distinct because of specific living environment with different selection pressure.

  19. Insulin stimulates uric acid reabsorption via regulating urate transporter 1 and ATP-binding cassette subfamily G member 2.

    Science.gov (United States)

    Toyoki, Daigo; Shibata, Shigeru; Kuribayashi-Okuma, Emiko; Xu, Ning; Ishizawa, Kenichi; Hosoyamada, Makoto; Uchida, Shunya

    2017-09-01

    Accumulating data indicate that renal uric acid (UA) handling is altered in diabetes and by hypoglycemic agents. In addition, hyperinsulinemia is associated with hyperuricemia and hypouricosuria. However, the underlying mechanisms remain unclear. In this study, we aimed to investigate how diabetes and hypoglycemic agents alter the levels of renal urate transporters. In insulin-depleted diabetic rats with streptozotocin treatment, both UA excretion and fractional excretion of UA were increased, suggesting that tubular handling of UA is altered in this model. In the membrane fraction of the kidney, the expression of urate transporter 1 (URAT1) was significantly decreased, whereas that of ATP-binding cassette subfamily G member 2 (ABCG2) was increased, consistent with the increased renal UA clearance. Administration of insulin to the diabetic rats decreased UA excretion and alleviated UA transporter-level changes, while sodium glucose cotransporter 2 inhibitor (SGLT2i) ipragliflozin did not change renal UA handling in this model. To confirm the contribution of insulin in the regulation of urate transporters, normal rats received insulin and separately, ipragliflozin. Insulin significantly increased URAT1 and decreased ABCG2 levels, resulting in increased UA reabsorption. In contrast, the SGLT2i did not alter URAT1 or ABCG2 levels, although blood glucose levels were similarly reduced. Furthermore, we found that insulin significantly increased endogenous URAT1 levels in the membrane fraction of NRK-52E cells, the kidney epithelial cell line, demonstrating the direct effects of insulin on renal UA transport mechanisms. These results suggest a previously unrecognized mechanism for the anti-uricosuric effects of insulin and provide novel insights into the renal UA handling in the diabetic state. Copyright © 2017 the American Physiological Society.

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

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    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

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

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    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.

  2. ATP-binding cassette transporter G26 is required for male fertility and pollen exine formation in Arabidopsis.

    Science.gov (United States)

    Quilichini, Teagen D; Friedmann, Michael C; Samuels, A Lacey; Douglas, Carl J

    2010-10-01

    The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires the transport of one or more sporopollenin constituents to the surface of developing microspores. Here, we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis (Arabidopsis thaliana). abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early pollen wall formation, sporopollenin biosynthesis, and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein was localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development and are consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls.

  3. Multidrug resistance protein 1 (MRP1, ABCC1), a "multitasking" ATP-binding cassette (ABC) transporter.

    Science.gov (United States)

    Cole, Susan P C

    2014-11-07

    The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. 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...

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

    Science.gov (United States)

    Yu, Fang; De Luca, Vincenzo

    2013-01-01

    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. PMID:24019465

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

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    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.

  7. Targeted Deletion of Adipocyte Abca1 (ATP-Binding Cassette Transporter A1) Impairs Diet-Induced Obesity.

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    Cuffe, Helen; Liu, Mingxia; Key, Chia-Chi C; Boudyguina, Elena; Sawyer, Janet K; Weckerle, Allison; Bashore, Alexander; Fried, Susan K; Chung, Soonkyu; Parks, John S

    2018-01-18

    Adipose tissue cholesterol increases with adipocyte triglyceride content and size during development of obesity. However, how adipocyte cholesterol affects adipocyte function is poorly understood. The aim of this study was to evaluate the role of the cellular cholesterol exporter, Abca1 (ATP-binding cassette transporter A1), on adipose tissue function during diet-induced obesity. Adiponectin Cre recombinase transgenic mice were crossed with Abca1flox/flox mice to generate ASKO (adipocyte-specific Abca1 knockout) mice. Control and ASKO mice were then fed a high-fat, high-cholesterol (45% calories as fat and 0.2% cholesterol) diet for 16 weeks. Compared with control mice, ASKO mice had a 2-fold increase in adipocyte plasma membrane cholesterol content and significantly lower body weight, epididymal fat pad weight, and adipocyte size. ASKO versus control adipose tissue had decreased PPARγ (peroxisome proliferator-activated receptor γ) and CCAAT/enhancer-binding protein expression, nuclear SREBP1 (sterol regulatory element-binding protein 1) protein, lipogenesis, and triglyceride accretion but similar Akt activation after acute insulin stimulation. Acute siRNA-mediated Abca1 silencing during 3T3L1 adipocyte differentiation reduced adipocyte Abca1 and PPARγ protein expression and triglyceride content. Systemic stimulated triglyceride lipolysis and glucose homeostasis was similar between control and ASKO mice. Adipocyte Abca1 is a key regulator of adipocyte lipogenesis and lipid accretion, likely because of increased adipose tissue membrane cholesterol, resulting in decreased activation of lipogenic transcription factors PPARγ and SREBP1. © 2018 American Heart Association, Inc.

  8. Functional analysis of an ATP-binding cassette transporter protein from Aspergillus fumigatus by heterologous expression in Saccharomyces cerevisiae.

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    Paul, Sanjoy; Moye-Rowley, W Scott

    2013-08-01

    Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Although A. fumigatus can be treated with many of the available antifungal drugs, including azole compounds, drug resistant isolates are being recovered at an increasing rate. In other fungal pathogens such as the Candida species, ATP-binding cassette (ABC) transporter proteins play important roles in development of clinically-significant azole resistance phenotypes. Central among these ABC transporter proteins are homologues of the Saccharomyces cerevisiae Pdr5 multidrug transporter. In this work, we test the two A. fumigatus genes encoding proteins sharing the highest degree of sequence similarity to S. cerevisiae Pdr5 for their ability to be function in a heterologous pdr5Δ strain of S. cerevisiae. Expression of full-length cDNAs for these two Afu proteins failed to suppress the drug sensitive phenotype of a pdr5Δ strain and no evidence could be obtained for their expression as green fluorescent protein (GFP) fusions. To improve the expression of one of these Afu ABC transporters (XP_755847), we changed the sequence of the cDNA to use codons corresponding to the major tRNA species in S. cerevisiae. This codon-optimized (CO Afu abcA) cDNA was efficiently expressed in pdr5Δ cells and able to be detected as a GFP fusion protein. The CO Afu abcA did not correct the drug sensitivity of the pdr5Δ strain and exhibited a high degree of perinuclear fluorescence suggesting that this fusion protein was localized to the S. cerevisiae ER. Interestingly, when these experiments were repeated at 37 °C, the CO Afu abcA was able to complement the drug sensitive phenotype of pdr5Δ cells and exhibited less intracellular fluorescence. Additionally, we found that the CO Afu abcA was able to reduce resistance to drugs like phytosphingosine that act via causing mislocalization of amino acid permeases in fungi. These data suggest that the Afu abcA protein can carry out two different functions of Pdr5: drug

  9. ATP-binding cassette transporter-1 induces rearrangement of actin cytoskeletons possibly through Cdc42/N-WASP.

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    Tsukamoto, K; Hirano, K; Tsujii, K; Ikegami, C; Zhongyan, Z; Nishida, Y; Ohama, T; Matsuura, F; Yamashita, S; Matsuzawa, Y

    2001-09-28

    Positional cloning approaches revealed that Tangier disease (TD), a genetic high density lipoprotein deficiency, is associated with mutations in the ATP-binding cassette transporter-1 (ABCA1) gene. However, the biological function of ABCA1 is still not fully investigated. Recently, we have reported that the cells from the patients with TD had abnormal actin cytoskeletons in association with decreased expression of Cdc42, a member of RhoGTPases family. In the present study, we have found that actin cytoskeletons were altered in HEK293 cells transfected with human ABCA1 (hABCA1) cDNA. Cells expressing hABCA1 were divided into the following two groups by the distinct morphology with altered actin cytoskeletons: one had increased formation of filopodia (designated as Type I) and the other had long protrusions (designated as Type II). Type I cells had morphology similar to that of cells transfected with dominant active form of Cdc42 (Cdc42-DA, V12Cdc42Hs-DA). Type II cells had morphology similar to that of cells transfected with neural Wiskott-Aldrich Syndrome Protein (N-WASP),one of the established downstream effector molecules of Cdc42. We have obtained the data showing a possible pathway of ABCA1/Cdc42/N-WASP by the following experiments. Introduction of mutant of Cdc42 (dominant negative form of Cdc42, N17Cdc42Hs-DN) and N-WASP (N-WASP lacking verprolin homology domain, N-WASPDeltaVPH), both of which are supposed to have potential to inhibit rearrangement of actin cytoskeletons, significantly inhibited the morphological changes induced by expression of hABCA1. Immunoprecipitation study with FLAG-tagged ABCA1 (hABCA1-FLAG) revealed that Cdc42 was coimmunoprecipitated with hABCA1-FLAG. In addition, we have demonstrated possible intracellular colocalization of these two molecules in the overexpressing cells by the confocal laser microscopy. These results may suggest that hABCA1 regulates actin organization through the possible interaction with Cdc42Hs. Copyright 2001

  10. Association of ATP binding cassette transporter G8 rs4148217 SNP and serum lipid levels in Mulao and Han nationalities.

    Science.gov (United States)

    Li, Qing; Wei, Xian-Liang; Yin, Rui-Xing

    2012-05-01

    The association of ATP binding cassette transporter G8 gene (ABCG8) rs4148217 single nucleotide polymorphism (SNP) and serum lipid profiles is still controversial in diverse racial/ethnic groups. Mulao nationality is an isolated minority in China. The aim of this study was to evaluate the association of ABCG8 rs4148217 SNP and several environmental factors with serum lipid levels in the Guangxi Mulao and Han populations. A total of 634 subjects of Mulao nationality and 717 participants of Han nationality were randomly selected from our previous samples. Genotyping of the ABCG8 rs4148217 SNP was performed by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. The genotypic and allelic frequencies of ABCG8 rs4148217 SNP were different between the two nationalities (P < 0.01 for each), the frequency of A allele was higher in Mulao than in Han. The A allele carriers in Han had lower high-density lipoprotein cholesterol (HDL-C) and apolipoprotein (Apo) A1 levels than the A allele noncarriers (P < 0.05 for each), whereas the A allele carriers in Mulao had lower ApoA1 levels than the A allele noncarriers (P < 0.05). Subgroup analyses showed that the A allele carriers in Han had lower HDL-C and higher triglyceride (TG) levels in females but not in males than the A allele noncarriers (P < 0.05 for each), and the A allele carriers in Mulao had lower ApoA1 levels in females but not in males than the A allele noncarriers (P < 0.05). The levels of TG and HDL-C in Han, and ApoA1 in Mulao were associated with genotypes in females but not in males (P < 0.05-0.01). Serum lipid parameters were also correlated with several environmental factors (P < 0.05-0.001). The ABCG8 rs4148217 SNP is associated with serum TG, HDL-C and ApoA1 levels in our study populations, but this association is different between the Mulao and Han populations. There is a sex (female

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

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    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.

  12. Fructose Uptake in Sinorhizobium meliloti Is Mediated by a High-Affinity ATP-Binding Cassette Transport System

    OpenAIRE

    Lambert, Annie; Østerås, Magne; Mandon, Karine; Poggi, Marie-Christine; Le Rudulier, Daniel

    2001-01-01

    By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding ca...

  13. 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...

  14. D-helix influences dimerization of the ATP-binding cassette (ABC transporter associated with antigen processing 1 (TAP1 nucleotide-binding domain.

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    Ahmet S Vakkasoglu

    Full Text Available ATP-binding cassette (ABC transporters form a large family of transmembrane importers and exporters. Using two nucleotide-binding domains (NBDs, which form a canonical ATP-sandwich dimer at some point within the transport cycle, the transporters harness the energy from ATP binding and hydrolysis to drive substrate transport. However the structural elements that enable and tune the dimerization propensity of the NBDs have not been fully elucidated. Here we compared the biochemical properties of the NBDs of human and rat TAP1, a subunit of the heterodimeric transporter associated with antigen processing (TAP. The isolated human TAP1 NBD was monomeric in solution, in contrast to the previously observed ATP-mediated homodimerization of the isolated rat TAP1 NBD. Using a series of human-rat chimeric constructs, we identified the D-helix, an α-helix N-terminal to the conserved D-loop motif, as an important determinant of NBD dimerization. The ATPase activity of our panel of TAP1 NBD constructs largely correlated with dimerization ability, indicating that the observed dimerization uses the canonical ATP-sandwich interface. The N-terminus of the D-helix from one protomer interacts with the ATP-binding Walker A motif of the second protomer at the ATP-sandwich interface. However, our mutational analysis indicated that residues farther from the interface, within the second and third turn of the D-helix, also influence dimerization. Overall, our data suggest that although the D-helix sequence is not conserved in ABC transporters, its precise positioning within the NBD structure has a critical role in NBD dimerization.

  15. Isolation and characterization of the ATP-binding cassette (ABC) transporter system genes from loofah witches' broom phytoplasma.

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    Huang, Chun-Lin; Ho, Kuo-Chieh

    2007-10-01

    A clone containing a 3903 bp EcoRI-restriction fragment was obtained from a lambda(ZAP) genomic library of loofah witches' broom (LfWB) phytoplasma by plaque hybridization using a PCR fragment as a probe. Sequence analysis revealed that this fragment contained three open reading frames (ORFs). The deduced amino acid sequences of ORF 1 and ORF 2 showed a high homology with the ATP-binding proteins of the ABC transporter system genes of prokaryotes and eukaryotes, and encoded proteins with a molecular mass of 36 and 30 kDa, respectively. Based on amino acid sequence similarity, secondary structure, hydrophilicity and a signal peptide sequence at the N-terminus, we predicted that ORF 3 might encode a specific solute-binding prolipoprotein of the ABC transporter system with a molecular mass of 62 kDa. The cleavage site of this prolipoprotein signal peptide was similar to those of gram-positive bacteria. In addition to nutrient uptake, ABC transporter systems of bacteria also play a role in signal transduction, drug-resistance and perhaps virulence. The possible implications of the system to the survival and the pathogenesis of phytoplasma were discussed.

  16. 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

    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...... overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100...... analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol....

  17. Functional roles of YPT31 and YPT32 in clotrimazole resistance of Saccharomyces cerevisiae through effects on vacuoles and ATP-binding cassette transporter(s).

    Science.gov (United States)

    Tsujimoto, Yoshiyuki; Takase, Daisuke; Okano, Hajime; Tomari, Naohiro; Watanabe, Kunihiko; Matsui, Hiroshi

    2013-01-01

    We identified YPT31, which is involved in Golgi traffic, as a clotrimazole (CTZ)-resistance gene in a multicopy library screen. Multicopies of the YPT31 homolog YPT32 also conferred resistance to CTZ, and single disruption of YPT31 or YPT32 resulted in sensitivity to CTZ. Pdr5p, an ATP-binding cassette (ABC) transporter at the plasma membrane, was the most important factor for mediating basal resistance to CTZ, suggesting that Ypt31p and Ypt32p might be involved in the trafficking of Pdr5p to the plasma membrane. However, the activity of Pdr5p was independent of YPT31 or YPT32, and multicopies of YPT31 or YPT32 still conferred resistance to CTZ in pdr5 cells. To elucidate the roles of YPT31 and YPT32 in CTZ resistance, we analyzed mutants of 11 genes that are involved in the following vesicular trafficking: Golgi traffic (kes1, trs33, trs65, gyp1, trs85, and gyp2), vacuole inheritance (ypt7), endocytosis (rcy1 and ypt51) and exocytosis (msb3 and msb4). All of the mutant cells except ypt51, msb3 and msb4 were sensitive to CTZ, indicating that vacuoles were involved in CTZ resistance, since vacuole formation requires proper Golgi-trafficking and endocytosis. Microscopic analysis showed abnormal vacuoles in ypt31 cells. Multicopies of YPT31 or YPT32 conferred resistance to CTZ in AD1-8 cells, which are defective in seven major drug transporters, and in pdr5 ypt7 cells, but not in ypt7 or AD1-8-7 (AD1-8/ypt7) cells. These results indicated that Ypt31p and Ypt32p played minor but compensatory roles in cellular resistance to CTZ through vacuoles and specific ABC transporter(s) other than Pdr5p. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  18. An ATP-binding cassette subfamily G full transporter is essential for the retention of leaf water in both wild barley and rice.

    Science.gov (United States)

    Chen, Guoxiong; Komatsuda, Takao; Ma, Jian Feng; Nawrath, Christiane; Pourkheirandish, Mohammad; Tagiri, Akemi; Hu, Yin-Gang; Sameri, Mohammad; Li, Xinrong; Zhao, Xin; Liu, Yubing; Li, Chao; Ma, Xiaoying; Wang, Aidong; Nair, Sudha; Wang, Ning; Miyao, Akio; Sakuma, Shun; Yamaji, Naoki; Zheng, Xiuting; Nevo, Eviatar

    2011-07-26

    Land plants have developed a cuticle preventing uncontrolled water loss. Here we report that an ATP-binding cassette (ABC) subfamily G (ABCG) full transporter is required for leaf water conservation in both wild barley and rice. A spontaneous mutation, eibi1.b, in wild barley has a low capacity to retain leaf water, a phenotype associated with reduced cutin deposition and a thin cuticle. Map-based cloning revealed that Eibi1 encodes an HvABCG31 full transporter. The gene was highly expressed in the elongation zone of a growing leaf (the site of cutin synthesis), and its gene product also was localized in developing, but not in mature tissue. A de novo wild barley mutant named "eibi1.c," along with two transposon insertion lines of rice mutated in the ortholog of HvABCG31 also were unable to restrict water loss from detached leaves. HvABCG31 is hypothesized to function as a transporter involved in cutin formation. Homologs of HvABCG31 were found in green algae, moss, and lycopods, indicating that this full transporter is highly conserved in the evolution of land plants.

  19. The yeast ATP-binding cassette (ABC) transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion.

    Science.gov (United States)

    Sasser, Terry L; Lawrence, Gus; Karunakaran, Surya; Brown, Christopher; Fratti, Rutilio A

    2013-06-21

    The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd(2+) transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p(K669M)) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.

  20. The Yeast ATP-binding Cassette (ABC) Transporter Ycf1p Enhances the Recruitment of the Soluble SNARE Vam7p to Vacuoles for Efficient Membrane Fusion*

    Science.gov (United States)

    Sasser, Terry L.; Lawrence, Gus; Karunakaran, Surya; Brown, Christopher; Fratti, Rutilio A.

    2013-01-01

    The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd2+ transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1pK669M) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion. PMID:23658021

  1. The ATP-binding cassette transporter ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

    Science.gov (United States)

    Higashikuni, Yasutomi; Sainz, Julie; Nakamura, Kazuto; Takaoka, Minoru; Enomoto, Soichiro; Iwata, Hiroshi; Tanaka, Kimie; Sahara, Makoto; Hirata, Yasunobu; Nagai, Ryozo; Sata, Masataka

    2012-03-01

    ATP-binding cassette transporter subfamily G member 2 (ABCG2), expressed in microvascular endothelial cells in the heart, has been suggested to regulate several tissue defense mechanisms. This study was performed to elucidate its role in pressure overload-induced cardiac hypertrophy. Pressure overload was induced in 8- to 12-week-old wild-type and Abcg2-/- mice by transverse aortic constriction (TAC). Abcg2-/- mice showed exaggerated cardiac hypertrophy and ventricular remodeling after TAC compared with wild-type mice. In the early phase after TAC, functional impairment in angiogenesis and antioxidant response in myocardium was found in Abcg2-/- mice. In vitro experiments demonstrated that ABCG2 regulates transport of glutathione, an important endogenous antioxidant, from microvascular endothelial cells. Besides, glutathione transported from microvascular endothelial cells in ABCG2-dependent manner ameliorated oxidative stress-induced cardiomyocyte hypertrophy. In vivo, glutathione levels in plasma and the heart were increased in wild-type mice but not in Abcg2-/- mice after TAC. Treatment with the superoxide dismutase mimetic ameliorated cardiac hypertrophy in Abcg2-/- mice after TAC to the same extent as that in wild-type mice, although cardiac dysfunction with impaired angiogenesis was observed in Abcg2-/- mice. ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

  2. ATP-binding cassette transporters A1 and G1, HDL metabolism, cholesterol efflux, and inflammation: important targets for the treatment of atherosclerosis.

    Science.gov (United States)

    Ye, Dan; Lammers, Bart; Zhao, Ying; Meurs, Illiana; Van Berkel, T J; Van Eck, Miranda

    2011-05-01

    Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries. Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. One central antiatherogenic role of HDL is believed to be its ability to remove excessive peripheral cholesterol back to the liver for subsequent catabolism and excretion, a physiologic process termed reverse cholesterol transport (RCT). Cholesterol efflux from macrophage foam cells, the initial step of RCT is the most relevant step with respect to atherosclerosis. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play crucial roles in the efflux of cellular cholesterol to HDL and its apolipoproteins. Moreover, ABCA1 and ABCG1 affect cellular inflammatory cytokine secretion by modulating cholesterol content in the plasma membrane and within intracellular compartments. In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excessive cholesterol in macrophages, and physiological manipulation of ABCA1 expression affects atherogenesis. Here we review recent advances in the role of ABCA1 and ABCG1 in HDL metabolism, macrophage cholesterol efflux, inflammation, and atherogenesis. Next, we summarize the structure, expression, and regulation of ABCA1 and ABCG1. Finally, we give an update on the progress and pitfalls of therapeutic approaches that target ABCA1 and ABCG1 to stimulate the flux of lipids through the RCT pathway.

  3. 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. PMID:25615936

  4. Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC Transporter Genes in Common Carp (Cyprinus carpio.

    Directory of Open Access Journals (Sweden)

    Xiang Liu

    Full Text Available The ATP-binding cassette (ABC gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp.

  5. Genome-Wide Identification, Characterization and Phylogenetic Analysis of ATP-Binding Cassette (ABC) Transporter Genes in Common Carp (Cyprinus carpio)

    Science.gov (United States)

    Peng, Wenzhu; Feng, Shuaisheng; Feng, Jianxin; Mahboob, Shahid; Al-Ghanim, Khalid A.

    2016-01-01

    The ATP-binding cassette (ABC) gene family is considered to be one of the largest gene families in all forms of prokaryotic and eukaryotic life. Although the ABC transporter genes have been annotated in some species, detailed information about the ABC superfamily and the evolutionary characterization of ABC genes in common carp (Cyprinus carpio) are still unclear. In this research, we identified 61 ABC transporter genes in the common carp genome. Phylogenetic analysis revealed that they could be classified into seven subfamilies, namely 11 ABCAs, six ABCBs, 19 ABCCs, eight ABCDs, two ABCEs, four ABCFs, and 11 ABCGs. Comparative analysis of the ABC genes in seven vertebrate species including common carp, showed that at least 10 common carp genes were retained from the third round of whole genome duplication, while 12 duplicated ABC genes may have come from the fourth round of whole genome duplication. Gene losses were also observed for 14 ABC genes. Expression profiles of the 61 ABC genes in six common carp tissues (brain, heart, spleen, kidney, intestine, and gill) revealed extensive functional divergence among the ABC genes. Different copies of some genes had tissue-specific expression patterns, which may indicate some gene function specialization. This study provides essential genomic resources for future studies in common carp. PMID:27058731

  6. ATP-Binding Cassette Transporter A1 Deficiency in Human Induced Pluripotent Stem Cell-Derived Hepatocytes Abrogates HDL Biogenesis and Enhances Triglyceride Secretion

    Directory of Open Access Journals (Sweden)

    Xin Bi

    2017-04-01

    Full Text Available Despite the recognized role of the ATP-binding Cassette Transporter A1 (ABCA1 in high-density lipoprotein (HDL metabolism, our understanding of ABCA1 deficiency in human hepatocytes is limited. To define the functional effects of human hepatocyte ABCA1 deficiency, we generated induced pluripotent stem cell (iPSC-derived hepatocyte-like cells (HLCs from Tangier disease (TD and matched control subjects. Control HLCs exhibited robust cholesterol efflux to apolipoprotein A-I (apoA-I and formed nascent HDL particles. ABCA1-deficient HLCs failed to mediate lipid efflux or nascent HDL formation, but had elevated triglyceride (TG secretion. Global transcriptome analysis revealed significantly increased ANGPTL3 expression in ABCA1-deficient HLCs. Angiopoietin-related protein 3 (ANGPTL3 was enriched in plasma of TD relative to control subjects. These results highlight the required role of ABCA1 in cholesterol efflux and nascent HDL formation by hepatocytes. Furthermore, our results suggest that hepatic ABCA1 deficiency results in increased hepatic TG and ANGPTL3 secretion, potentially underlying the elevated plasma TG levels in TD patients.

  7. Peroxisome Proliferator-Activated Receptor α Activates Human Multidrug Resistance Transporter 3/ATP-Binding Cassette Protein Subfamily B4 Transcription and Increases Rat Biliary Phosphatidylcholine Secretion

    Science.gov (United States)

    Ghonem, Nisanne S.; Ananthanarayanan, Meenakshisundaram; Soroka, Carol J.; Boyer, James L.

    2014-01-01

    Multidrug resistance transporter 3/ATP-binding cassette protein subfamily B4 (MDR3/ABCB4) is a critical determinant of biliary phosphatidylcholine (PC) secretion. Clinically, mutations and partial deficiencies in MDR3 result in cholestatic liver injury. Thus, MDR3 is a potential therapeutic target for cholestatic liver disease. Fenofibrate is a peroxisome proliferator-activated receptor (PPAR) α ligand that has antiinflammatory actions and regulates bile acid detoxification. Here we examined the mechanism by which fenofibrate regulates MDR3 gene expression. Fenofibrate significantly up-regulated MDR3 messenger RNA (mRNA) and protein expression in primary cultured human hepatocytes, and stimulated MDR3 promoter activity in HepG2 cells. In silico analysis of 5′-upstream region of human MDR3 gene revealed a number of PPARα response elements (PPRE). Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated specific binding of PPARα to the human MDR3 promoter. Targeted mutagenesis of three novel PPREs reduced inducibility of the MDR3 promoter by fenofibrate. In collagen sandwich cultured rat hepatocytes, treatment with fenofibrate increased secretion of fluorescent PC into bile canaliculi. Conclusion Fenofibrate transactivates MDR3 gene transcription by way of the binding of PPARα to three novel and functionally critical PPREs in the MDR3 promoter. Fenofibrate treatment further stimulates biliary phosphatidylcholine secretion in rat hepatocytes, thereby providing a functional correlate. We have established a molecular mechanism that may contribute to the beneficial use of fenofibrate therapy in human cholestatic liver disease. PMID:24122873

  8. Molecular phylogenetic study and expression analysis of ATP-binding cassette transporter gene family in Oryza sativa in response to salt stress.

    Science.gov (United States)

    Saha, Jayita; Sengupta, Atreyee; Gupta, Kamala; Gupta, Bhaskar

    2015-02-01

    ATP-binding cassette (ABC) transporter is a large gene superfamily that utilizes the energy released from ATP hydrolysis for transporting myriad of substrates across the biological membranes. Although many investigations have been done on the structural and functional analysis of the ABC transporters in Oryza sativa, much less is known about molecular phylogenetic and global expression pattern of the complete ABC family in rice. In this study, we have carried out a comprehensive phylogenetic analysis constructing neighbor-joining and maximum-likelihood trees based on various statistical methods of different ABC protein subfamily of five plant lineages including Chlamydomonas reinhardtii (green algae), Physcomitrella patens (moss), Selaginella moellendorffii (lycophyte), Arabidopsis thaliana (dicot) and O. sativa (monocot) to explore the origin and evolutionary patterns of these ABC genes. We have identified several conserved motifs in nucleotide binding domain (NBD) of ABC proteins among all plant lineages during evolution. Amongst the different ABC protein subfamilies, 'ABCE' has not yet been identified in lower plant genomes (algae, moss and lycophytes). The result indicated that gene duplication and diversification process acted upon these genes as a major operative force creating new groups and subgroups and functional divergence during evolution. We have demonstrated that rice ABCI subfamily consists of only half size transporters that represented highly dynamic members showing maximum sequence variations among the other rice ABC subfamilies. The evolutionary and the expression analysis contribute to a deep insight into the evolution and diversity of rice ABC proteins and their roles in response to salt stress that facilitate our further understanding on rice ABC transporters. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Localized Induction of the ATP-Binding Cassette B19 Auxin Transporter Enhances Adventitious Root Formation in Arabidopsis1[C][W][OA

    Science.gov (United States)

    Sukumar, Poornima; Maloney, Gregory S.; Muday, Gloria K.

    2013-01-01

    Adventitious roots emerge from aerial plant tissues, and the induction of these roots is essential for clonal propagation of agriculturally important plant species. This process has received extensive study in horticultural species but much less focus in genetically tractable model species. We have explored the role of auxin transport in this process in Arabidopsis (Arabidopsis thaliana) seedlings in which adventitious root initiation was induced by excising roots from low-light-grown hypocotyls. Inhibition of auxin transport from the shoot apex abolishes adventitious root formation under these conditions. Root excision was accompanied by a rapid increase in radioactive indole-3-acetic acid (IAA) transport and its accumulation in the hypocotyl above the point of excision where adventitious roots emerge. Local increases in auxin-responsive gene expression were also observed above the site of excision using three auxin-responsive reporters. These changes in auxin accumulation preceded cell division events, monitored by a cyclin B1 reporter (pCYCB1;1:GUS), and adventitious root initiation. We examined excision-induced adventitious root formation in auxin influx and efflux mutants, including auxin insensitive1, pin-formed1 (pin1), pin2, pin3, and pin7, with the most profound reductions observed in ATP-binding cassette B19 (ABCB19). An ABCB19 overexpression line forms more adventitious roots than the wild type in intact seedlings. Examination of transcriptional and translational fusions between ABCB19 and green fluorescent protein indicates that excision locally induced the accumulation of ABCB19 transcript and protein that is temporally and spatially linked to local IAA accumulation leading to adventitious root formation. These experiments are consistent with localized synthesis of ABCB19 protein after hypocotyl excision leads to enhanced IAA transport and local IAA accumulation driving adventitious root formation. PMID:23677937

  10. 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

    2014-10-06

    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.

  11. Implications of cerebrovascular ATP-binding cassette transporter G1 (ABCG1) and apolipoprotein M in cholesterol transport at the blood-brain barrier.

    Science.gov (United States)

    Kober, Alexandra Carmen; Manavalan, Anil Paul Chirackal; Tam-Amersdorfer, Carmen; Holmér, Andreas; Saeed, Ahmed; Fanaee-Danesh, Elham; Zandl, Martina; Albrecher, Nicole Maria; Björkhem, Ingemar; Kostner, Gerhard M; Dahlbäck, Björn; Panzenboeck, Ute

    2017-06-01

    Impaired cholesterol/lipoprotein metabolism is linked to neurodegenerative diseases such as Alzheimer's disease (AD). Cerebral cholesterol homeostasis is maintained by the highly efficient blood-brain barrier (BBB) and flux of the oxysterols 24(S)-hydroxycholesterol and 27-hydroxycholesterol, potent liver-X-receptor (LXR) activators. HDL and their apolipoproteins are crucial for cerebral lipid transfer, and loss of ATP binding cassette transporters (ABC)G1 and G4 results in toxic accumulation of oxysterols in the brain. The HDL-associated apolipoprotein (apo)M is positively correlated with pre-β HDL formation in plasma; its presence and function in the brain was thus far unknown. Using an in vitro model of the BBB, we examined expression, regulation, and functions of ABCG1, ABCG4, and apoM in primary porcine brain capillary endothelial cells (pBCEC). RT Q-PCR analyses and immunoblotting revealed that in addition to ABCA1 and scavenger receptor, class B, type I (SR-BI), pBCEC express high levels of ABCG1, which was up-regulated by LXR activation. Immunofluorescent staining, site-specific biotinylation and immunoprecipitation revealed that ABCG1 is localized both to early and late endosomes and on apical and basolateral plasma membranes. Using siRNA interference to silence ABCG1 (by 50%) reduced HDL-mediated [3H]-cholesterol efflux (by 50%) but did not reduce [3H]-24(S)-hydroxycholesterol efflux. In addition to apoA-I, pBCEC express and secrete apoM mainly to the basolateral (brain) compartment. HDL enhanced expression and secretion of apoM by pBCEC, apoM-enriched HDL promoted cellular cholesterol efflux more efficiently than apoM-free HDL, while apoM-silencing diminished cellular cholesterol release. We suggest that ABCG1 and apoM are centrally involved in regulation of cholesterol metabolism/turnover at the BBB. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. 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.

  13. Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

    Science.gov (United States)

    Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

    2014-01-01

    Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

  14. ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58.

    Science.gov (United States)

    Wichelecki, Daniel J; Vetting, Matthew W; Chou, Liyushang; Al-Obaidi, Nawar; Bouvier, Jason T; Almo, Steven C; Gerlt, John A

    2015-11-27

    Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Polymorphisms in ATP-binding cassette transporters associated with maternal methylmercury disposition and infant neurodevelopment in mother-infant pairs in the Seychelles Child Development Study.

    Science.gov (United States)

    Engström, Karin; Love, Tanzy M; Watson, Gene E; Zareba, Grazyna; Yeates, Alison; Wahlberg, Karin; Alhamdow, Ayman; Thurston, Sally W; Mulhern, Maria; McSorley, Emeir M; Strain, J J; Davidson, Philip W; Shamlaye, Conrad F; Myers, G J; Rand, Matthew D; van Wijngaarden, Edwin; Broberg, Karin

    2016-09-01

    ATP-binding cassette (ABC) transporters have been associated with methylmercury (MeHg) toxicity in experimental animal models. To evaluate the association of single nucleotide polymorphisms (SNPs) in maternal ABC transporter genes with 1) maternal hair MeHg concentrations during pregnancy and 2) child neurodevelopmental outcomes. Nutrition Cohort 2 (NC2) is an observational mother-child cohort recruited in the Republic of Seychelles from 2008-2011. Total mercury (Hg) was measured in maternal hair growing during pregnancy as a biomarker for prenatal MeHg exposure (N=1313) (mean 3.9ppm). Infants completed developmental assessments by Bayley Scales of Infant Development II (BSID-II) at 20months of age (N=1331). Genotyping for fifteen SNPs in ABCC1, ABCC2 and ABCB1 was performed for the mothers. Seven of fifteen ABC SNPs (ABCC1 rs11075290, rs212093, and rs215088; ABCC2 rs717620; ABCB1 rs10276499, rs1202169, and rs2032582) were associated with concentrations of maternal hair Hg (p<0.001 to 0.013). One SNP (ABCC1 rs11075290) was also significantly associated with neurodevelopment; children born to mothers with rs11075290 CC genotype (mean hair Hg 3.6ppm) scored on average 2 points lower on the Mental Development Index (MDI) and 3 points lower on the Psychomotor Development Index (PDI) than children born to mothers with TT genotype (mean hair Hg 4.7ppm) while children with the CT genotype (mean hair Hg 4.0ppm) had intermediate BSID scores. Genetic variation in ABC transporter genes was associated with maternal hair Hg concentrations. The implications for MeHg dose in the developing child and neurodevelopmental outcomes need to be further investigated. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    Science.gov (United States)

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

    2017-09-01

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

  17. 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.

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    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.

  18. 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

    2012-01-01

    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. PMID:22761424

  19. 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.

  20. ATP-Binding Cassette Transporter G2 Activity in the Bovine Spermatozoa Is Modulated Along the Epididymal Duct and at Ejaculation1

    Science.gov (United States)

    Caballero, Julieta; Frenette, Gilles; D'Amours, Olivier; Dufour, Maurice; Oko, Richard; Sullivan, Robert

    2012-01-01

    During their epididymal maturation, stabilizing factors such as cholesterol sulfate are associated with the sperm plasma membrane. Cholesterol is sulfated in epididymal spermatozoa by the enzyme estrogen sulfotransferase. Because of its role in the efflux of sulfate conjugates formed intracellularly by sulfotransferases, the ATP-binding cassette membrane transporter G2 (ABCG2) might have a role in the translocation of this compound across the plasma membrane. In the present study we showed that ABCG2 is present in the plasma membrane overlaying the acrosomal region of spermatozoa recovered from testis, epididymis, and after ejaculation. Although ABCG2 is also present in epididymosomes, the transporter is not transferred to spermatozoa via this mechanism. Furthermore, although epididymal sperm ABCG2 was shown to be functional, as determined by its ability to extrude Hoechst 33342 in the presence of the specific inhibitor Fumitremorgin C, ABCG2 present in ejaculated sperm was found to be nonfunctional. Additional experiments demonstrated that phosphorylation of ABCG2 tyrosyl residues, but not its localization in lipid rafts, is the mechanism responsible for its functionality. Dephosphorylation of ABCG2 in ejaculated spermatozoa is proposed to cause a partial protein relocalization to other intracellular compartments. Prostasomes are proposed to have a role in this process because incubation with this fraction of seminal plasma induces a decrease in the amount of ABCG2 in the associated sperm membrane fraction. These results demonstrate that ABCG2 plays a role in epididymal sperm maturation, but not after ejaculation. The loss of ABCG2 function after ejaculation is proposed to be regulated by prostasomes. PMID:22441796

  1. RCN1/OsABCG5, an ATP-binding cassette (ABC) transporter, is required for hypodermal suberization of roots in rice (Oryza sativa).

    Science.gov (United States)

    Shiono, Katsuhiro; Ando, Miho; Nishiuchi, Shunsaku; Takahashi, Hirokazu; Watanabe, Kohtaro; Nakamura, Motoaki; Matsuo, Yuichi; Yasuno, Naoko; Yamanouchi, Utako; Fujimoto, Masaru; Takanashi, Hideki; Ranathunge, Kosala; Franke, Rochus B; Shitan, Nobukazu; Nishizawa, Naoko K; Takamure, Itsuro; Yano, Masahiro; Tsutsumi, Nobuhiro; Schreiber, Lukas; Yazaki, Kazufumi; Nakazono, Mikio; Kato, Kiyoaki

    2014-10-01

    Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C(28) and C(30) fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  2. 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

    Science.gov (United States)

    Lee, Yung-Kuo; Lin, Tsung-Hsien; Chang, Chuan-Fa; Lo, Yu-Li

    2013-01-01

    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. PMID:24303084

  3. Multidrug Resistance Protein 1 (MRP1, ABCC1), a “Multitasking” ATP-binding Cassette (ABC) Transporter*

    Science.gov (United States)

    Cole, Susan P. C.

    2014-01-01

    The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases. PMID:25281745

  4. Cholesterol transporter ATP-binding cassette A1 (ABCA1) is elevated in prion disease and affects PrPC and PrPSc concentrations in cultured cells.

    Science.gov (United States)

    Kumar, Rajeev; McClain, Denise; Young, Rebecca; Carlson, George A

    2008-06-01

    Prion diseases are transmissible neurodegenerative disorders of prion protein (PrP) conformation. Prion replication by conversion of benign PrPC isoforms into disease-specific PrPSc isoforms is intimately involved in prion disease pathogenesis and may be initiated in cholesterol-rich caveolae-like domains (CLD). Concentrations of the cholesterol transporter ATP-binding cassette A1 protein (ABCA1) are elevated in pre-clinical scrapie prion-infected mice and in prion-infected cells in vitro. Elevation of ABCA1 in prion-infected brain is not a direct consequence of local PrPSc accumulation, indeed levels of ABCA1 are comparable in brain regions that differ dramatically in the amount of PrPSc. Similarly, ABCA1 concentrations are identical in normal mice, transgenic mice overexpressing PrP and PrP knockout mice. In contrast, PrPC and PrPSc levels, but not Prnp mRNA, were increased by overexpression of ABCA1 in N2a neuroblastoma cells and scrapie prion-infected N2a cells (ScN2a). Conversely, RNAi-mediated knock down of Abca1 expression decreased the concentrations of PrPC in N2a cells and of PrPSc in ScN2a cells. These results suggest that ABCA1's effects on PrPC levels are post-translational and may reflect an increase in of PrPC stability, mediated either indirectly by increasing membrane cholesterol and CLD formation or by other functions of ABCA1. The increased supply of PrPC available for conversion would lead to increased PrPSc formation.

  5. MicroRNA-144 regulates hepatic ATP binding cassette transporter A1 and plasma high-density lipoprotein after activation of the nuclear receptor farnesoid X receptor.

    Science.gov (United States)

    de Aguiar Vallim, Thomas Q; Tarling, Elizabeth J; Kim, Tammy; Civelek, Mete; Baldán, Ángel; Esau, Christine; Edwards, Peter A

    2013-06-07

    The bile acid receptor farnesoid X receptor (FXR) regulates many aspects of lipid metabolism by variouscomplex and incompletely understood molecular mechanisms. We set out to investigate the molecular mechanisms for FXR-dependent regulation of lipid and lipoprotein metabolism. To identify FXR-regulated microRNAs that were subsequently involved in regulating lipid metabolism. ATP binding cassette transporter A1 (ABCA1) is a major determinant of plasma high-density lipoprotein (HDL)-cholesterol levels. Here, we show that activation of the nuclear receptor FXR in vivo increases hepatic levels of miR-144, which in turn lowers hepatic ABCA1 and plasma HDL levels. We identified 2 complementary sequences to miR-144 in the 3' untranslated region of ABCA1 mRNA that are necessary for miR-144-dependent regulation. Overexpression of miR-144 in vitro decreased both cellular ABCA1 protein and cholesterol efflux to lipid-poor apolipoprotein A-I protein, whereas overexpression in vivo reduced hepatic ABCA1 protein and plasma HDL-cholesterol. Conversely, silencing miR-144 in mice increased hepatic ABCA1 protein and HDL-cholesterol. In addition, we used tissue-specific FXR-deficient mice to show that induction of miR-144 and FXR-dependent hypolipidemia requires hepatic, but not intestinal, FXR. Finally, we identified functional FXR response elements upstream of the miR-144 locus, consistent with direct FXR regulation. We have identified a novel pathway involving FXR, miR-144, and ABCA1 that together regulate plasma HDL-cholesterol.

  6. SALL4, a stem cell factor, affects the side population by regulation of the ATP-binding cassette drug transport genes.

    Directory of Open Access Journals (Sweden)

    Ha-Won Jeong

    Full Text Available Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2-4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP, quantitative reverse transcription polymerase chain reaction (qRT-PCR and electrophoretic mobility shift assay(EMSA, we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.

  7. Hydrogen Sulfide Up-Regulates the Expression of ATP-Binding Cassette Transporter A1 via Promoting Nuclear Translocation of PPARα

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    Dong Li

    2016-04-01

    Full Text Available ATP binding cassette transporter A1 (ABCA1 plays a key role in atherogenesis. Hydrogen sulfide (H2S, a gasotransmitter, has been reported to play an anti-atherosclerotic role. However, the underlying mechanisms are largely unknown. In this study we examined whether and how H2S regulates ABCA1 expression. The effect of H2S on ABCA1 expression and lipid metabolism were assessed in vitro by cultured human hepatoma cell line HepG2, and in vivo by ApoE−/− mice with a high-cholesterol diet. NaHS (an exogenous H2S donor treatment significantly increased the expression of ABCA1, ApoA1, and ApoA2 and ameliorated intracellular lipid accumulation in HepG2 cells. Depletion of the endogenous H2S generator cystathionine γ-lyase (CSE by small RNA interference (siRNA significantly decreased the expression of ABCA1 and resulted in the accumulation of lipids in HepG2 cells. In vivo NaHS treatment significantly reduced the serum levels of total cholesterol (TC, triglycerides (TG, and low-density lipoproteins (LDL, diminished atherosclerotic plaque size, and increased hepatic ABCA1 expression in fat-fed ApoE−/− mice. Further study revealed that NaHS upregulated ABCA1 expression by promoting peroxisome proliferator-activated receptor α (PPARα nuclear translocation. H2S up-regulates the expression of ABCA1 by promoting the nuclear translocation of PPARα, providing a fundamental mechanism for the anti-atherogenic activity of H2S. H2S may be a promising potential drug candidate for the treatment of atherosclerosis.

  8. Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth.

    Science.gov (United States)

    Glass, Lisa N; Swapna, Ganduri; Chavadi, Sivagami Sundaram; Tufariello, JoAnn M; Mi, Kaixia; Drumm, Joshua E; Lam, TuKiet T; Zhu, Guofeng; Zhan, Chenyang; Vilchéze, Catherine; Arcos, Jesus; Chen, Yong; Bi, Lijun; Mehta, Simren; Porcelli, Steven A; Almo, Steve C; Yeh, Syun-Ru; Jacobs, William R; Torrelles, Jordi B; Chan, John

    2017-07-01

    We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv

  9. 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.

  10. Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC transporter Rv1747 to regulate mycobacterial growth.

    Directory of Open Access Journals (Sweden)

    Lisa N Glass

    2017-07-01

    Full Text Available We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT, a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623 displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs, while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747

  11. Mycobacterium tuberculosis universal stress protein Rv2623 interacts with the putative ATP binding cassette (ABC) transporter Rv1747 to regulate mycobacterial growth

    Science.gov (United States)

    Tufariello, JoAnn M.; Mi, Kaixia; Zhu, Guofeng; Zhan, Chenyang; Vilchéze, Catherine; Arcos, Jesus; Chen, Yong; Bi, Lijun; Porcelli, Steven A.; Almo, Steve C.; Yeh, Syun-Ru; Jacobs, William R.; Torrelles, Jordi B.

    2017-01-01

    We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (ΔRv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ΔRv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ΔRv2623 is hypervirulent, while ΔRv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ΔRv2623 and ΔRv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv

  12. Diosgenin inhibits atherosclerosis via suppressing the MiR-19b-induced downregulation of ATP-binding cassette transporter A1.

    Science.gov (United States)

    Lv, Yun-cheng; Yang, Jing; Yao, Feng; Xie, Wei; Tang, Yan-yan; Ouyang, Xin-ping; He, Ping-ping; Tan, Yu-lin; Li, Liang; Zhang, Min; Liu, Dan; Cayabyab, Francisco S; Zheng, Xi-Long; Tang, Chao-ke

    2015-05-01

    Diosgenin (Dgn), a structural analogue of cholesterol, has been reported to have the hypolipidemic and antiatherogenic properties, but the underlying mechanisms are not fully understood. Given the key roles of macrophages in cholesterol metabolism and atherogenesis, it is critical to investigate macrophage cholesterol efflux and development of atherosclerotic lesion after Dgn treatment. This study was designed to evaluate the potential effects of Dgn on macrophage cholesterol metabolism and the development of aortic atherosclerosis, and to explore its underlying mechanisms. Dgn significantly up-regulated the expression of ATP-binding cassette transporter A1 (ABCA1) protein, but didn't affect liver X receptor α levels in foam cells derived from human THP-1 macrophages and mouse peritoneal macrophages (MPMs) as determined by western blotting. The miR-19b levels were markedly down-regulated in Dgn-treated THP-1 macrophages/MPM-derived foam cells. Cholesterol transport assays revealed that treatment with Dgn alone or together with miR-19b inhibitor notably enhanced ABCA1-dependent cholesterol efflux, resulting in the reduced levels of total cholesterol, free cholesterol and cholesterol ester as determined by high-performance liquid chromatography. The fecal 3H-sterol originating from cholesterol-laden MPMs was increased in apolipoprotein E knockout mice treated with Dgn or both Dgn and antagomiR-19b. Treatment with Dgn alone or together with antagomiR-19b elevated plasma high-density lipoprotein levels, but reduced plasma low-density lipoprotein levels. Accordingly, aortic lipid deposition and plaque area were reduced, and collagen content and ABCA1 expression were increased in mice treated with Dgn alone or together with antagomiR-19b. However, miR-19b overexpression abrogated the lipid-lowering and atheroprotective effects induced by Dgn. The present study demonstrates that Dgn enhances ABCA1-dependent cholesterol efflux and inhibits aortic atherosclerosis

  13. Mycophenolic acid induces ATP-binding cassette transporter A1 (ABCA1) expression through the PPAR{gamma}-LXR{alpha}-ABCA1 pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yanni; Lai, Fangfang; Xu, Yang; Wu, Yexiang; Liu, Qi; Li, Ni; Wei, Yuzhen; Feng, Tingting; Zheng, Zhihui; Jiang, Wei; Yu, Liyan; Hong, Bin [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China); Si, Shuyi, E-mail: sisyimb@hotmail.com [Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050 (China)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Using an ABCA1p-LUC HepG2 cell line, we found that MPA upregulated ABCA1 expression. Black-Right-Pointing-Pointer MPA induced ABCA1 and LXR{alpha} protein expression in HepG2 cells. Black-Right-Pointing-Pointer PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. Black-Right-Pointing-Pointer The effect of MPA upregulating ABCA1 was due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 pathway. -- Abstract: ATP-binding cassette transporter A1 (ABCA1) promotes cholesterol and phospholipid efflux from cells to lipid-poor apolipoprotein A-I and plays an important role in atherosclerosis. In a previous study, we developed a high-throughput screening method using an ABCA1p-LUC HepG2 cell line to find upregulators of ABCA1. Using this method in the present study, we found that mycophenolic acid (MPA) upregulated ABCA1 expression (EC50 = 0.09 {mu}M). MPA upregulation of ABCA1 expression was confirmed by real-time quantitative reverse transcription-PCR and Western blot analysis in HepG2 cells. Previous work has indicated that MPA is a potent agonist of peroxisome proliferator-activated receptor gamma (PPAR{gamma}; EC50 = 5.2-9.3 {mu}M). Liver X receptor {alpha} (LXR{alpha}) is a target gene of PPAR{gamma} and may directly regulate ABCA1 expression. Western blot analysis showed that MPA induced LXR{alpha} protein expression in HepG2 cells. Addition of PPAR{gamma} antagonist GW9662 markedly inhibited MPA-induced ABCA1 and LXR{alpha} protein expression. These data suggest that MPA increased ABCA1 expression mainly through activation of PPAR{gamma}. Thus, the effects of MPA on upregulation of ABCA1 expression were due mainly to activation of the PPAR{gamma}-LXR{alpha}-ABCA1 signaling pathway. This is the first report that the antiatherosclerosis activity of MPA is due to this mechanism.

  14. 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

    The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco--(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria...... 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...

  15. Rosuvastatin activates ATP-binding cassette transporter A1-dependent efflux ex vivo and promotes reverse cholesterol transport in macrophage cells in mice fed a high-fat diet.

    Science.gov (United States)

    Shimizu, Tomohiko; Miura, Shin-ichiro; Tanigawa, Hiroyuki; Kuwano, Takashi; Zhang, Bo; Uehara, Yoshinari; Saku, Keijiro

    2014-10-01

    It is controversial whether statins improve high-density lipoprotein (HDL) function, which plays an important role in reverse cholesterol transport in vivo. The aim of the present study was to clarify the effects of rosuvastatin and atorvastatin on reverse cholesterol transport in macrophage cells in vivo and their underlying mechanisms. Male C57BL mice were divided into 3 groups (rosuvastatin, atorvastatin, and control groups) and orally administered rosuvastatin, atorvastatin, or placebo for 6 weeks under feeding with a 0.5% cholesterol+10% coconut oil diet. After administration, although there were no changes in plasma HDL cholesterol levels among the groups, plasma from the rosuvastatin group showed an increased ability to promote ATP-binding cassette transporter A1-mediated cholesterol efflux ex vivo. In addition, capillary electrophoresis revealed a shift in HDL toward the pre-β HDL fraction only in the rosuvastatin group. Mice in all 3 groups were intraperitoneally injected with (3)H-cholesterol-labeled and cholesterol-loaded macrophages and then were monitored for the appearance of (3)H-tracer in plasma and feces. The amount of (3)H-tracer excreted into feces during 48 hours in the rosuvastatin group was greater than that in the control group. Finally, (3)H-cholesteryl oleate-HDL was intravenously injected into all groups, blood samples were taken, and the count of (3)H-cholesterol was analyzed. Plasma (3)H-cholesteryl oleate-HDL changed similarly, and no differences in fractional catabolic rates were observed. Rosuvastatin enhanced the ATP-binding cassette transporter A1-dependent HDL efflux function of reverse cholesterol transport, and this finding highlights the potential of rosuvastatin for the regression of atherosclerosis. © 2014 American Heart Association, Inc.

  16. Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2)

    NARCIS (Netherlands)

    Zelcer, Noam; Huisman, Maarten T.; Reid, Glen; Wielinga, Peter; Breedveld, Pauline; Kuil, Annemieke; Knipscheer, Puck; Schellens, Jan H. M.; Schinkel, Alfred H.; Borst, Piet

    2003-01-01

    Multidrug resistance protein 2 (MRP2) belongs to the ATP binding cassette family of transporters. Its substrates include organic anions and anticancer drugs. We have used transport assays with vesicles derived from Sf9 insect cells overproducing MRP2 to study the interactions of drugs, organic

  17. 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...

  18. Human ATP-binding cassette transporter 1 (ABC1): Genomic organization and identification of the genetic defect in the original Tangier disease kindred

    Science.gov (United States)

    Remaley, Alan T.; Rust, Stephan; Rosier, Marie; Knapper, Cathy; Naudin, Laurent; Broccardo, Cyril; Peterson, Katherine M.; Koch, Christine; Arnould, Isabelle; Prades, Catherine; Duverger, Nicholas; Funke, Harald; Assman, Gerd; Dinger, Maria; Dean, Michael; Chimini, Giovanna; Santamarina-Fojo, Silvia; Fredrickson, Donald S.; Denefle, Patrice; Brewer, H. Bryan

    1999-01-01

    Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease. PMID:10535983

  19. Opposing Gatekeepers of Apical Sterol Transport: Niemann-Pick C1-Like 1 (NPC1L1) and ATP-Binding Cassette Transporters G5 and G8 (ABCG5/ABCG8)

    Science.gov (United States)

    Brown, J. Mark; Yu, Liqing

    2010-01-01

    Cholesterol is essential for the growth and function of all mammalian cells, but abnormally elevated levels of circulating low-density lipoprotein cholesterol (LDL-C) are a major risk factor for the development of atherosclerotic cardiovascular disease (ASCVD). For many years, statin drugs have been used to effectively lower LDL-C, but ASCVD still persists in most of the world. Hence, additional LDL-C lowering is now recommended, and the search for therapeutic strategies that work in synergy with statins has now begun. Intestinal absorption and biliary excretion of cholesterol represent two major pathways and continue to show promise as druggable processes. Importantly, both of these complex physiological pathways are tightly regulated by key proteins located at the apical surface of the small intestine and the liver. One of these proteins, the target of ezetimibe Niemann-Pick C1-Like 1 (NPC1L1), was recently identified to be essential for intestinal cholesterol absorption and protect against excessive biliary sterol loss. In direct opposition of NPC1L1, the heterodimer of ATP-binding cassette transporters G5 and G8 (ABCG5/ABCG8) has been shown to be critical for promoting biliary cholesterol secretion in the liver, and has also been proposed to play a direct role in intestinal disposal of sterols. The purpose of this review is to summarize the current state of knowledge regarding the function of these opposing apical cholesterol transporters, and provide a framework for future studies examining these proteins. PMID:20174593

  20. Selective substrate uptake: The role of ATP-binding cassette (ABC) importers in pathogenesis.

    Science.gov (United States)

    Tanaka, Kari J; Song, Saemee; Mason, Kevin; Pinkett, Heather W

    2018-04-01

    The uptake of nutrients, including metals, amino acids and peptides are required for many biological processes. Pathogenic bacteria scavenge these essential nutrients from microenvironments to survive within the host. Pathogens must utilize a myriad of mechanisms to acquire these essential nutrients from the host while mediating the effects of toxicity. Bacteria utilize several transport proteins, including ATP-binding cassette (ABC) transporters to import and expel substrates. ABC transporters, conserved across all organisms, are powered by the energy from ATP to move substrates across cellular membranes. In this review, we will focus on nutrient uptake, the role of ABC importers at the host-pathogen interface, and explore emerging therapies to combat pathogenesis. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  1. 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.

  2. Effect of ATP-binding cassette subfamily B member 1 on bovine blastocyst implantation.

    Science.gov (United States)

    Mori, M; Kuwano, T; Kamori, T; Isozaki, Y; Nishihara, T; Yamauchi, N; Hattori, M-A

    2014-03-15

    The ATP-binding cassette subfamily B member 1 (ABCB1) is an efflux transporter that excretes xenobiotics and waste matter. High expression of ABCB1 induced by forskolin (FSK) and rifampicin (RIF) in the bovine blastocysts reportedly improves the cellular quality. In the present study, interferon-α, similar to FSK and RIF, was highly potent in inducing the expression of ABCB1 in the bovine blastocysts but did not exhibit an additive effect with FSK and RIF. Bovine blastocysts stimulated by the combined treatment with FSK, RIF, and interferon-α to express high levels of ABCB1 displayed better freezing resistance as indicated by higher cell numbers in post thawing cultures. On transfer to recipients, such embryos established pregnancies with significantly higher frequencies in repeat breeder cows rather than normal ones. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. 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.

  4. Structural elucidation of transmembrane domain zero (TMD0) of EcdL: A multidrug resistance-associated protein (MRP) family of ATP-binding cassette transporter protein revealed by atomistic simulation.

    Science.gov (United States)

    Bera, Krishnendu; Rani, Priyanka; Kishor, Gaurav; Agarwal, Shikha; Kumar, Antresh; Singh, Durg Vijay

    2017-09-20

    ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 μs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.

  5. 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.

    Science.gov (United States)

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

    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 to assess whether polymorphisms in ABCB1, ABCC2 and ABCG2 were associated with risk of colorectal cancer (CRC) and to investigate gene-environment (dietary factors, smoking and use of non-steroidal anti-inflammatory drugs) and gene-gene interactions between previously studied polymorphisms in IL1B and IL10 and ABC transporter genes in relation to CRC risk. We used a Danish prospective case-cohort study of 1010 CRC cases and 1829 randomly selected participants from the Danish Diet, Cancer and Health cohort. Incidence rate ratios were calculated based on Cox' proportional hazards model. None of the polymorphisms were associated with CRC, but ABCB1 and ABCG2 haplotypes were associated with risk of CRC. ABCB1/rs1045642 interacted with intake of cereals and fiber (p-Value for interaction (P(int)) = 0.001 and 0.01, respectively). In a three-way analysis, both ABCB1/rs1045642 and ABCG2/rs2231137 in combination with IL10/rs3024505 interacted with fiber intake in relation to risk of CRC (P(int) = 0.0007 and 0.009). Our results suggest that the ABC transporters P-glycoprotein/multidrug resistance 1 and BRCP, in cooperation with IL-10, are involved in the biological mechanism underlying the protective effect of fiber intake in relation to CRC. These results should be replicated in other cohorts to rule out chance findings.

  6. A common highly conserved cadmium detoxification mechanism from bacteria to humans: heavy metal tolerance conferred by the ATP-binding cassette (ABC) transporter SpHMT1 requires glutathione but not metal-chelating phytochelatin peptides.

    Science.gov (United States)

    Prévéral, Sandra; Gayet, Landry; Moldes, Cristina; Hoffmann, Jonathan; Mounicou, Sandra; Gruet, Antoine; Reynaud, Florie; Lobinski, Ryszard; Verbavatz, Jean-Marc; Vavasseur, Alain; Forestier, Cyrille

    2009-02-20

    Cadmium poses a significant threat to human health due to its toxicity. In mammals and in bakers' yeast, cadmium is detoxified by ATP-binding cassette transporters after conjugation to glutathione. In fission yeast, phytochelatins constitute the co-substrate with cadmium for the transporter SpHMT1. In plants, a detoxification mechanism similar to the one in fission yeast is supposed, but the molecular nature of the transporter is still lacking. To investigate further the relationship between SpHMT1 and its co-substrate, we overexpressed the transporter in a Schizosaccharomyces pombe strain deleted for the phytochelatin synthase gene and heterologously in Saccharomyces cerevisiae and in Escherichia coli. In all organisms, overexpression of SpHMT1 conferred a markedly enhanced tolerance to cadmium but not to Sb(III), AgNO(3), As(III), As(V), CuSO(4), or HgCl(2). Abolishment of the catalytic activity by expression of SpHMT1(K623M) mutant suppressed the cadmium tolerance phenotype independently of the presence of phytochelatins. Depletion of the glutathione pool inhibited the SpHMT1 activity but not that of AtHMA4, a P-type ATPase, indicating that GSH is necessary for the SpHMT1-mediated cadmium resistance. In E. coli, SpHMT1 was targeted to the periplasmic membrane and led to an increased amount of cadmium in the periplasm. These results demonstrate that SpHMT1 confers cadmium tolerance in the absence of phytochelatins but depending on the presence of GSH and ATP. Our results challenge the dogma of the two separate cadmium detoxification pathways and demonstrate that a common highly conserved mechanism has been selected during the evolution from bacteria to humans.

  7. ATP-binding cassette (ABC) proteins in aquatic invertebrates: Evolutionary significance and application in marine ecotoxicology.

    Science.gov (United States)

    Jeong, Chang-Bum; Kim, Hui-Su; Kang, Hye-Min; Lee, Jae-Seong

    2017-04-01

    The ATP-binding cassette (ABC) protein superfamily is known to play a fundamental role in biological processes and is highly conserved across animal taxa. The ABC proteins function as active transporters for multiple substrates across the cellular membrane by ATP hydrolysis. As this superfamily is derived from a common ancestor, ABC genes have evolved via lineage-specific duplications through the process of adaptation. In this review, we summarized information about the ABC gene families in aquatic invertebrates, considering their evolution and putative functions in defense mechanisms. Phylogenetic analysis was conducted to examine the evolutionary significance of ABC gene families in aquatic invertebrates. Particularly, a massive expansion of multixenobiotic resistance (MXR)-mediated efflux transporters was identified in the absence of the ABCG2 (BCRP) gene in Ecdysozoa and Platyzoa, suggesting that a loss of Abcg2 gene occurred sporadically in these species during divergence of Protostome to Lophotrochozoa. Furthermore, in aquatic invertebrates, the ecotoxicological significance of MXR is discussed while considering the role of MXR-mediated efflux transporters in response to various environmental pollutants. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. 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

  9. Luminescence resonance energy transfer spectroscopy of ATP-binding cassette proteins.

    Science.gov (United States)

    Zoghbi, Maria E; Altenberg, Guillermo A

    2018-04-01

    The ATP-binding cassette (ABC) superfamily includes regulatory and transport proteins. Most human ABC exporters pump substrates out of cells using energy from ATP hydrolysis. Although major advances have been made toward understanding the molecular mechanism of ABC exporters, there are still many issues unresolved. During the last few years, luminescence resonance energy transfer has been used to detect conformational changes in real time, with atomic resolution, in isolated ABC nucleotide binding domains (NBDs) and full-length ABC exporters. NBDs are particularly interesting because they provide the power stroke for substrate transport. Luminescence resonance energy transfer (LRET) is a spectroscopic technique that can provide dynamic information with atomic-resolution of protein conformational changes under physiological conditions. Using LRET, it has been shown that NBD dimerization, a critical step in ABC proteins catalytic cycle, requires binding of ATP to two nucleotide binding sites. However, hydrolysis at just one of the sites can drive dissociation of the NBD dimer. It was also found that the NBDs of the bacterial ABC exporter MsbA reconstituted in a lipid bilayer membrane and studied at 37°C never separate as much as suggested by crystal structures. This observation stresses the importance of performing structural/functional studies of ABC exporters under physiologic conditions. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. The non-synonymous Arg230Cys variant (R230C) of the ATP-binding cassette transporter A1 is associated with low HDL cholesterol concentrations in Mexican adults: a population based nation wide study.

    Science.gov (United States)

    Aguilar-Salinas, Carlos A; Canizales-Quinteros, Samuel; Rojas-Martínez, Rosalba; Mehta, Roopa; Rodriguez-Guillén, Rosario; Ordoñez-Sanchez, María Luisa; Riba, Laura; Tusié-Luna, María Teresa

    2011-05-01

    To search for an association between the non-synonymous Arg230Cys variant (R230C) of the ATP-binding cassette transporter A1 and low HDL cholesterol levels in a Mexican, population-based nation wide survey. The 2000 National Health Survey is a cross sectional study that included individuals from 400 cities. All individuals who had a 9-12-h fasted blood sample and a DNA sample were selected (n = 1729). These cases were randomly distributed; no bias was detected for sex, education, region or socioeconomic status. The R230C variant was genotyped using TaqMan assays. In individuals with the R230C/C230C genotypes (39.03 mg/dl (36.63-41.43)) lower HDL-C levels (p cholesterol levels between alleles was 5.73 ± 1.4 mg/dl. The magnitude of the effect was significantly greater in males. The C230 allele of ABCA1 was associated with an increased risk for hypoalphalipoproteinemia (OR 1.66 (95%CI 1.08-2.54), p effect of waist circumference and gender, was 12.2% (95%CI 1.4-24.2%). The non-synonymous Arg230Cys variant of ABCA1 is associated with low levels of HDL cholesterol levels in Mexican adults. The HDL cholesterol lowering effect of the variant is greater in males. The size of the effect is greater compared to that reported for other ABCA1 variants. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  12. Divergent action of calcium channel blockers on ATP-binding cassette protein expression.

    Science.gov (United States)

    Hasegawa, Kazuhiro; Wakino, Shu; Kanda, Takeshi; Yoshioka, Kyoko; Tatematsu, Satoru; Homma, Koichiro; Takamatsu, Ichiro; Sugano, Naoki; Hayashi, Koichi

    2005-12-01

    Calcium channel blockers (CCBs) are widely used in clinical practice, and have been reported to be effective in preventing the progression of atherosclerosis. We examined whether various types of calcium channel blockers affected the expression of ATP binding cassette transporter A1 (ABCA1), a factor contributing to anti-atherogenesis. Undifferentiated monocytic cell line, THP-1 cells were maintained in RPMI 1640 medium and treated with different kinds of calcium channel blockers. Among the calcium channel blockers tested, aranidipine and efonidipine increased ABCA1 protein expression without an increase in ABCA1 mRNA expression, whereas other calcium channel blockers (eg, nifedipine, amlodipine, and nicardipine) or T-type calcium channel blockers (eg, mibefradil and nickel chloride) failed to upregulate ABCA1 expression. H89, a protein kinase A inhibitor inhibited the aranidipine-induced ABCA1 protein expression, whereas genistein (a tyrosine kinase inhibitor), or AG490 (a JAK-2 inhibitor) had no effects. Neither of these inhibitors suppressed the efonidipine-induced ABCA1 protein expression. Intracellular cAMP levels were elevated only by aranidipine, but not by efonidipine. In conclusion, aranidipine and efonidipine have the ability to induce ABCA1 protein by distinct mechanisms; protein kinase A is involved in the aranidipine-induced ABCA1 upregulation. This non-class effect of calcium channel blockers may potentially offer beneficial action in the treatment of hypertensive subjects with atherosclerosis.

  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. Downregulation of ATP-binding cassette subfamily C member 4 increases sensitivity to neoadjuvant radiotherapy for locally advanced rectal carcinoma.

    Science.gov (United States)

    Yu, Zhi-Qi; Zhang, Chang; Wang, Hao; Lao, Xin-Yuan; Chai, Rui; Gao, Xian-Hua; Cao, Guang-Wen; Fu, Chuan-Gang

    2013-05-01

    This study was designed to verify the effect of ATP-binding cassette subfamily C member 4 on radiosensitivity of locally advanced rectal carcinoma. The expression of ATP-binding cassette subfamily C member 4 protein in 121 pretreatment tissue samples from locally advanced rectal carcinoma patients was detected by immunohistochemistry. Pathological response to radiotherapy was evaluated according to tumor regression grading by postoperative histological examinations after they received long-course preoperative neoadjuvant radiotherapy, and the association between clinicopathological data and tumor regression grading was analyzed retrospectively. For further validation, short hairpin RNA was constructed and transfected into colorectal carcinoma cell line HT29. The knockdown efficiency was confirmed at both RNA and protein levels. The altered radiosensitivity was evaluated by methylthiazolyl tetrazolium assay, colony formation assay, flow cytometry, and Hoechst 33258 staining. Univariate analysis revealed that ATP-binding cassette subfamily C member 4 expression (p member 4 expression (p member 4 expression efficiently and persistently. Downregulation of ATP-binding cassette subfamily C member 4 expression significantly enhanced inhibition of cell proliferation, decreased colony formation capacity, and increased cell apoptosis induced by irradiation, as examined by a series of experiments in vitro. In addition, radiobiological parameters calculated according to the single-hit multitarget model were also decreased significantly. Our data indicate that ATP-binding cassette subfamily C member 4 may be a useful molecular marker in predicting radiosensitivity, and a potential target in improving the response to neoadjuvant radiotherapy in locally advanced rectal carcinoma patients.

  15. Cell and molecular biology of ATP-binding cassette proteins in plants.

    Science.gov (United States)

    Yazaki, Kazufumi; Shitan, Nobukazu; Sugiyama, Akifumi; Takanashi, Kojiro

    2009-01-01

    ATP-binding cassette (ABC) proteins constitute a large and diverse superfamily of membrane-bound and soluble proteins, which are involved in a wide range of biological processes in all organisms from prokaryotes to eukaryotes. Genome analyses of model plants, for example, Arabidopsis and rice, have revealed that plants have more than double numbers of this family member in their genomes compared to animals and insects. In recent years, various biochemical and physiological functions of ABC proteins in plants have been reported. Some are relevant for the defense mechanisms to biotic and abiotic stresses, whereas others are involved in the basic functions necessary for maintaining the plant life. Here, we provide an updated inventory of plant ABC proteins and summarize their tissue specificities, membrane localizations, and physiological functions.

  16. Endothelial ATP-binding cassette G1 in mouse endothelium protects against hemodynamic-induced atherosclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Shanshan [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Department of Pediatrics, Baodi District People’s Hospital of Tianjin City, Tianjin, 301800 (China); Wang, Jiaxing [Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); Zhang, Xu; Shi, Ying; Li, Bochuan; Bao, Qiankun [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Pang, Wei [Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); Ai, Ding [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Zhu, Yi [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); He, Jinlong, E-mail: hejinlong@tmu.edu.cn [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China)

    2016-08-19

    Activated vascular endothelium inflammation under persistent hyperlipidemia is the initial step of atherogenesis. ATP-binding cassette G1 (ABCG1) is a crucial factor maintaining sterol and lipid homeostasis by transporting cholesterol efflux to high-density lipoprotein. In this study, we investigated the protective effects of ABCG1 in endothelial inflammation activation during early-stage atherogenesis in mice and the underlying mechanisms. Endothelial cell (EC)-specific ABCG1 transgenic (EC-ABCG1-Tg) mice were generated and cross-bred with low-density lipoprotein receptor–deficient (Ldlr{sup −/−}) mice. After a 4-week Western-type diet, the mice were sacrificed for assessing atherosclerosis. Human umbilical vein ECs were treated with different flows, and ABCG1 was adenovirally overexpressed to investigate the mechanism in vitro. Compared with Ldlr{sup −/−} mouse aortas, EC-ABCG1-Tg/Ldlr{sup −/−} aortas showed decreased early-stage lesions. Furthermore, the lesion area in the EC-ABCG1-Tg/Ldlr{sup −/−} mouse aortic arch but not thoracic aorta was significantly reduced, which suggests a protective role of ABCG1 under atheroprone flow. In vitro, overexpression of ABCG1 attenuated EC activation caused by oscillatory shear stress. Overexpression of ABCG1 blunted cholesterol-activated ECs in vitro. In exploring the mechanisms of ABCG1 attenuating endothelial inflammation, we found that ABCG1 inhibited oscillatory flow-activated nuclear factor kappa B and NLRP3 inflammasome in ECs. ABCG1 may play a protective role in early-stage atherosclerosis by reducing endothelial activation induced by oscillatory shear stress via suppressing the inflammatory response. - Highlights: • EC-ABCG1-Tg mice in a Ldlr{sup −/−} background showed decreased atherosclerosis. • Overexpression of ABCG1 in ECs decreased OSS-induced EC activation. • NLRP3 and NF-κB might be an underlying mechanism of ABCG1 protective role.

  17. 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

  18. ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei

    Directory of Open Access Journals (Sweden)

    Titball Richard W

    2007-03-01

    Full Text Available Abstract Background ATP binding cassette (ABC systems are responsible for the import and export of a wide variety of molecules across cell membranes and comprise one of largest protein superfamilies found in prokarya, eukarya and archea. ABC systems play important roles in bacterial lifestyle, virulence and survival. In this study, an inventory of the ABC systems of Burkholderia pseudomallei strain K96243 and Burkholderia mallei strain ATCC 23344 has been compiled using bioinformatic techniques. Results The ABC systems in the genomes of B. pseudomallei and B. mallei have been reannotated and subsequently compared. Differences in the number and types of encoded ABC systems in belonging to these organisms have been identified. For example, ABC systems involved in iron acquisition appear to be correlated with differences in genome size and lifestyles between these two closely related organisms. Conclusion The availability of complete inventories of the ABC systems in B. pseudomallei and B. mallei has enabled a more detailed comparison of the encoded proteins in this family. This has resulted in the identification of ABC systems which may play key roles in the different lifestyles and pathogenic properties of these two bacteria. This information has the potential to be exploited for improved clinical identification of these organisms as well as in the development of new vaccines and therapeutics targeted against the diseases caused by these organisms.

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

    Directory of Open Access Journals (Sweden)

    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.

  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

    2017-05-01

    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. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. A selective ATP-binding cassette subfamily G member 2 efflux inhibitor revealed via high-throughput flow cytometry.

    Science.gov (United States)

    Strouse, J Jacob; Ivnitski-Steele, Irena; Khawaja, Hadya M; Perez, Dominique; Ricci, Jerec; Yao, Tuanli; Weiner, Warren S; Schroeder, Chad E; Simpson, Denise S; Maki, Brooks E; Li, Kelin; Golden, Jennifer E; Foutz, Terry D; Waller, Anna; Evangelisti, Annette M; Young, Susan M; Chavez, Stephanie E; Garcia, Matthew J; Ursu, Oleg; Bologa, Cristian G; Carter, Mark B; Salas, Virginia M; Gouveia, Kristine; Tegos, George P; Oprea, Tudor I; Edwards, Bruce S; Aubé, Jeffrey; Larson, Richard S; Sklar, Larry A

    2013-01-01

    Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)-driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

  2. Expression and regulation of prostaglandin transporters, ATP-binding cassette, subfamily C, member 1 and 9, and solute carrier organic anion transporter family, member 2A1 and 5A1 in the uterine endometrium during the estrous cycle and pregnancy in pigs.

    Science.gov (United States)

    Jang, Hwanhee; Choi, Yohan; Yoo, Inkyu; Han, Jisoo; Kim, Minjeong; Ka, Hakhyun

    2017-05-01

    Prostaglandins (PGs) function in various reproductive processes, including luteolysis, maternal pregnancy recognition, conceptus development, and parturition. Our earlier study has shown that PG transporters ATP-binding cassette, subfamily C, member 4 ( ABCC4 ) and solute carrier organic anion transporter family, member 2A1 ( SLCO2A1 ) are expressed in the uterine endometrium in pigs. Since several other PG transporters such as ABCC1 , ABCC9 , SLCO4C1 , and SLCO5A1 are known to be present in the uterine endometrium, this study investigated the expression of these PG transporters in the porcine uterine endometrium and placenta. Uterine endometrial tissues were obtained from gilts on day (D) 12 and D15 of the estrous cycle and days 12, 15, 30, 60, 90, and 114 of pregnancy. ABCC1 , ABCC9 , SLCO4C1 , and SLCO5A1 mRNAs were expressed in the uterine endometrium, and levels of expression changed during the estrous cycle and pregnancy. Expression of ABCC1 and ABCC9 mRNAs was localized mainly to luminal and glandular epithelial cells in the uterine endometrium, and chorionic epithelial cells during pregnancy. Conceptuses during early pregnancy and chorioallantoic tissues from mid to late pregnancy also expressed these PG transporters. Estradiol-17β increased the expression of ABCC1 and SLCO5A1 , but not ABCC9 and SLCO4C1 mRNAs and increasing doses of interleukin-1β induced the expression of ABCC9 , SLCO4C1 , and SLCO5A1 mRNAs in endometrial explant tissues. These data showed that several PG transporters such as ABCC1 , ABCC9 , SLCO4C1 , and SLCO5A1 were expressed at the maternal-conceptus interface, suggesting that these PG transporters may play an important role in the establishment and maintenance of pregnancy by regulating PG transport in the uterine endometrium and placenta in pigs.

  3. Hydrolysis at One of the Two Nucleotide-binding Sites Drives the Dissociation of ATP-binding Cassette Nucleotide-binding Domain Dimers*

    Science.gov (United States)

    Zoghbi, Maria E.; Altenberg, Guillermo A.

    2013-01-01

    The functional unit of ATP-binding cassette (ABC) transporters consists of two transmembrane domains and two nucleotide-binding domains (NBDs). ATP binding elicits association of the two NBDs, forming a dimer in a head-to-tail arrangement, with two nucleotides “sandwiched” at the dimer interface. Each of the two nucleotide-binding sites is formed by residues from the two NBDs. We recently found that the prototypical NBD MJ0796 from Methanocaldococcus jannaschii dimerizes in response to ATP binding and dissociates completely following ATP hydrolysis. However, it is still unknown whether dissociation of NBD dimers follows ATP hydrolysis at one or both nucleotide-binding sites. Here, we used luminescence resonance energy transfer to study heterodimers formed by one active (donor-labeled) and one catalytically defective (acceptor-labeled) NBD. Rapid mixing experiments in a stop-flow chamber showed that NBD heterodimers with one functional and one inactive site dissociated at a rate indistinguishable from that of dimers with two hydrolysis-competent sites. Comparison of the rates of NBD dimer dissociation and ATP hydrolysis indicated that dissociation followed hydrolysis of one ATP. We conclude that ATP hydrolysis at one nucleotide-binding site drives NBD dimer dissociation. PMID:24129575

  4. 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

    to assess whether polymorphisms in ABCB1, ABCC2 and ABCG2 were associated with risk of colorectal cancer (CRC) and to investigate gene-environment (dietary factors, smoking and use of non-steroidal anti-inflammatory drugs) and gene-gene interactions between previously studied polymorphisms in IL1B and IL10...... and ABC transporter genes in relation to CRC risk. We used a Danish prospective case-cohort study of 1010 CRC cases and 1829 randomly selected participants from the Danish Diet, Cancer and Health cohort. Incidence rate ratios were calculated based on Cox' proportional hazards model. None...

  5. Apolipoprotein A-I and HDL Have Anti-Inflammatory Effects on Adipocytes via Cholesterol Transporters: ATP-Binding Cassette (ABC) A-1, ABCG-1 and Scavenger Receptor B-1(SRB-1)

    Science.gov (United States)

    Umemoto, Tomio; Han, Chang Yeop; Mitra, Poulami; Averill, Michelle M.; Tang, Chongren; Goodspeed, Leela; Omer, Mohamed; Subramanian, Savitha; Wang, Shari; Den Hartigh, Laura J.; Wei, Hao; Kim, Eung Ju; Kim, Jinkyu; O'Brien, Kevin D.; Chait, Alan

    2013-01-01

    Rationale Macrophage accumulation in adipose tissue associates with insulin resistance and increased cardiovascular disease risk. We previously have shown that generation of reactive oxygen species (ROS) and monocyte chemotactic factors after exposure of adipocytes to saturated fatty acids (SFAs) such as palmitate occurs via translocation of NADPH oxidase 4 (NOX4) into lipid rafts (LRs). The anti-inflammatory effects of apolipoprotein A-I (apoA-I) and HDL on macrophages and endothelial cells appears to occur via cholesterol depletion of LRs. However, little is known concerning anti-inflammatory effects of HDL and apoA-I on adipocytes. Objective To determine whether apoA-I and HDL inhibit inflammation in adipocytes and adipose tissue, and whether this is dependent on LRs. Methods and Results In 3T3L-1 adipocytes, apoA-I, HDL and methyl-β-cyclodextrin inhibited chemotactic factor expression. ApoA-I and HDL also disrupted LRs, reduced plasma membrane cholesterol content, inhibited NOX4 translocation into LRs, and reduced palmitate-induced ROS generation and monocyte chemotactic factor expression. Silencing ABCA-1 abrogated the effect of apoA-I, but not HDL, while silencing ABCG-1 or SRB-1 abrogated the effect of HDL but not apoA-I. In vivo, apoA-I transgenic mice fed a high fat, high sucrose, cholesterol-containing diet showed reduced chemotactic factor and pro-inflammatory cytokine expression and reduced macrophage accumulation in adipose tissue. Conclusion ApoA-I and HDL have anti-inflammatory effects in adipocytes and adipose tissue similar to their effects in other cell types. These effects are consistent with disruption and removal of cholesterol from LRs, which are regulated by cholesterol transporters such as ABCA-1, ABCG-1 and SRB-1. PMID:23501697

  6. Flavone Glucoside Uptake into Barley Mesophyll and Arabidopsis Cell Culture Vacuoles. Energization Occurs by H+-Antiport and ATP-Binding Cassette-Type Mechanisms1

    Science.gov (United States)

    Frangne, Nathalie; Eggmann, Thomas; Koblischke, Carsten; Weissenböck, Gottfried; Martinoia, Enrico; Klein, Markus

    2002-01-01

    In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a Km of 50 to 100 μm. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H+ antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta. PMID:11842175

  7. Acridone suppresses the proliferation of human breast cancer cellsin vitrovia ATP-binding cassette subfamily G member 2.

    Science.gov (United States)

    Xu, Licheng; Li, Shuyan; Liang, Zhi; Lin, Haixia; Fu, Rongzhan

    2018-02-01

    In the past decades, the tricyclic acridone ring system has become a focus of major research by medicinal chemists due to the biological significance of this moiety in drug design and discovery. Acridone has substantial bio-potential since it performs crucial functions, including antibacterial, antimalarial, antiviral and anti-neoplastic activities. However, the anticancer effect and the underlying mechanisms of acridone on breast cancer cells remains unclear. In the present study, the anti-tumor function and the underlying mechanisms of acridone were evaluated in vitro . Firstly, an MTT assay was used to evaluate the inhibitory effect of acridone. Subsequently, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to investigate whether ATP binding cassette subfamily G member 2 (ABCG2) was associated with the function of acridone. Finally, western blotting was used to confirm the results of RT-qPCR. The present study demonstrated that acridone may decrease the proliferation of MDA-MB-231 cells dose-dependently. Further experiments revealed that acridone may downregulate the mRNA and protein expression levels of ABCG2, supporting the potential application of acridone in breast cancer treatment. These findings suggested that acridone is a potential agent in the treatment of human breast cancer.

  8. Sorafenib modulates the gene expression of multi-drug resistance mediating ATP-binding cassette proteins in experimental hepatocellular carcinoma.

    Science.gov (United States)

    Hoffmann, Katrin; Franz, Clemens; Xiao, Zhi; Mohr, Elvira; Serba, Susanne; Büchler, Markus W; Schemmer, Peter

    2010-11-01

    High ATP-binding cassette (ABC) protein expression leads to intrinsic drug resistance of hepatocellular carcinoma (HCC). The aim of this study was to investigate the potential chemosensitizing effects of sorafenib on the multi-drug resistance (MDR) phenotype. The ABC-protein gene expression and the cellular survival were determined by RT-PCR analysis and MTT assay in HUH7 cells. Sorafenib inhibits MDR. The ABC-protein mRNA expression decreased by up to 51% (p ≤ 0.01). Addition of sorafenib to conventional chemotherapy restored the chemosensitivity. Combination of gemcitabine plus sorafenib decreased the ABC-protein mRNA levels by up to 77%, compared to gemcitabine monotherapy (p ≤ 0.001). Doxorubicin plus sorafenib decreased the ABC-protein mRNA levels up to 74% compared to doxorubicin monotherapy (p ≤ 0.001). This study provides evidence that the MDR phenotype of HCC cells can be modulated by the multi-kinase inhibitor sorafenib and consequentially may lead towards personalized therapies in patients with highly resistant tumors.

  9. ATP-binding cassette G5/G8 deficiency causes hypertriglyceridemia by affecting multiple metabolic pathways.

    Science.gov (United States)

    Méndez-González, Jesús; Julve, Josep; Rotllan, Noemí; Llaverias, Gemma; Blanco-Vaca, Francisco; Escolà-Gil, Joan Carles

    2011-12-01

    Mutations in ABCG5 or ABCG8 transporters are responsible for sitosterolemia, an autosomal recessive disease characterized by the accumulation of plant sterols. The aim of this study was to investigate the effects of ABCG5 and ABCG8 deficiency on TG metabolism in mice. Experiments were carried out in wild-type (G5/G8+/+) mice, mice heterozygous for ABCG5 and ABCG8 deficiency (G5/G8+/-) and ABCG5/G8-deficient (G5/G8-/-) mice fed a chow diet. Plasma TG were 2.6 and 4.3-fold higher in fasted G5/G8+/- and G5/G8-/- mice, respectively, than in G5/G8+/+ mice. Postprandial TG were 5-fold higher in G5/G8-/- mice. TG metabolism studies indicate that: first, the fractional catabolic rate was significantly lower in G5/G8+/- (1.3-fold) and G5/G8-/- mice (1.5-fold) compared to G5/G8+/+ and postheparin plasma lipoprotein lipase activities were significantly lower in G5/G8+/- (1.8-fold) and G5/G8-/- mice (5.4-fold) than in G5/G8+/+. Second, liver TG secretion was 1.3-fold higher in G5/G8+/- and G5/G8-/- than in G5/G8+/+ mice and this was associated with an increase in liver LXR, FAS, ACAC and CD36 gene expression. Third, TG intestinal secretion, determined after an oral fat gavage of glycerol tri[9,10(n)-(3)H] oleate, was 5.8-fold higher in G5/G8-/- than in G5/G8+/+ mice. Also, the HOMA index was 2.6-fold higher in G5/G8-/- than in G5/G8+/+ mice, reflecting a degree of insulin resistance. In conclusion, ABCG5/G8 deficiency in mice fed a chow diet markedly raises TG levels by impairing TG catabolism and by increasing liver and intestinal TG secretion. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ngu Njei Abanda

    2017-02-01

    Full Text Available 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.

  11. Pravastatin Modulate Niemann-Pick C1-Like 1 and ATP-Binding Cassette G5 and G8 to Influence Intestinal Cholesterol Absorption.

    Science.gov (United States)

    Kawase, Atsushi; Hata, Seiji; Takagi, Mai; Iwaki, Masahiro

    2015-01-01

    Niemann-Pick C1-like 1 (NPC1L1), ATP-binding cassette (ABC)G5, and ABCG8 mediate intestinal cholesterol absorption. It is unclear whether pravastatin (PR) or ezetimibe (EZ) affect expression of these transporters. We examined the effects of PR and EZ on NPC1L1, ABCG5, and ABCG8 expression in human hepatoma HepG2 cells and the murine small intestine. We also assessed expression of the transcription factors liver X receptor (LXR)a, LXRb and sterol regulatory element-binding protein. Transporter mRNA levels were determined in murine small intestines 6 and 24 h after oral PR and EZ administration by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). In PR- and EZ-treated HepG2 cells, transporter and transcription factor mRNA and protein levels were examined by RT-PCR and western blot, respectively. Significant decreases in NPC1L1, ABCG5, and ABCG8 mRNA expression were observed in the duodenum, but not jejunum and ileum, of mice 24 h after treatment with PR, but not EZ. In HepG2 cells, PR but not EZ treatment for 24 h also significantly decreased NPC1L1 protein and ABCG5, and ABCG8 mRNA expression, while increasing LXRa mRNA levels. PR but not EZ treatment reduced duodenal cholesterol transporter expression in mice. PR-induced increases in LXRa mRNA levels may be involved in attenuation of NPC1L1 expression, subsequently decreasing intestinal cholesterol absorption.

  12. Evaluation of the in vitro expression of ATP binding-cassette (ABC) proteins in an Ixodes ricinus cell line exposed to ivermectin.

    Science.gov (United States)

    Mangia, Carlo; Vismarra, Alice; Kramer, Laura; Bell-Sakyi, Lesley; Porretta, Daniele; Otranto, Domenico; Epis, Sara; Grandi, Giulio

    2016-04-18

    Ticks are among the most important vectors of pathogens causing human and animal disease. Acaricides are used to control tick infestation, although there are increasing reports of resistance. Recently, over-expression of ATP-binding cassette (ABC) transporter proteins (P-glycoproteins, PgP) has been implicated in resistance to the acaricide ivermectin in the ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus sanguineus sensu lato. Ixodid tick cell lines have been used to investigate drug resistance mechanisms. The aim of the present study was to evaluate expression of several PgPs in the Ixodes ricinus-derived cell line IRE/CTVM19 and to determine modulation of expression following treatment with ivermectin. IRE/CTVM19 cells were treated with different concentrations of ivermectin (0, 11, 22 or 33 μM) and incubated for 10 days. Evaluation of viability and relative expression of ABCB1, ABCB6, ABCB8 and ABCB10 genes were carried out at day 10 post treatment. Cell viability ranged between 84% and 92% with no significant differences between untreated and treated cells. qRT-PCR showed that ABC pump expression was not significantly modulated by ivermectin treatment. Expression of the ABCB8 PgP subfamily revealed a biphasic trend, based on the ivermectin concentration. ABCB6 and ABCB10 gene expression was not modulated by ivermectin treatment and ABCB1 expression was not detected. This is the first report of PgP expression in an I. ricinus-derived tick cell line. Development of an in vitro model for the study of acaricide resistance mechanisms would greatly facilitate screening for drug resistance in ticks.

  13. A role for calcium in the regulation of ATP-binding cassette, sub-family C, member 3 (ABCC3) gene expression in a model of epidermal growth factor-mediated breast cancer epithelial-mesenchymal transition.

    Science.gov (United States)

    Stewart, Teneale A; Azimi, Iman; Thompson, Erik W; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2015-03-13

    Epithelial-mesenchymal transition (EMT), a process implicated in cancer metastasis, is associated with the transcriptional regulation of members of the ATP-binding cassette superfamily of efflux pumps, and drug resistance in breast cancer cells. Epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells is calcium signal dependent. In this study induction of EMT was shown to result in the transcriptional up-regulation of ATP-binding cassette, subfamily C, member 3 (ABCC3), a member of the ABC transporter superfamily, which has a recognized role in multidrug resistance. Buffering of cytosolic free calcium inhibited EGF-mediated ABCC3 increases, indicating a calcium-dependent mode of regulation. Silencing of TRPM7 (an ion channel involved in EMT associated vimentin induction) did not inhibit ABCC3 up-regulation. Silencing of the store operated calcium entry (SOCE) pathway components ORAI1 and STIM1 also did not alter ABCC3 induction by EGF. However, the calcium permeable ion channel transient receptor potential cation channel, subfamily C, member 1 (TRPC1) appears to contribute to the regulation of both basal and EGF-induced ABCC3 mRNA. Improved understanding of the relationship between calcium signaling, EMT and the regulation of genes important in therapeutic resistance may help identify novel therapeutic targets for breast cancer. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. 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.

  15. Regression of atherosclerosis with apple procyanidins by activating the ATP-binding cassette subfamily A member 1 in a rabbit model.

    Science.gov (United States)

    Wang, Liang; Fumoto, Toshio; Masumoto, Saeko; Shoji, Toshihiko; Miura, Tomisato; Naraoka, Masato; Matsuda, Naoya; Imaizumi, Tadaatsu; Ohkuma, Hiroki

    2017-03-01

    Apple polyphenol contains abundant procyanidins, which have been associated with an anti-atherosclerosis and cholesterol-lowering effect. The aim of this study was to investigate whether apple procyanidins (APCs) feature therapeutic efficacy in terms of regressing atherosclerosis and whether this efficacy is due to mechanisms other than a cholesterol-lowering effect. After eight weeks on an atherogenic diet, rabbits were given a normal diet for another eight weeks to normalize the increased serum lipids level. The rabbits in the baseline group were sacrificed at this stage. The control group was subsequently fed a normal diet for eight weeks, while the APCs group was administrated 50 mg/kg/day of APCs in addition to the normal diet. Serum lipids and aortic intimal-medial thickness (IMT) were serially examined, and the resected aorta was examined histologically and through molecular biology. Aortic IMT on ultrasonography and the lipid accumulation area examined using Sudan IV staining were significantly reduced in the APCs group as compared to the control group. Serum lipid profiles were not different between the groups. Immunohistochemistry showed significantly decreased staining of an oxidative stress marker and significantly increased staining of ATP-binding cassette subfamily A member 1 (ABCA1) in the APCs group. Western blotting and RT-PCR also showed increased expression of ABCA1 mRNA and its protein in the APCs group. This study revealed that APCs administration causes a regression of atherosclerosis. APCs might hold promise as an anti-atherosclerotic agent. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The human ATP binding cassette gene ABCA13, located on chromosome 7p12.3, encodes a 5058 amino acid protein with an extracellular domain encoded in part by a 4.8-kb conserved exon.

    Science.gov (United States)

    Prades, C; Arnould, I; Annilo, T; Shulenin, S; Chen, Z Q; Orosco, L; Triunfol, M; Devaud, C; Maintoux-Larois, C; Lafargue, C; Lemoine, C; Denèfle, P; Rosier, M; Dean, M

    2002-01-01

    The ABCA subfamily of ATP-binding cassette (ABC) transporters includes eleven members to date. In this study, we describe a new, unusually large gene on chromosome 7p12.3, ABCA13. This gene spans over 450 kb and is split into 62 exons. The predicted ABCA13 protein consists of 5,058 ami- no acid residues making it the largest ABC protein described to date. Like the other ABCA subfamily members, ABCA13 contains a hydrophobic, predicted transmembrane segment at the N-terminus, followed by a large hydrophilic region. In the case of ABCA13, the hydrophilic region is unexpectedly large, more than 3,500 amino acids, encoded by 30 exons, two of which are 4.8 and 1.7 kb in length. These two large exons are adjacent to each other and are conserved in the mouse Abca13 gene. Tissue profiling of the major transcript reveals the highest expression in human trachea, testis, and bone marrow. The expression of the gene was also determined in 60 tumor cell lines and the highest expression was detected in the SR leukemia, SNB-19 CNS tumor and DU-145 prostate tumor cell lines. ABCA13 has high similarity with other ABCA subfamily genes which are associated with human inherited diseases: ABCA1 with the cholesterol transport disorders Tangier disease and familial hypoalphalipoproteinemia, and ABCA4 with several retinal degeneration disorders. The ABCA13 gene maps to chromosome 7p12.3, a region that contains an inherited disorder affecting the pancreas (Shwachman-Diamond syndrome) as well as a locus involved in T-cell tumor invasion and metastasis (INM7), and therefore is a positional candidate for these pathologies. Copyright 2002 S. Karger AG, Basel

  17. Adenosine triphosphate-binding cassette transporter genes up-regulation in untreated hepatocellular carcinoma is mediated by cellular microRNAs

    NARCIS (Netherlands)

    Borel, Florie; Han, Ruiqi; Visser, Allerdien; Petry, Harald; van Deventer, Sander J. H.; Jansen, Peter L. M.; Konstantinova, Pavlina

    2012-01-01

    Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are drug efflux pumps responsible for the multidrug resistance phenotype causing hepatocellular carcinoma (HCC) treatment failure. Here we studied the expression of 15 ABC transporters relevant for multidrug resistance in 19 paired HCC

  18. ATP Binding and Hydrolysis Properties of ABCB10 and Their Regulation by Glutathione

    Science.gov (United States)

    Qiu, Wei; Liesa, Marc; Carpenter, Elizabeth P.; Shirihai, Orian S.

    2015-01-01

    ABCB10 (ATP binding cassette sub-family B10) is a mitochondrial inner-membrane ABC transporter. ABCB10 has been shown to protect the heart from the impact of ROS during ischemia-reperfusion and to allow for proper hemoglobin synthesis during erythroid development. ABC transporters are proteins that increase ATP binding and hydrolysis activity in the presence of the transported substrate. However, molecular entities transported by ABCB10 and its regulatory mechanisms are currently unknown. Here we characterized ATP binding and hydrolysis properties of ABCB10 by using the 8-azido-ATP photolabeling technique. This technique can identify potential ABCB10 regulators, transported substrates and amino-acidic residues required for ATP binding and hydrolysis. We confirmed that Gly497 and Lys498 in the Walker A motif, Glu624 in the Walker B motif and Gly602 in the C-Loop motif of ABCB10 are required for proper ATP binding and hydrolysis activity, as their mutation changed ABCB10 8-Azido-ATP photo-labeling. In addition, we show that the potential ABCB10 transported entity and heme precursor delta-aminolevulinic acid (dALA) does not alter 8-azido-ATP photo-labeling. In contrast, oxidized glutathione (GSSG) stimulates ATP hydrolysis without affecting ATP binding, whereas reduced glutathione (GSH) inhibits ATP binding and hydrolysis. Indeed, we detectABCB10 glutathionylation in Cys547 and show that it is one of the exposed cysteine residues within ABCB10 structure. In all, we characterize essential residues for ABCB10 ATPase activity and we provide evidence that supports the exclusion of dALA as a potential substrate directly transported by ABCB10. Last, we show the first molecular mechanism by which mitochondrial oxidative status, through GSH/GSSG, can regulate ABCB10. PMID:26053025

  19. Evidence for direct physical association between a K+ channel (Kir6.2) and an ATP-binding cassette protein (SUR1) which affects cellular distribution and kinetic behavior of an ATP-sensitive K+ channel.

    Science.gov (United States)

    Lorenz, E; Alekseev, A E; Krapivinsky, G B; Carrasco, A J; Clapham, D E; Terzic, A

    1998-03-01

    Structurally unique among ion channels, ATP-sensitive K+ (KATP) channels are essential in coupling cellular metabolism with membrane excitability, and their activity can be reconstituted by coexpression of an inwardly rectifying K+ channel, Kir6.2, with an ATP-binding cassette protein, SUR1. To determine if constitutive channel subunits form a physical complex, we developed antibodies to specifically label and immunoprecipitate Kir6.2. From a mixture of Kir6.2 and SUR1 in vitro-translated proteins, and from COS cells transfected with both channel subunits, the Kir6.2-specific antibody coimmunoprecipitated 38- and 140-kDa proteins corresponding to Kir6.2 and SUR1, respectively. Since previous reports suggest that the carboxy-truncated Kir6.2 can form a channel independent of SUR, we deleted 114 nucleotides from the carboxy terminus of the Kir6.2 open reading frame (Kir6.2deltaC37). Kir6.2deltaC37 still coimmunoprecipitated with SUR1, suggesting that the distal carboxy terminus of Kir6.2 is unnecessary for subunit association. Confocal microscopic images of COS cells transfected with Kir6.2 or Kir6.2deltaC37 and labeled with fluorescent antibodies revealed unique honeycomb patterns unlike the diffuse immunostaining observed when cells were cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1. Membrane patches excised from COS cells cotransfected with Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 exhibited single-channel activity characteristic of pancreatic KATP channels. Kir6.2deltaC37 alone formed functional channels with single-channel conductance and intraburst kinetic properties similar to those of Kir6.2-SUR1 or Kir6.2deltaC37-SUR1 but with reduced burst duration. This study provides direct evidence that an inwardly rectifying K+ channel and an ATP-binding cassette protein physically associate, which affects the cellular distribution and kinetic behavior of a KATP channel.

  20. Population-Specific Resequencing Associates the ATP-Binding Cassette Subfamily C Member 4 Gene With Gout in New Zealand Māori and Pacific Men.

    Science.gov (United States)

    Tanner, Callum; Boocock, James; Stahl, Eli A; Dobbyn, Amanda; Mandal, Asim K; Cadzow, Murray; Phipps-Green, Amanda J; Topless, Ruth K; Hindmarsh, Jennie Harré; Stamp, Lisa K; Dalbeth, Nicola; Choi, Hyon K; Mount, David B; Merriman, Tony R

    2017-07-01

    There is no evidence for a genetic association between organic anion transporters 1-3 (SLC22A6, SLC22A7, and SLC22A8) and multidrug resistance protein 4 (MRP4; encoded by ABCC4) with the levels of serum urate or gout. The Māori and Pacific (Polynesian) population of New Zealand has the highest prevalence of gout worldwide. The aim of this study was to determine whether any Polynesian population-specific genetic variants in SLC22A6-8 and ABCC4 are associated with gout. All participants had ≥3 self-reported Māori and/or Pacific grandparents. Among the total sample set of 1,808 participants, 191 hyperuricemic and 202 normouricemic individuals were resequenced over the 4 genes, and the remaining 1,415 individuals were used for replication. Regression analyses were performed, adjusting for age, sex, and Polynesian ancestry. To study the functional effect of nonsynonymous variants of ABCC4, transport assays were performed in Xenopus laevis oocytes. A total of 39 common variants were detected, with an ABCC4 variant (rs4148500) significantly associated with hyperuricemia and gout. This variant was monomorphic for the urate-lowering allele in Europeans. There was evidence for an association of rs4148500 with gout in the resequenced samples (odds ratio [OR] 1.62 [P = 0.012]) that was replicated (OR 1.25 [P = 0.033]) and restricted to men (OR 1.43 [P = 0.001] versus OR 0.98 [P = 0.89] in women). The gout risk allele was associated with fractional excretion of uric acid in male individuals (β = -0.570 [P = 0.01]). A rare population-specific allele (P1036L) with predicted strong functional consequence reduced the uric acid transport activity of ABCC4 by 30%. An association between ABCC4 and gout and fractional excretion of uric acid is consistent with the established role of MRP4 as a unidirectional renal uric acid efflux pump. © 2017, American College of Rheumatology.

  1. 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

  2. Impact of genetic variants of ATP binding cassette B1, AICAR transformylase/IMP cyclohydrolase, folyl-polyglutamatesynthetase, and methylenetetrahydrofolatereductase on methotrexate toxicity.

    Science.gov (United States)

    Sala-Icardo, Luis; Lamana, Amalia; Ortiz, Ana María; García Lorenzo, Elena; Moreno Fresneda, Pablo; García-Vicuña, Rosario; González-Álvaro, Isidoro

    To analyze the effect of single nucleotide polymorphisms (SNPs) with well-known functional impact of methylenetetrahydrofolatereductase (MTHFR; rs1801131 and rs1801133), the membrane transporter ABCB1 (rs1045642), the AICAR transformylase/IMP cyclohydrolase (ATIC; rs2372536) and folyl-polyglutamatesynthetase (FPGS; rs1544105), on liver and bone marrow toxicity of methotrexate (MTX). We analyzed 1415 visits from 350 patients of the PEARL (Princesa Early Arthritis Register Longitudinal) study: (732 with MTX, 683 without MTX). The different SNPs were genotyped using specific TaqMan probes (Applied Biosystems). Multivariate analyzes were performed using generalized linear models in which the dependent variables were the levels of serum alanine aminotransferase (liver toxicity), leukocytes, platelets or hemoglobin (hematologic toxicity) and adjusted for clinical variables (disease activity, etc.), analytical (renal function, etc.), sociodemographic (age, sex, etc.) and genetic variants of MTHFR, ABCB1, ATIC and FPGS. The effect of these variables on the MTX doses prescribed throughout follow-up was also analyzed through multivariate analysis nested by visit and patient. When taking MTX, those patients carrying the CC genotype of rs1045642 in ABCB1 showed significantly higher GPT levels (7.1±2.0 U/L; P<.001). Carrying at least one G allele of rs1544105 in FPGS was associated with lower leukocyte (-0.67±0.32; 0.038), hemoglobin (-0.34±0.11g/dL; P=.002), and platelet (-11.8±4.7; P=.012) levels. The presence of the G allele of rs1544105 in FPGS, and the T allele of rs1801133 in MTHFR, was significantly associated with the use of lower doses of MTX. Our data suggest that genotyping functional variants in FGPS and MTHFR enzymes and the transporter ABCB1 could help to identify patients with increased risk of MTX toxicity. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

  3. 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.

  4. 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...

  5. MicroRNA-320a and microRNA-4496 attenuate Helicobacter pylori cytotoxin-associated gene A (CagA)-induced cancer-initiating potential and chemoresistance by targeting β-catenin and ATP-binding cassette, subfamily G, member 2.

    Science.gov (United States)

    Kang, Dong Woo; Yang, Eun Sun; Noh, Yu Na; Hwang, Won Chan; Jo, Se-Young; Suh, Young-Ah; Park, Won Sang; Choi, Kang-Yell; Min, Do Sik

    2017-04-01

    Infection with Helicobacter pylori is closely linked to an increased risk of gastric cancer. Although cytotoxin-associated gene A (CagA), a major virulence factor of H. pylori, is known to be a causal factor for gastric carcinogenesis, the molecular link between CagA and gastric cancer-initiating cell (CIC)-like properties remains elusive. Here, we demonstrate that CagA is required for increased expression of β-catenin and its target CIC markers via downregulation of microRNA (miR)-320a and miR-4496. CagA promoted gastric CIC properties and was responsible for chemoresistance. miR-320a and miR-4496 attenuated the in vitro self-renewal and tumour-initiating capacity of CagA-expressing CICs by targeting β-catenin. Moreover, miR-320a and miR-4496 decreased CagA-induced chemoresistance by targeting ATP-binding cassette, subfamily G, member 2 (ABCG2) at the transcriptional and post-transcriptional levels, respectively. Combination therapy with 5-fluorouracil and miR-320a/miR-4496 suppressed gastric tumourigenesis and metastatic potential in an orthotopic mouse model, probably via suppression of CagA-induced CIC properties and chemoresistance. Our results provide novel evidence that CIC properties, chemoresistance and tumourigenesis associated with H. pylori are linked to CagA-induced upregulation of β-catenin and ABCG2. These data provide novel insights into the molecular mechanisms of CagA-induced carcinogenisis and the therapeutic potential of of miR-320a and miR-4496. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  6. 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

    2015-01-20

    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.

  7. Membrane topology of the lactococcal bacteriocin ATP-binding cassette transporter protein LcnC : Involvement of LcnC in lactococcin A maturation

    NARCIS (Netherlands)

    Franke, CM; Tiemersma, J; Venema, G; Kok, J

    1999-01-01

    Many non-lantibiotic bacteriocins of lactic acid bacteria are produced as precursors with N-terminal leader peptides different from those present in preproteins exported by the general sec-dependent (type II) secretion pathway. These bacteriocins utilize a dedicated (type I) secretion system for

  8. Versatile inhibitory effects of the flavonoid-derived PI3K/Akt inhibitor, LY294002, on ATP-binding cassette transporters that characterize stem cells

    OpenAIRE

    Imai, Yasuo; Yamagishi, Hidetsugu; Ono, Yuko; Ueda, Yoshihiko

    2012-01-01

    Stem cells are undifferentiated cells capable of proliferation, self-renewal, and production of a large number of differentiated progeny. Stem cells exist even in malignancies. They are called cancer stem cells, which may represent the origin of these tumors and may be one of the reasons of chemoresistance. The phosphatidylinositol-3-kinase (PI3K)/Akt pathway is important for the maintenance of pluripotency in stem cells. Flow cytometry assay for identifying stem cells defines a side populati...

  9. Deficiency of ATP-Binding Cassette Transporters A1 and G1 in Macrophages Increases Inflammation and Accelerates Atherosclerosis in Mice

    NARCIS (Netherlands)

    Westerterp, Marit; Murphy, Andrew J.; Wang, Mi; Pagler, Tamara A.; Vengrenyuk, Yuliya; Kappus, Mojdeh S.; Gorman, Darren J.; Nagareddy, Prabhakara R.; Zhu, Xuewei; Abramowicz, Sandra; Parks, John S.; Welch, Carrie; Fisher, Edward A.; Wang, Nan; Yvan-Charvet, Laurent; Tall, Alan R.

    2013-01-01

    Rationale: Plasma high-density lipoprotein levels are inversely correlated with atherosclerosis. Although it is widely assumed that this is attributable to the ability of high-density lipoprotein to promote cholesterol efflux from macrophage foam cells, direct experimental support for this

  10. 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.

  11. Diversity of transport mechanisms: common structural principles

    NARCIS (Netherlands)

    Driessen, A.J.M.; Rosen, B.P.; Konings, W.N

    2000-01-01

    Traditionally, prokaryotic solute transport systems are classified into major groups based on the energetic requirement of the transport process. These include the secondary transporters that are driven by a proton or sodium motive force, and the ATP-binding cassette (ABC) primary transporters,

  12. 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

  13. ABC and MFS transporters from Botrytis cinerea involved in sensitivity to fungicides and natural toxic compounds

    NARCIS (Netherlands)

    Hayashi, K.

    2003-01-01

    ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters are two major classes of proteins involved in drug resistance. ABC transporter proteins are primary transporters that use the energy generated by ATP hydrolysis to transport drugs over membranes, while MFS transport

  14. Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

    NARCIS (Netherlands)

    Slotboom, Dirk J.

    2014-01-01

    Energy-coupling factor (ECF) transporters belong to the ATP-binding cassette (ABC)-transporter family and mediate the uptake of essential micronutrients in many prokaryotic species. Two crystal structures of bacterial ECF transporters have recently been obtained and suggest that transport involves

  15. 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

  16. Metabolism and transport of the citrus flavonoid hesperetin in Caco-2 cell monolayers

    NARCIS (Netherlands)

    Brand, W.; Wel, van der P.A.I.; Rein, M.J.; Barron, D.; Williamson, G.; Bladeren, van P.J.; Rietjens, I.M.C.M.

    2008-01-01

    Metabolism and transport from intestinal cells back into the lumen by ATP-binding cassette (ABC) transporters is believed to limit the bioavailability of flavonoids. We studied metabolism and transport of the citrus flavonoid hesperetin, the aglycone of hesperidin, using a two-compartment transwell

  17. ABC of oral bioavailability: transporters as gatekeepers in the gut

    NARCIS (Netherlands)

    Dietrich, C. G.; Geier, A.; Oude Elferink, R. P. J.

    2003-01-01

    MDR1 (ABCB1), MRP2 (ABCC2), and BCRP (ABCG2) are members of the family of ATP binding cassette (ABC) transporters. These are plasma membrane transporters that are expressed in various organs. The role of MDR1 and MRP2 in the hepatobiliary system is well defined; both contribute to bile formation by

  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

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

  4. Drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p

    NARCIS (Netherlands)

    Kolaczkowski, M; vanderRest, M; CybularzKolaczkowska, A; Soumillion, JP; Konings, WN; Goffeau, A

    1996-01-01

    Pdr5p is the yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Its high overproduction in Pdr1p transcription factor mutants allows us to study the molecular mechanism of multidrug transport and substrate specificity. We have developed

  5. 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; Knudsen, Lina Almind

    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...... with glucocorticoids. The evidence for the involvement of ABCC2 and ABCG2 in colonic pathophysiology was weak. CONCLUSION: ABCB1, diet, and gut microbes mutually interact in colonic inflammation, a well-known risk factor for CRC. Further insight may be translated into preventive and treatment strategies....

  6. 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 ta...

  7. 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

  8. ABC transporter genes and risk of type 2 diabetes

    DEFF Research Database (Denmark)

    Schou, Jesper; Tybjærg-Hansen, Anne; Møller, Holger Jon

    2012-01-01

    Alterations of pancreatic β-cell cholesterol content may contribute to β-cell dysfunction. Two important determinants of intracellular cholesterol content are the ATP-binding cassette (ABC) transporters A1 (ABCA1) and -G1 (ABCG1). Whether genetic variation in ABCA1 and ABCG1 predicts risk of type 2...... diabetes in the general population is unknown....

  9. 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

  10. Functional Analysis of Detergent‐Solubilized and Membrane‐Reconstituted ATP‐Binding Cassette Transporters

    NARCIS (Netherlands)

    Poolman, Bert; Doeven, Mark K.; Geertsma, Eric R.; Biemans‐Oldehinkel, Esther; Konings, Wil N.; Rees, Douglas C.

    2005-01-01

    ATP‐binding cassette (ABC) transporters are vital to any living system and are involved in the translocation of a wide variety of substances, from ions and nutrients to high molecular weight proteins. This chapter describes methods used to purify and membrane reconstitute ABC transporters in a fully

  11. 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

  12. Drosophila ABC Transporter DmHMT-1 Confers Tolerance to Cadmium.

    Science.gov (United States)

    Half molecule ATP-binding cassette transporters of the HMT1(heavy metal tolerance factor 1)subfamily are required for Cd2+ tolerance in Schizosaccharomyces pombe, Caenorhabditis elegans and Chlamydomonas reinhardtii, and have homologs in other species, including plants and humans. Based on studies i...

  13. Identification of intra- and intermolecular disulfide bridges in the multidrug resistance transporter ABCG2

    DEFF Research Database (Denmark)

    Henriksen, Ulla Birk; Fog, Jacob U; Litman, Thomas

    2005-01-01

    ABCG2 is an ATP binding cassette (ABC) half-transporter that plays a key role in multidrug resistance to chemotherapy. ABCG2 is believed to be a functional homodimer that has been proposed to be linked by disulfide bridges. We have investigated the structural and functional role of the only three...

  14. ABC transporter expression in hematopoietic stem cells and the role in AML drug resistance

    NARCIS (Netherlands)

    de Jonge-Peeters, Susan D. P. W. M.; Kuipers, Folkert; de Vries, Elisabeth G. E.; Vellenga, Edo

    ATP-binding cassette (ABC) transporters are known to play an important role in human physiology, toxicology, pharmacology, and numerous disorders including acute myeloid leukemia (AML). In AML only a few cells have properties allowing for ongoing proliferation and for expansion of this malignant

  15. In vitro functional characterization of BtuCD-F, the Escherichia coli ABC transporter for vitamin B-12 uptake

    NARCIS (Netherlands)

    Borths, EL; Poolman, B; Hvorup, RN; Locher, KP; Rees, DC; Hvorup, Rikki N.; Locher, Kaspar P.; Rees, Douglas C.

    2005-01-01

    BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B-12 into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in

  16. Functional Analysis of Detergent‐Solubilized and Membrane‐Reconstituted ATP‐Binding Cassette Transporters

    OpenAIRE

    Poolman, Bert; Doeven, Mark K.; Geertsma, Eric R.; Biemans‐Oldehinkel, Esther; Konings, Wil N.; Rees, Douglas C.

    2005-01-01

    ATP‐binding cassette (ABC) transporters are vital to any living system and are involved in the translocation of a wide variety of substances, from ions and nutrients to high molecular weight proteins. This chapter describes methods used to purify and membrane reconstitute ABC transporters in a fully functional state. The procedures are largely based on our experience with substrate‐binding protein‐dependent ABC uptake systems from bacteria, but the approaches should be applicable to multisubu...

  17. ABCA Transporter Gene Expression and Poor Outcome in Epithelial Ovarian Cancer

    DEFF Research Database (Denmark)

    Hedditch, Ellen L; Gao, Bo; Russell, Amanda J

    2014-01-01

    BACKGROUND: ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. METHODS: The relationship between clinical outcomes and ABC transporter gene expression in two in...... cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. CONCLUSIONS: Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid trafficking as a potentially important process in EOC....

  18. Tissue Distribution, Gender-Divergent Expression, Ontogeny, and Chemical Induction of Multidrug Resistance Transporter Genes (Mdr1a, Mdr1b, Mdr2) in Mice

    OpenAIRE

    Cui, Yue Julia; Cheng, Xingguo; Weaver, Yi Miao; Klaassen, Curtis D.

    2008-01-01

    Multidrug resistance (Mdr) transporters are ATP-binding cassette transporters that efflux amphipathic cations from cells and protect tissues from xenobiotics. Unfortunately, Mdr transporters also efflux anticancer drugs from some tumor cells, resulting in multidrug resistance. There are two groups of Mdrs in mice: group I includes Mdr1a and Mdr1b that transport xenobiotics, whereas group II is Mdr2, a flipase that facilitates phospholipid excretion into bile. Little is...

  19. Kinetics of signaling-DNA-aptamer-ATP binding

    Science.gov (United States)

    Nakamura, Issei; Shi, An-Chang; Nutiu, Razvan; Yu, Jasmine M. Y.; Li, Yingfu

    2009-03-01

    DNA aptamers are molecular biosensors consisting of single functionalized DNA molecules, which can bind to specific targets or complementary DNA sequences. The binding kinetics of DNA aptamers is studied by fluorescence quenching at 23°C . A kinetic model for the binding reaction of DNA aptamer, antisense DNA, and ATP target is developed to describe experimental observations. The approach leads to a simple procedure to deduce relevant kinetic reactions and their rate constants. A comparison between theory and experiments indicates that the previously established bimolecular DNA-ATP binding does not provide a complete description of the experimental data. Side reactions such as trimolecular complexation are proposed. Rate constants of the model are determined by comparing the model predictions and experiments. Good agreements between the model and experiments have been obtained. Possible blocking reactions by the misfolded DNA aptamer are also discussed.

  20. Purification and biochemical characterisation of the yeast ABC transporter Pdr11p

    DEFF Research Database (Denmark)

    Laub, Katrine Rude

    Sterols constitute an essential lipid class in eukaryotic membranes where intracellular distributions are highly regulated. In the yeast Saccharomyces cerevisiae sterol uptake has been attributed to the two plasma membrane-localised ATP-binding cassette (ABC) transporters, Aus1p and Pdr11p. These....../reconstitution protocol and adding new insight to the biochemical behaviour of Pdr11p this work paves for future studies to unravel the sterol transport mechanism....

  1. Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation

    OpenAIRE

    Ziegler, J?rg; Schmidt, Stephan; Strehmel, Nadine; Scheel, Dierk; Abel, Steffen

    2017-01-01

    The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and ...

  2. Inactivation of the Ecs ABC Transporter of Staphylococcus aureus Attenuates Virulence by Altering Composition and Function of Bacterial Wall

    OpenAIRE

    Jonsson, Ing-Marie; Juuti, Jarmo T.; Fran?ois, Patrice; AlMajidi, Rana; Pieti?inen, Milla; Girard, Myriam; Lindholm, Catharina; Saller, Manfred J.; Driessen, Arnold J. M.; Kuusela, Pentti; Bokarewa, Maria; Schrenzel, Jacques; Kontinen, Vesa P.

    2010-01-01

    BACKGROUND: Ecs is an ATP-binding cassette (ABC) transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s) transported by Ecs is (are) still unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characte...

  3. La multiplicité de transport de la P-glycoprotéine : Etudes de modélisation comparative et de docking au sein de la famille des protéines ABC

    OpenAIRE

    Bessadok, Anis

    2011-01-01

    Some ATP-binding-cassette (ABC) transporters confer multidrug resistance (MDR) to pathogenic microorganisms and human tumor cells by mediating the extrusion of a variety of chemotherapeutic drugs out of the cell. One major resistance to chemotherapy has been correlated with the presence of these ABC transporters, acting as molecular " pumps " that actively transport drugs out of the cell, the most prevalent of these being P-glycoprotein (P-gp). Although the X-ray structures of two bacterial A...

  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...... transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1...... pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid...

  5. Bone marrow transplantation of CD117+ (c-Kit) stem cells and investigation of the bile acid transporter regulation in Abcb4-/- mice, a model of sclerosing cholangitis

    OpenAIRE

    Pasupuleti, Sravanthi

    2014-01-01

    Abcb4 (ATP-binding cassette sub family-b) or Mdr2 (multidrug resistance protein 2) is a gene which encodes for ABCB4 protein that mediates the transportation of phospholipids across the canalicular membrane of hepatocytes into the bile. Functional loss of the ABCB4 transporter disturbs the excretion of phospholipids into bile, leading to toxic bile composition, bile duct alterations, and damaged bile duct epithelia resembling sclerosing cholangitis (1). Long term consequences are biliary cirr...

  6. 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

    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...... 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...... provided will hopefully contribute to more successful transport engineering in synthetic biology....

  7. 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...

  8. 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...

  9. Mechanistic determinants of the directionality and energetics of active export by a heterodimeric ABC transporter

    Science.gov (United States)

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; Abele, Rupert; Gaudet, Rachelle; Tampé, Robert

    2014-11-01

    The ATP-binding cassette (ABC) transporter associated with antigen processing (TAP) participates in immune surveillance by moving proteasomal products into the endoplasmic reticulum (ER) lumen for major histocompatibility complex class I loading and cell surface presentation to cytotoxic T cells. Here we delineate the mechanistic basis for antigen translocation. Notably, TAP works as a molecular diode, translocating peptide substrates against the gradient in a strict unidirectional way. We reveal the importance of the D-loop at the dimer interface of the two nucleotide-binding domains (NBDs) in coupling substrate translocation with ATP hydrolysis and defining transport vectoriality. Substitution of the conserved aspartate, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. Our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  10. Plant ABC transporters enable many unique aspects of a terrestrial plant's lifestyle

    DEFF Research Database (Denmark)

    Hwang, Jae-Ung; Song, Won-Yong; Hong, Daewoong

    2016-01-01

    Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental...... processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us...

  11. Structural organization of essential iron-sulfur clusters in the evolutionarily highly conserved ATP-binding cassette protein ABCE1

    NARCIS (Netherlands)

    Barthelme, Dominik; Scheele, Urte; Dinkelaker, Stephanie; Janoschka, Adam; MacMillan, Fraser; Albers, Sonja-Verena; Driessen, Arnold J. M.; Stagni, Marco Salamone; Bill, Eckhard; Meyer-Klaucke, Wolfram; Schuenemann, Volker; Tampe, Robert; Schünemann, Volker

    2007-01-01

    The ABC protein ABCE1, formerly named RNase L inhibitor RLI1, is one of the most conserved proteins in evolution and is expressed in all organisms except eubacteria. Because of its fundamental role in translation initiation and/or ribosome biosynthesis, ABCE1 is essential for life. Its molecular

  12. Facile conversion of ATP-binding RNA aptamer to quencher-free molecular aptamer beacon.

    Science.gov (United States)

    Park, Yoojin; Nim-Anussornkul, Duangrat; Vilaivan, Tirayut; Morii, Takashi; Kim, Byeang Hyean

    2018-01-15

    We have developed RNA-based quencher-free molecular aptamer beacons (RNA-based QF-MABs) for the detection of ATP, taking advantage of the conformational changes associated with ATP binding to the ATP-binding RNA aptamer. The RNA aptamer, with its well-defined structure, was readily converted to the fluorescence sensors by incorporating a fluorophore into the loop region of the hairpin structure. These RNA-based QF-MABs exhibited fluorescence signals in the presence of ATP relative to their low background signals in the absence of ATP. The fluorescence emission intensity increased upon formation of a RNA-based QF-MAB·ATP complex. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. The importance of drug transporters in human pluripotent stem cells and in early tissue differentiation.

    Science.gov (United States)

    Apáti, Ágota; Szebényi, Kornélia; Erdei, Zsuzsa; Várady, György; Orbán, Tamás I; Sarkadi, Balázs

    2016-01-01

    Drug transporters are large transmembrane proteins which catalyse the movement of a wide variety of chemicals, including drugs as well as xeno- and endobiotics through cellular membranes. The major groups of these proteins include the ATP-binding cassette transporters which in eukaryotes work as ATP-fuelled drug 'exporters' and the Solute Carrier transporters, with various transport directions and mechanisms. In this review, we discuss the key ATP-binding cassette and Solute Carrier drug transporters which have been reported to contribute to the function and/or protection of undifferentiated human stem cells and during tissue differentiation. We review the various techniques for studying transporter expression and function in stem cells, and the role of drug transporters in foetal and placental tissues is also discussed. We especially focus on the regulation of transporter expression by factors modulating cell differentiation properties and on the function of the transporters in adjustment to environmental challenges. The relatively new and as yet unexplored territory of transporters in stem cell biology may rapidly expand and bring important new information regarding the metabolic and epigenetic regulation of 'stemness' and the early differentiation properties. Drug transporters are clearly important protective and regulatory components in stem cells and differentiation.

  14. Functional analysis of candidate ABC transporter proteins for sitosterol transport

    DEFF Research Database (Denmark)

    Albrecht, C; Elliott, J I; Sardini, A

    2002-01-01

    Two ATP-binding cassette (ABC) proteins, ABCG5 and ABCG8, have recently been associated with the accumulation of dietary cholesterol in the sterol storage disease sitosterolemia. These two 'half-transporters' are assumed to dimerize to form the complete sitosterol transporter which reduces...... the absorption of sitosterol and related molecules in the intestine by pumping them back into the lumen. Although mutations altering ABCG5 and ABCG8 are found in affected patients, no functional demonstration of sitosterol transport has been achieved. In this study, we investigated whether other ABC transporters......-specific ABC transporters have acquired specificity to exclude sitosterol and related sterols like cholesterol presumably because the abundance of cholesterol in the membrane would interfere with their action; in consequence, specific transporters have evolved to handle these sterols....

  15. ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins.

    Science.gov (United States)

    Oram, J F; Vaughan, A M

    2000-06-01

    Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.

  16. Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus

    Science.gov (United States)

    Salzer, Ralf; Herzberg, Martin; Nies, Dietrich H.; Joos, Friederike; Rathmann, Barbara; Thielmann, Yvonne; Averhoff, Beate

    2014-01-01

    The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions. PMID:25202014

  17. Mechanisms of Membrane Transport of Folates into Cells and Across Epithelia

    Science.gov (United States)

    Zhao, Rongbao; Diop-Bove, Ndeye; Visentin, Michele; Goldman, I. David

    2013-01-01

    Until recently, the transport of folates into cells and across epithelia has been interpreted primarily within the context of two transporters with high affinity and specificity for folates, the reduced folate carrier and the folate receptors. However, there were discrepancies between the properties of these transporters and characteristics of folate transport in many tissues, most notably the intestinal absorption of folates, in terms of pH dependency and substrate specificity. With the recent cloning of the proton-coupled folate transporter (PCFT) and the demonstration that this transporter is mutated in hereditary folate malabsorption, an autosomal recessive disorder, the molecular basis for this low-pH transport activity is now understood. This review focuses on the properties of PCFT and briefly addresses the two other folate-specific transporters along with other facilitative and ATP-binding cassette (ABC) transporters with folate transport activities. The role of these transporters in the vectorial transport of folates across epithelia is considered. PMID:21568705

  18. The structural basis of a high affinity ATP binding ε subunit from a bacterial ATP synthase.

    Directory of Open Access Journals (Sweden)

    Alexander Krah

    Full Text Available The ε subunit from bacterial ATP synthases functions as an ATP sensor, preventing ATPase activity when the ATP concentration in bacterial cells crosses a certain threshold. The R103A/R115A double mutant of the ε subunit from thermophilic Bacillus PS3 has been shown to bind ATP two orders of magnitude stronger than the wild type protein. We use molecular dynamics simulations and free energy calculations to derive the structural basis of the high affinity ATP binding to the R103A/R115A double mutant. Our results suggest that the double mutant is stabilized by an enhanced hydrogen-bond network and fewer repulsive contacts in the ligand binding site. The inferred structural basis of the high affinity mutant may help to design novel nucleotide sensors based on the ε subunit from bacterial ATP synthases.

  19. Overview: ABC transporters and human disease.

    Science.gov (United States)

    Gottesman, M M; Ambudkar, S V

    2001-12-01

    ABC transporters are found in all known organisms, and approximately 1,100 different transporters belonging to this family have been described in the literature. The family is defined by homology within the ATP-binding cassette (ABC) region, which extends outside of the more typical Walker motifs found in all ATP-binding proteins. Most family members also contain transmembrane domains involved in recognition of substrates, which are transported across, into, and out of cell membranes, but some members utilize ABCs as engines to regulate ion channels. There are approximately 50 known ABC transporters in the human, and there are currently 13 genetic diseases associated with defects in 14 of these transporters. The most common genetic disease conditions include cystic fibrosis, Stargardt disease, age-related macular degeneration, adrenoleukodystrophy, Tangier disease, Dubin-Johnson syndrome and progressive familial intrahepatic cholestasis. At least 8 members of this family are involved in the transport of a variety of amphipathic compounds, including anticancer drugs, and some appear to contribute to the resistance of cancer cells to chemotherapy.

  20. SwissProt search result: AK120079 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK120079 J013012G06 (O95477) ATP-binding cassette sub-family A member 1 (ATP-binding casset...te transporter 1) (ATP-binding cassette 1) (ABC-1) (Cholesterol efflux regulatory protein) ABCA1_HUMAN 2e-11 ...

  1. SwissProt search result: AK059549 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK059549 001-029-F10 (O95477) ATP-binding cassette sub-family A member 1 (ATP-binding casset...te transporter 1) (ATP-binding cassette 1) (ABC-1) (Cholesterol efflux regulatory protein) ABCA1_HUMAN 9e-11 ...

  2. SwissProt search result: AK070374 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK070374 J023051G10 (O95477) ATP-binding cassette sub-family A member 1 (ATP-binding casset...te transporter 1) (ATP-binding cassette 1) (ABC-1) (Cholesterol efflux regulatory protein) ABCA1_HUMAN 6e-12 ...

  3. SwissProt search result: AK119274 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK119274 001-130-B06 (O95477) ATP-binding cassette sub-family A member 1 (ATP-binding casset...te transporter 1) (ATP-binding cassette 1) (ABC-1) (Cholesterol efflux regulatory protein) ABCA1_HUMAN 8e-55 ...

  4. Sphingosine-1-phosphate transport and its role in immunology

    Directory of Open Access Journals (Sweden)

    Vera Reitsema

    2014-12-01

    Full Text Available 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 family, ABCA1, ABCC1, ABCG2 and potentially ABCA7 have been identified as S1P transporters. In addition spinster 2 (SPNS2, a protein from the major facilitator superfamily (MFS, was identified as a S1P transporter. Here we review the current knowledge on S1P transport and discuss how this process creates S1P gradients in the body that are important in various functions of the immune system.

  5. ATP binding by NLRP7 is required for inflammasome activation in response to bacterial lipopeptides.

    Science.gov (United States)

    Radian, Alexander D; Khare, Sonal; Chu, Lan H; Dorfleutner, Andrea; Stehlik, Christian

    2015-10-01

    Nucleotide-binding oligimerization domain (NOD)-like receptors (NLRs) are pattern recognition receptors (PRRs) involved in innate immune responses. NLRs encode a central nucleotide-binding domain (NBD) consisting of the NAIP, CIITA, HET-E and TP1 (NACHT) domain and the NACHT associated domain (NAD), which facilitates receptor oligomerization and downstream inflammasome signaling. The NBD contains highly conserved regions, known as Walker motifs, that are required for nucleotide binding and hydrolysis. The NLR containing a PYRIN domain (PYD) 7 (NLRP7) has been recently shown to assemble an ASC and caspase-1-containing high molecular weight inflammasome complex in response to microbial acylated lipopeptides and Staphylococcus aureus infection. However, the molecular mechanism responsible for NLRP7 inflammasome activation is still elusive. Here we demonstrate that the NBD of NLRP7 is an ATP binding domain and has ATPase activity. We further show that an intact nucleotide-binding Walker A motif is required for NBD-mediated nucleotide binding and hydrolysis, oligomerization, and NLRP7 inflammasome formation and activity. Accordingly, THP-1 cells expressing a mutated Walker A motif display defective NLRP7 inflammasome activation, interleukin (IL)-1β release and pyroptosis in response to acylated lipopeptides and S. aureus infection. Taken together, our results provide novel insights into the mechanism of NLRP7 inflammasome assembly. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis.

    Science.gov (United States)

    Dunbar, Kyle L; Chekan, Jonathan R; Cox, Courtney L; Burkhart, Brandon J; Nair, Satish K; Mitchell, Douglas A

    2014-10-01

    Despite intensive research, the cyclodehydratase responsible for azoline biogenesis in thiazole/oxazole-modified microcin (TOMM) natural products remains enigmatic. The collaboration of two proteins, C and D, is required for cyclodehydration. The C protein is homologous to E1 ubiquitin-activating enzymes, whereas the D protein is within the YcaO superfamily. Recent studies have demonstrated that TOMM YcaOs phosphorylate amide carbonyl oxygens to facilitate azoline formation. Here we report the X-ray crystal structure of an uncharacterized YcaO from Escherichia coli (Ec-YcaO). Ec-YcaO harbors an unprecedented fold and ATP-binding motif. This motif is conserved among TOMM YcaOs and is required for cyclodehydration. Furthermore, we demonstrate that the C protein regulates substrate binding and catalysis and that the proline-rich C terminus of the D protein is involved in C protein recognition and catalysis. This study identifies the YcaO active site and paves the way for the characterization of the numerous YcaO domains not associated with TOMM biosynthesis.

  7. Transport processes of radiopharmaceuticals and -modulators

    Directory of Open Access Journals (Sweden)

    Langguth Peter

    2011-06-01

    Full Text Available Abstract Radiotherapy and radiology have been indispensable components in cancer care for many years. The detection limit of small tumor foci as well as the development of radio-resistance and severe side effects towards normal tissues led to the development of strategies to improve radio-diagnostic and -therapeutic approaches by pharmaceuticals. The term "radiopharmaceutical" has been used for drugs labeled with radioactive tracers for therapy or diagnosis. In addition, drugs have been described to sensitize tumor cells to radiotherapy (radiosensitizers or to protect normal tissues from detrimental effects of radiation (radioprotectors. The present review summarizes recent concepts on the transport of radiopharmaceuticals, radiosensitizers, and radioprotectors in cells and tissues, e.g. by ATP-binding cassette transporters such as P-glycoprotein. Strengths and weaknesses of current strategies to improve transport-based processes are discussed.

  8. Nucleotide-induced conformational dynamics in ABC transporters from structure-based coarse grained modelling.

    Science.gov (United States)

    Flechsig, Holger

    2016-02-01

    ATP-binding cassette (ABC) transporters are integral membrane proteins which mediate the exchange of diverse substrates across membranes powered by ATP molecules. Our understanding of their activity is still hampered since the conformational dynamics underlying the operation of such proteins cannot yet be resolved in detailed molecular dynamics studies. Here a coarse grained model which allows to mimic binding of nucleotides and follow subsequent conformational motions of full-length transporter structures in computer simulations is proposed and implemented. To justify its explanatory quality, the model is first applied to the maltose transporter system for which multiple conformations are known and we find that the model predictions agree remarkably well with the experimental data. For the MalK subunit the switching from open to the closed dimer configuration upon ATP binding is reproduced and, moreover, for the full-length maltose transporter, progression from inward-facing to the outward-facing state is correctly obtained. For the heme transporter HmuUV, for which only the free structure could yet be determined, the model was then applied to predict nucleotide-induced conformational motions. Upon binding of ATP-mimicking ligands the structure changed from a conformation in which the nucleotide-binding domains formed an open shape, to a conformation in which they were found in tight contact, while, at the same time, a pronounced rotation of the transmembrane domains was observed. This finding is supported by normal mode analysis, and, comparison with structural data of the homologous vitamin B12 transporter BtuCD suggests that the observed rotation mechanism may contribute a common functional aspect for this class of ABC transporters. Although in HmuuV noticeable rearrangement of essential transmembrane helices was detected, there are no indications from our simulations that ATP binding alone may facilitate propagation of substrate molecules in this transporter

  9. Microchamber Device for Detection of Transporter Activity of Adherent Cells

    Directory of Open Access Journals (Sweden)

    Mamiko eTsugane

    2015-03-01

    Full Text Available We present a method to detect the transporter activity of intact adherent cells using a microchamber device. When adherent cells are seeded onto the poly-di-methyl siloxane (PDMS substrate having microchambers with openings smaller than the size of a cell, the cells form a confluent layer that covers the microchambers, creating minute, confined spaces. As substances exported across the cell membrane accumulate, transporter activity can be detected by observing the fluorescence intensity increase in the microchamber. We tested the microchamber device with HeLa cells overexpressing MDR1, an ATP-binding cassette (ABC transporter, and succeeded in detecting the transport of fluorescence-conjugated paclitaxel, the anti-cancer drug, at the single-cell level.

  10. [Plant ABC transporters--the family with tradition].

    Science.gov (United States)

    Jasiński, Michał; Figlerowicz, Marek

    2006-01-01

    ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. They have been thoroughly studied in mammals, where they became the center of interest for clinical reasons related to the resistance of tumor cells to chemotherapy treatment. Less is known about plant members of the ABC family, however, growing number of reports on their role in different physiological processes attract attention. The vacuolar ABC transporters in plants characterized to date are involved in the intracellular sequestration of cytotoxins (e.g. herbicides), as well as the products of endogenous metabolism like chlorophyll catabolites. Others localized within plasma membrane are active in the transport of secondary metabolites or phytohormones. Finally certain transporters are present in cell organelles and play a role in such processes as P oxidation. Here, we briefly introduce these proteins, and describe structural characteristic and physiological aspect of their activity in a plant cell.

  11. Structure and mechanism of ATP-dependent phospholipid transporters

    DEFF Research Database (Denmark)

    Lopez Marques, Rosa Laura; Poulsen, Lisbeth Rosager; Bailly, Aurélien

    2015-01-01

    , despite differences in overall architecture, both appear to operate by an alternating access mechanism and during transport they might allow access of phospholipids to the internal part of the transmembrane domain. The latter feature is obvious for ABC transporters, but phospholipids and other hydrophobic......Background ATP-binding cassette (ABC) transporters and P4-ATPases are two large and seemingly unrelated families of primary active pumps involved in moving phospholipids from one leaflet of a biological membrane to the other. Scope of review This review aims to identify common mechanistic features...... in the way phospholipid flipping is carried out by two evolutionarily unrelated families of transporters. Major conclusions Both protein families hydrolyze ATP, although they employ different mechanisms to use it, and have a comparable size with twelve transmembrane segments in the functional unit. Further...

  12. The effects of ADF/cofilin and profilin on the conformation of the ATP-binding cleft of monomeric actin.

    Science.gov (United States)

    Kardos, Roland; Pozsonyi, Kinga; Nevalainen, Elisa; Lappalainen, Pekka; Nyitrai, Miklós; Hild, Gábor

    2009-03-18

    Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer's ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound epsilon-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound epsilon-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound epsilon-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.

  13. Long-range coupling between ATP-binding and lever-arm regions in myosin via dielectric allostery

    Science.gov (United States)

    Sato, Takato; Ohnuki, Jun; Takano, Mitsunori

    2017-12-01

    A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.

  14. Long-range coupling between ATP-binding and lever-arm regions in myosin via dielectric allostery.

    Science.gov (United States)

    Sato, Takato; Ohnuki, Jun; Takano, Mitsunori

    2017-12-07

    A protein molecule is a dielectric substance, so the binding of a ligand is expected to induce dielectric response in the protein molecule, considering that ligands are charged or polar in general. We previously reported that binding of adenosine triphosphate (ATP) to molecular motor myosin actually induces such a dielectric response in myosin due to the net negative charge of ATP. By this dielectric response, referred to as "dielectric allostery," spatially separated two regions in myosin, the ATP-binding region and the actin-binding region, are allosterically coupled. In this study, from the statistically stringent analyses of the extensive molecular dynamics simulation data obtained in the ATP-free and the ATP-bound states, we show that there exists the dielectric allostery that transmits the signal of ATP binding toward the distant lever-arm region. The ATP-binding-induced electrostatic potential change observed on the surface of the main domain induced a movement of the converter subdomain from which the lever arm extends. The dielectric response was found to be caused by an underlying large-scale concerted rearrangement of the electrostatic bond network, in which highly conserved charged/polar residues are involved. Our study suggests the importance of the dielectric property for molecular machines in exerting their function.

  15. Functional characterization of the Shigella dysenteriae heme ABC transporter.

    Science.gov (United States)

    Burkhard, Kimberly A; Wilks, Angela

    2008-08-05

    The heme ATP binding cassette (ABC) transporter, ShuUV, of Shigella dysenteriae has been incorporated into proteoliposomes. Functional characterization of ShuUV revealed that ATP hydrolysis and transport of heme from the periplasmic binding protein, ShuT, to the cytoplasmic binding protein, ShuS, are coupled. Site-directed mutagenesis of ShuT residues proposed to be required for stabilization of the complex abolished heme transport. Furthermore, residues His-252 and His-262, located in the translocation channel of ShuU, were required for the release of heme from ShuT and translocation to ShuS. The initial functional characterization of an in vitro heme uptake system provides a platform for future spectroscopic studies.

  16. Computer-Aided Recognition of ABC Transporters Substrates and Its Application to the Development of New Drugs for Refractory Epilepsy.

    Science.gov (United States)

    Couyoupetrou, Manuel; Gantner, Melisa E; Di Ianni, Mauricio E; Palestro, Pablo H; Enrique, Andrea V; Gavernet, Luciana; Ruiz, Maria E; Pesce, Guido; Bruno-Blanch, Luis E; Talevi, Alan

    2017-01-01

    Despite the introduction of more than 15 third generation antiepileptic drugs to the market from 1990 to the moment, about one third of the epileptic patients still suffer from refractory to intractable epilepsy. Several hypotheses seek to explain the failure of drug treatments to control epilepsy symptoms in such patients. The most studied one proposes that drug resistance might be related with regional overactivity of efflux transporters from the ATP-Binding Cassette (ABC) superfamily at the blood-brain barrier and/or the epileptic foci in the brain. Different strategies have been conceived to address the transporter hypothesis, among them inhibiting or down-regulating the efflux transporters or bypassing them through a diversity of artifices. Here, we review scientific evidence supporting the transporter hypothesis along with its limitations, as well as computer-assisted early recognition of ABC transporter substrates as an interesting strategy to develop novel antiepileptic drugs capable of treating refractory epilepsy linked to ABC transporters overactivity.

  17. Crystal Structure of a Group I Energy Coupling Factor Vitamin Transporter S Component in Complex with Its Cognate Substrate.

    Science.gov (United States)

    Josts, Inokentijs; Almeida Hernandez, Yasser; Andreeva, Antonina; Tidow, Henning

    2016-07-21

    Energy coupling factor (ECF) transporters are responsible for the uptake of essential scarce nutrients in prokaryotes. This ATP-binding cassette transporter family comprises two subgroups that share a common architecture forming a tripartite membrane protein complex consisting of a translocation component and ATP hydrolyzing module and a substrate-capture (S) component. Here, we present the crystal structure of YkoE from Bacillus subtilis, the S component of the previously uncharacterized group I ECF transporter YkoEDC. Structural and biochemical analyses revealed the constituent residues of the thiamine-binding pocket as well as an unexpected mode of vitamin recognition. In addition, our experimental and bioinformatics data demonstrate major differences between YkoE and group II ECF transporters and indicate how group I vitamin transporter S components have diverged from other group I and group II ECF transporters. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Stender, Stefan; Frikke-Schmidt, Ruth; Nordestgaard, Børge G; Tybjaerg-Hansen, Anne

    2011-02-01

    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 in the general population, we studied 62,279 white individuals from The Copenhagen City Heart Study and The Copenhagen General Population Study, randomly selected to reflect the adult Danish population aged 20 to 80+ years. Endpoints were recorded from January 1976 through May 2009. During a mean follow-up of, respectively, 31 and 4.4 years, 3124 participants developed symptomatic gallstone disease and 30 developed biliary cancer. The multifactorially adjusted hazard ratio for symptomatic gallstone disease was 1.9 (95% confidence interval, 1.7-2.1) in DH heterozygotes (prevalence, 12%), and 3.3 (2.3-4.6) in HH homozygotes (0.4%) versus noncarriers (P for trend <0.001). Mean age at onset of symptomatic gallstone disease was 56 years for noncarriers, 54 for DH heterozygotes, and 52 for HH homozygotes (P for trend <0.001). The fraction of all gallstones attributed to D19H was 11%. The multifactorially adjusted hazard ratio for biliary cancer was 4.0 (1.9-8.4) in DH heterozygotes and HH homozygotes combined versus noncarriers (P < 0.001). The fraction of all biliary cancers attributed to the D19H genotype was 27%. Finally, D19H genotype associated with stepwise increases in plasma levels of alanine aminotransferase and gamma glutamyltransferase of up to 14% and 25% in HH homozygotes, and with corresponding stepwise reductions in plasma levels of total and low-density lipoprotein cholesterol of up to 5% versus noncarriers (all comparisons, P for trend <0.001). In this general population cohort, ABCG8 D19H genotype was an important predictor of both symptomatic gallstone disease and biliary cancer. Copyright © 2010 American Association for the Study of Liver Diseases.

  19. 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.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  1. Design and synthesis of a heterocyclic compound collection for probing the spatial charactistics of ATP binding sites

    CSIR Research Space (South Africa)

    Kenyon, CP

    2006-02-28

    Full Text Available stream_source_info parkinson_2006.pdf.txt stream_content_type text/plain stream_size 3323 Content-Encoding UTF-8 stream_name parkinson_2006.pdf.txt Content-Type text/plain; charset=UTF-8 Design and Synthesis of a... Heterocyclic Compound Collection for Probing the Spatial Charactistics of ATP Binding Sites Presented at the CSIR Conference Centre CSIR Biosciences C.P. Kenyon, P.M. Matlaba, C.J. Parkinson, A.L. Rousseau and C.W. van der Westhuyzen February 28, 2006...

  2. 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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. 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

    2015-01-01

    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 [3H]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. PMID:25991724

  4. 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.

  5. Intracellular trafficking of MDR transporters and relevance of SNPs.

    Science.gov (United States)

    Porcelli, Letizia; Lemos, Clara; Peters, Godefridus J; Paradiso, Angelo; Azzariti, Amalia

    2009-01-01

    Multi-drug resistance (MDR) frequently contributes to the failure of chemotherapeutic treatments in cancer patients. Mechanisms underlying the development of MDR have been extensively studied and are considered multifactorial. Among them, the ATP-Binding Cassette (ABC) family of proteins plays a pivotal role. Processes of cellular distribution and subcellular localization of MDR-ABC proteins are not yet well explored and to enlighten these topics could be crucial to understand cellular drug uptake and retention. In this review, we analysed literature data concerning i) intracellular trafficking of MDR-ABC proteins (BCRP, P-gp and MRP1) and ii) mechanisms altering their cellular localization and trafficking. Moreover, we describe single nucleotide polymorphisms (SNP) that have been reported for some multidrug resistance (MDR) transporters, such as BCRP and P-gp, emphasizing their ability to affect the expression, function and localization of the transporters, with implications on drug resistance phenotypes.

  6. 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.

  7. Involvement of a cyclic adenosine monophosphate-dependent signal in the diet-induced canalicular trafficking of adenosine triphosphate-binding cassette transporter g5/g8.

    Science.gov (United States)

    Yamazaki, Yasuhiro; Yasui, Kenta; Hashizume, Takahiro; Suto, Arisa; Mori, Ayaka; Murata, Yuzuki; Yamaguchi, Masahiko; Ikari, Akira; Sugatani, Junko

    2015-10-01

    The adenosine triphosphate-binding cassette (ABC) half-transporters Abcg5 and Abcg8 promote the secretion of neutral sterol into bile. Studies have demonstrated the diet-induced gene expression of these transporters, but the regulation of their trafficking when the nutritional status changes in the liver remains to be elucidated. Here, we generated a novel in vivo kinetic analysis that can monitor the intracellular trafficking of Abcg5/Abcg8 in living mouse liver by in vivo transfection of the genes of fluorescent protein-tagged transporters and investigated how hypernutrition affects the canalicular trafficking of these transporters. The kinetic analysis showed that lithogenic diet consumption accelerated the translocation of newly synthesized fluorescent-tagged transporters to intracellular pools in an endosomal compartment and enhanced the recruitment of these pooled gene products into the bile canalicular membrane in mouse liver. Because some ABC transporters are reported to be recruited from intracellular pools to the bile canaliculi by cyclic adenosine monophosphate (cAMP) signaling, we next evaluated the involvement of this machinery in a diet-induced event. Administration of a protein kinase A inhibitor, N-(2-{[3-(4-bromophenyl)-2-propenyl]amino}ethyl)-5-isoquinolinesulfonamide, decreased the canalicular expression of native Abcg5/Abcg8 in lithogenic diet-fed mice, and injection of a cAMP analog, dibutyryl cAMP, transiently increased their levels in standard diet-fed mice, indicating the involvement of cAMP signaling. Indeed, canalicular trafficking of the fluorescent-tagged Abcg5/Abcg8 was enhanced by dibutyryl cAMP administration. These observations suggest that diet-induced lipid loading into liver accelerates the trafficking of Abcg5/Abcg8 to the bile canalicular membrane through cAMP signaling machinery. © 2015 by the American Association for the Study of Liver Diseases.

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

    Science.gov (United States)

    Jiang, Zhao-Yan; Cai, Qu; Chen, Er-Zhen

    2014-01-01

    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. 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. 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. 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.

  9. 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 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.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.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.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.

  10. 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.

  11. ATP-binding motifs play key roles in Krp1p, kinesin-related protein 1, function for bi-polar growth control in fission yeast.

    Science.gov (United States)

    Rhee, Dong Keun; Cho, Bon A; Kim, Hyong Bai

    2005-06-03

    Kinesin is a microtubule-based motor protein with various functions related to the cell growth and division. It has been reported that Krp1p, kinesin-related protein 1, which belongs to the kinesin heavy chain superfamily, localizes on microtubules and may play an important role in cytokinesis. However, the function of Krp1p has not been fully elucidated. In this study, we overexpressed an intact form and three different mutant forms of Krp1p in fission yeast constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif (LZiP). We observed hyper-extended microtubules and the aberrant nuclear shape in Krp1p-overexpressed fission yeast. As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered cell polarity. Additionally, truncation of the leucine zipper-like domain (LZiP) at the C-terminal of Krp1p showed a normal nuclear division. Taken together, we suggest that krp1p is involved in regulation of cell-polarized growth through ATP-binding motifs in fission yeast.

  12. Identification of the magnesium-binding domain of the high affinity ATP binding-site of the Bacillus subtilis and Escherichia coli seca protein

    NARCIS (Netherlands)

    van der Wolk, J.P.W.; Klose, M; de Wit, Janny; Blaauwen, T.den; Freudl, R; Driessen, A.J.M.

    1995-01-01

    The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain

  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. Uric acid transporters hiding in the intestine.

    Science.gov (United States)

    Xu, Xianxiang; Li, Canghai; Zhou, Pan; Jiang, Tingliang

    2016-12-01

    Hyperuricaemia is known as an abnormally increased uric acid level in the blood. Although it was observed many years ago, since uric acid excretion via the intestine pathway accounted for approximately one-third of total elimination of uric acid, the molecular mechanism of 'extra-renal excretion' was poorly understood until the finding of uric acid transporters. The objective of this study was to gather all information related to uric acid transporters in the intestine and present this information as a comprehensive and systematic review article. A literature search was performed from various databases (e.g., Medline, Science Direct, Springer Link, etc.). The key terms included uric acid, transporter and intestine. The period for the search is from the 1950s to the present. The bibliographies of papers relating to the review subject were also searched for further relevant references. The uric acid transporters identified in the intestine are discussed in this review. The solute carrier (SLC) transporters include GLUT9, MCT9, NPT4, NPT homolog (NPT5) and OAT10. The ATP binding cassette (ABC) transporters include ABCG2 (BCRP), MRP2 and MRP4. Bacterial transporter YgfU is a low-affinity and high-capacity transporter for uric acid. The present review may be helpful for further our understanding of hyperuricaemia and be of value in designing future studies on novel therapeutic pathways.

  15. 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.

  16. L1198F Mutation Resensitizes Crizotinib to ALK by Altering the Conformation of Inhibitor and ATP Binding Sites.

    Science.gov (United States)

    Li, Jian; Sun, Rong; Wu, Yuehong; Song, Mingzhu; Li, Jia; Yang, Qianye; Chen, Xiaoyi; Bao, Jinku; Zhao, Qi

    2017-02-24

    The efficacy of anaplastic lymphoma kinase (ALK) positive non-small-cell lung cancer (NSCLC) treatment with small molecule inhibitors is greatly challenged by acquired resistance. A recent study reported the newest generation inhibitor resistant mutation L1198F led to the resensitization to crizotinib, which is the first Food and Drug Administration (FDA) approved drug for the treatment of ALK-positive NSCLC. It is of great importance to understand how this extremely rare event occurred for the purpose of overcoming the acquired resistance of such inhibitors. In this study, we exploited molecular dynamics (MD) simulation to dissect the molecular mechanisms. Our MD results revealed that L1198F mutation of ALK resulted in the conformational change at the inhibitor site and altered the binding affinity of ALK to crizotinib and lorlatinib. L1198F mutation also affected the autoactivation of ALK as supported by the identification of His1124 and Tyr1278 as critical amino acids involved in ATP binding and phosphorylation. Our findings are valuable for designing more specific and potent inhibitors for the treatment of ALK-positive NSCLC and other types of cancer.

  17. 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.

  18. L1198F Mutation Resensitizes Crizotinib to ALK by Altering the Conformation of Inhibitor and ATP Binding Sites

    Directory of Open Access Journals (Sweden)

    Jian Li

    2017-02-01

    Full Text Available The efficacy of anaplastic lymphoma kinase (ALK positive non-small-cell lung cancer (NSCLC treatment with small molecule inhibitors is greatly challenged by acquired resistance. A recent study reported the newest generation inhibitor resistant mutation L1198F led to the resensitization to crizotinib, which is the first Food and Drug Administration (FDA approved drug for the treatment of ALK-positive NSCLC. It is of great importance to understand how this extremely rare event occurred for the purpose of overcoming the acquired resistance of such inhibitors. In this study, we exploited molecular dynamics (MD simulation to dissect the molecular mechanisms. Our MD results revealed that L1198F mutation of ALK resulted in the conformational change at the inhibitor site and altered the binding affinity of ALK to crizotinib and lorlatinib. L1198F mutation also affected the autoactivation of ALK as supported by the identification of His1124 and Tyr1278 as critical amino acids involved in ATP binding and phosphorylation. Our findings are valuable for designing more specific and potent inhibitors for the treatment of ALK-positive NSCLC and other types of cancer.

  19. 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....

  20. 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

  1. 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.

  2. Isolation of an ABA Transporter-Like 1 Gene from Arachis hypogaea That Affects ABA Import and Reduces ABA Sensitivity in Arabidopsis.

    Science.gov (United States)

    Ge, Kui; Liu, Xing; Li, Xiaoyun; Hu, Bo; Li, Ling

    2017-01-01

    Abscisic acid (ABA) transporters are essential for the transport of ABA from its sites of synthesis to its multiple sites of action within plants and are key players in plant stress responses. Despite their importance, there is limited information on ABA transporters in crop plants. In this study, we isolated and characterized an ABA transporter-like 1 (AhATL1) gene from peanut (Arachis hypogaea L.) whose cognate protein, AhATL1, is a member of the ATP-binding cassette transporter G subfamily and localizes to the plasma membrane. The expression of both the AhATL1 transcript and the corresponding protein were upregulated by water stress and treatment with exogenous ABA. Overexpression of AhATL1 in ecotype Columbia (Col) Arabidopsis (AhATL1-OX) plants reduced ABA sensitivity. When AhATL1-OX and Arabidopsis Col plants were subjected to dehydration stress, the expression of 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3) and responsive to desiccation 29 A (AtRD29A) accumulated rapidly in rosette leaves of both lines. In contrast, while expression of ATP-binding cassette G 40 (AtABCG40) was increased in Col rosette leaves, there was no change in expression of AtABCG40 in AhATL1-OX leaves. Similarly, water loss from detached leaves of AhATL1-OX plants was more rapid than from Col leaves. Therefore, we suggest that the function of AhATL1 is probably to modulate ABA sensitivity by specifically influencing ABA import into cells.

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

    DEFF Research Database (Denmark)

    Nakai, Hiroyuki; Baumann, Martin; Petersen, B.O.

    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...... 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......-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...

  4. 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

  5. A Nostoc punctiforme sugar transporter necessary to establish a Cyanobacterium-plant symbiosis.

    Science.gov (United States)

    Ekman, Martin; Picossi, Silvia; Campbell, Elsie L; Meeks, John C; Flores, Enrique

    2013-04-01

    In cyanobacteria-plant symbioses, the symbiotic nitrogen-fixing cyanobacterium has low photosynthetic activity and is supplemented by sugars provided by the plant partner. Which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis, however, is unknown. Mutants of the symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme were constructed bearing a neomycin resistance gene cassette replacing genes in a putative sugar transport gene cluster. Results of transport activity assays using (14)C-labeled fructose and glucose and tests of heterotrophic growth with these sugars enabled the identification of an ATP-binding cassette-type transporter for fructose (Frt), a major facilitator permease for glucose (GlcP), and a porin needed for the optimal uptake of both fructose and glucose. Analysis of green fluorescent protein fluorescence in strains of N. punctiforme bearing frt::gfp fusions showed high expression in vegetative cells and akinetes, variable expression in hormogonia, and no expression in heterocysts. The symbiotic efficiency of N. punctiforme sugar transport mutants was investigated by testing their ability to infect a nonvascular plant partner, the hornwort Anthoceros punctatus. Strains that were specifically unable to transport glucose did not infect the plant. These results imply a role for GlcP in establishing symbiosis under the conditions used in this work.

  6. The crustacean gill (Na+,K+)-ATPase: allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium.

    Science.gov (United States)

    Masui, D C; Silva, E C C; Mantelatto, F L M; McNamara, J C; Barrabin, H; Scofano, H M; Fontes, C F L; Furriel, R P M; Leone, F A

    2008-11-15

    The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na+,K+)-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na+ and K+ of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na+,K+)-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na+ and K+. However, in contrast to Na+, a threshold K+ concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations.Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na+,K+)-ATPase compared to the vertebrate enzyme.

  7. Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chun-Liang; Mermoud, James C.; Paul, Lake N.; Steussy, Calvin Nicklaus; Stauffacher, Cynthia V. (Purdue)

    2017-10-12

    The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis. The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDDEF). The MDDEF crystal structure in complex with ATP (MDDEF–ATP) revealed that the phosphate-binding loop (amino acids 97–105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDDEF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDDEF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDDEF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

  8. 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.

  9. Structure of a bacterial energy-coupling factor transporter.

    Science.gov (United States)

    Wang, Tingliang; Fu, Guobin; Pan, Xiaojing; Wu, Jianping; Gong, Xinqi; Wang, Jiawei; Shi, Yigong

    2013-05-09

    The energy-coupling factor (ECF) transporters constitute a novel family of conserved membrane transporters in prokaryotes that have a similar domain organization to the ATP-binding cassette transporters. Each ECF transporter comprises a pair of cytosolic ATPases (the A and A' components, or EcfA and EcfA'), a membrane-embedded substrate-binding protein (the S component, or EcfS) and a transmembrane energy-coupling component (the T component, or EcfT) that links the EcfA-EcfA' subcomplex to EcfS. The structure and transport mechanism of the quaternary ECF transporter remain largely unknown. Here we report the crystal structure of a nucleotide-free ECF transporter from Lactobacillus brevis at a resolution of 3.5 Å. The T component has a horseshoe-shaped open architecture, with five α-helices as transmembrane segments and two cytoplasmic α-helices as coupling modules connecting to the A and A' components. Strikingly, the S component, thought to be specific for hydroxymethyl pyrimidine, lies horizontally along the lipid membrane and is bound exclusively by the five transmembrane segments and the two cytoplasmic helices of the T component. These structural features suggest a plausible working model for the transport cycle of the ECF transporters.

  10. 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.

  11. 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. Copyright © 2016. Published by Elsevier B.V.

  12. Natural polyphenols: Influence on membrane transporters

    Directory of Open Access Journals (Sweden)

    Saad Abdulrahman Hussain

    2016-03-01

    Full Text Available Accumulated evidences have focused on the use of natural polyphenolic compounds as nutraceuticals, since they showed a wide range of bioactivities and exhibited protection against variety of age related disorders. Polyphenols have variable potencies to interact, and hence alter the activities of various transporter proteins, many of them classified as ATP-Binding Cassette transporters, like multidrug resistance protein (MDRP, and p-glycoprotein (P-gp. Some of the efflux transporters are generally linked with anticancer and antiviral drug resistance; in this context, polyphenols may be beneficial in modulating drug resistance by increasing the efficacy of anticancer and antiviral drugs. Additionally, these effects were implicated to explain the influence of dietary polyphenols on drug efficacy as result of food-drug interactions. However, limited data are available about the influence of these components on uptake transporters. Therefore, the objective of this article is to review the potential efficacies of polyphenols in modulating the functional integrity of uptake transporter proteins, including those terminated the effect of neurotransmitters, and their possible influence in neuropharmacology. [J Intercult Ethnopharmacol 2016; 5(1.000: 97-104

  13. NCBI nr-aa BLAST: CBRC-DNOV-01-0896 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-0896 ref|XP_001258991.1| ATP-binding cassette transporter [Neosartorya fischer...i NRRL 181] gb|EAW17094.1| ATP-binding cassette transporter [Neosartorya fischeri NRRL 181] XP_001258991.1 3.5 26% ...

  14. A Nostoc punctiforme Sugar Transporter Necessary to Establish a Cyanobacterium-Plant Symbiosis1[C][W

    Science.gov (United States)

    Ekman, Martin; Picossi, Silvia; Campbell, Elsie L.; Meeks, John C.; Flores, Enrique

    2013-01-01

    In cyanobacteria-plant symbioses, the symbiotic nitrogen-fixing cyanobacterium has low photosynthetic activity and is supplemented by sugars provided by the plant partner. Which sugars and cyanobacterial sugar uptake mechanism(s) are involved in the symbiosis, however, is unknown. Mutants of the symbiotically competent, facultatively heterotrophic cyanobacterium Nostoc punctiforme were constructed bearing a neomycin resistance gene cassette replacing genes in a putative sugar transport gene cluster. Results of transport activity assays using 14C-labeled fructose and glucose and tests of heterotrophic growth with these sugars enabled the identification of an ATP-binding cassette-type transporter for fructose (Frt), a major facilitator permease for glucose (GlcP), and a porin needed for the optimal uptake of both fructose and glucose. Analysis of green fluorescent protein fluorescence in strains of N. punctiforme bearing frt::gfp fusions showed high expression in vegetative cells and akinetes, variable expression in hormogonia, and no expression in heterocysts. The symbiotic efficiency of N. punctiforme sugar transport mutants was investigated by testing their ability to infect a nonvascular plant partner, the hornwort Anthoceros punctatus. Strains that were specifically unable to transport glucose did not infect the plant. These results imply a role for GlcP in establishing symbiosis under the conditions used in this work. PMID:23463784

  15. Diversity of ABC transporter genes across the plant kingdom and their potential utility in biotechnology.

    Science.gov (United States)

    Lane, Thomas S; Rempe, Caroline S; Davitt, Jack; Staton, Margaret E; Peng, Yanhui; Soltis, Douglas Edward; Melkonian, Michael; Deyholos, Michael; Leebens-Mack, James H; Chase, Mark; Rothfels, Carl J; Stevenson, Dennis; Graham, Sean W; Yu, Jun; Liu, Tao; Pires, J Chris; Edger, Patrick P; Zhang, Yong; Xie, Yinlong; Zhu, Ying; Carpenter, Eric; Wong, Gane Ka-Shu; Stewart, C Neal

    2016-05-31

    The ATP-binding cassette (ABC) transporter gene superfamily is ubiquitous among extant organisms and prominently represented in plants. ABC transporters act to transport compounds across cellular membranes and are involved in a diverse range of biological processes. Thus, the applicability to biotechnology is vast, including cancer resistance in humans, drug resistance among vertebrates, and herbicide and other xenobiotic resistance in plants. In addition, plants appear to harbor the highest diversity of ABC transporter genes compared with any other group of organisms. This study applied transcriptome analysis to survey the kingdom-wide ABC transporter diversity in plants and suggest biotechnology applications of this diversity. We utilized sequence similarity-based informatics techniques to infer the identity of ABC transporter gene candidates from 1295 phylogenetically-diverse plant transcriptomes. A total of 97,149 putative (approximately 25 % were full-length) ABC transporter gene members were identified; each RNA-Seq library (plant sample) had 88 ± 30 gene members. As expected, simpler organisms, such as algae, had fewer unique members than vascular land plants. Differences were also noted in the richness of certain ABC transporter subfamilies. Land plants had more unique ABCB, ABCC, and ABCG transporter gene members on average (p red algae, and bryophytes had significantly more ABCF transporter gene members (p biotechnology, with an emphasis on crops.

  16. ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates.

    Science.gov (United States)

    Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M; Bianco, Piero R; Surtees, Jennifer A

    2014-06-01

    In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3' non-homologous tail removal (3'NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3'NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3'NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3'NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. 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.

    Directory of Open Access Journals (Sweden)

    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.

  18. ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2-Msh3 are differentially modulated by Mismatch and Double-strand Break Repair DNA substrates

    Science.gov (United States)

    Kumar, Charanya; Eichmiller, Robin; Wang, Bangchen; Williams, Gregory M.; Bianco, Piero R.; Surtees, Jennifer A.

    2014-01-01

    In Saccharomyces cerevisiae, Msh2-Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2-Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2-Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2-Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′ NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2-Msh3 and Msh2-msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′ NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2-Msh3, indicating that the MMR and 3′ NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2-Msh3. Msh2-msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2-Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype. PMID:24746922

  19. Comparison of mechanistic transport cycle models of ABC exporters.

    Science.gov (United States)

    Szöllősi, Dániel; Rose-Sperling, Dania; Hellmich, Ute A; Stockner, Thomas

    2018-04-01

    ABC (ATP binding cassette) transporters, ubiquitous in all kingdoms of life, carry out essential substrate transport reactions across cell membranes. Their transmembrane domains bind and translocate substrates and are connected to a pair of nucleotide binding domains, which bind and hydrolyze ATP to energize import or export of substrates. Over four decades of investigations into ABC transporters have revealed numerous details from atomic-level structural insights to their functional and physiological roles. Despite all these advances, a comprehensive understanding of the mechanistic principles of ABC transporter function remains elusive. The human multidrug resistance transporter ABCB1, also referred to as P-glycoprotein (P-gp), is one of the most intensively studied ABC exporters. Using ABCB1 as the reference point, we aim to compare the dominating mechanistic models of substrate transport and ATP hydrolysis for ABC exporters and to highlight the experimental and computational evidence in their support. In particular, we point out in silico studies that enhance and complement available biochemical data. "This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain." Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Sphingosine-1-phosphate transporters as targets for cancer therapy.

    Science.gov (United States)

    Nagahashi, Masayuki; Takabe, Kazuaki; Terracina, Krista P; Soma, Daiki; Hirose, Yuki; Kobayashi, Takashi; Matsuda, Yasunobu; Wakai, Toshifumi

    2014-01-01

    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.

  1. 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.

  2. Structure of ABC transporters.

    Science.gov (United States)

    Zolnerciks, Joseph K; Andress, Edward J; Nicolaou, Michael; Linton, Kenneth J

    2011-09-07

    ABC (ATP-binding cassette) transporters are primary active membrane proteins that translocate solutes (allocrites) across lipid bilayers. The prototypical ABC transporter consists of four domains: two cytoplasmic NBDs (nucleotide-binding domains) and two TMDs (transmembrane domains). The NBDs, whose primary sequence is highly conserved throughout the superfamily, bind and hydrolyse ATP to power the transport cycle. The TMDs, whose primary sequence and protein fold can be quite disparate, form the translocation pathway across the membrane and generally (but not always) determine allocrite specificity. Structure determination of ABC proteins initially took advantage of the relative ease of expression and crystallization of the hydrophilic bacterial NBDs in isolation from the transporter complex, and revealed detailed information on the structural fold of these domains, the amino acids involved in the binding and hydrolysis of nucleotide, and the head-to-tail arrangement of the NBD-NBD dimer interface. More recently, several intact transporters have been crystallized and three types have, so far, been characterized: type I and II ABC importers, and ABC exporters. All three are present in prokaryotes, but only the ABC exporters appear to be present in eukaryotes. Their structural determination has provided insight into the mechanisms of energy and signal transduction between the NBDs and TMDs (i.e. between the ATP- and allocrite-binding sites) and, for some, the nature of the allocrite-binding site(s) within the TMDs. In this chapter, we focus primarily on the ABC exporters and describe the structural, biochemical and biophysical evidence for and against the controversial bellows-like mechanism proposed for allocrite efflux.

  3. Genomic data mining reveals a rich repertoire of transport proteins in Streptomyces.

    Science.gov (United States)

    Zhou, Zhan; Sun, Ning; Wu, Shanshan; Li, Yong-Quan; Wang, Yufeng

    2016-08-22

    Streptomycetes are soil-dwelling Gram-positive bacteria that are best known as the major producers of antibiotics used in the pharmaceutical industry. The evolution of exceptionally powerful transporter systems in streptomycetes has enabled their adaptation to the complex soil environment. Our comparative genomic analyses revealed that each of the eleven Streptomyces species examined possesses a rich repertoire of from 761-1258 transport proteins, accounting for 10.2 to 13.7 % of each respective proteome. These transporters can be divided into seven functional classes and 171 transporter families. Among them, the ATP-binding Cassette (ABC) superfamily and the Major Facilitator Superfamily (MFS) represent more than 40 % of all the transport proteins in Streptomyces. They play important roles in both nutrient uptake and substrate secretion, especially in the efflux of drugs and toxicants. The evolutionary flexibility across eleven Streptomyces species is seen in the lineage-specific distribution of transport proteins in two major protein translocation pathways: the general secretory (Sec) pathway and the twin-arginine translocation (Tat) pathway. Our results present a catalog of transport systems in eleven Streptomyces species. These expansive transport systems are important mediators of the complex processes including nutrient uptake, concentration balance of elements, efflux of drugs and toxins, and the timely and orderly secretion of proteins. A better understanding of transport systems will allow enhanced optimization of production processes for both pharmaceutical and industrial applications of Streptomyces, which are widely used in antibiotic production and heterologous expression of recombinant proteins.

  4. 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.

  5. 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)

    Herrera-Moyano, Emilia; Moriel-Carretero, María; Montelone, Beth A; Aguilera, Andrés

    2014-12-01

    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.

  6. The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

    Directory of Open Access Journals (Sweden)

    Emilia Herrera-Moyano

    2014-12-01

    Full Text Available 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.

  7. Zinc and ATP binding of the hexameric AAA-ATPase PilF from Thermus thermophilus: role in complex stability, piliation, adhesion, twitching motility, and natural transformation.

    Science.gov (United States)

    Salzer, Ralf; Herzberg, Martin; Nies, Dietrich H; Joos, Friederike; Rathmann, Barbara; Thielmann, Yvonne; Averhoff, Beate

    2014-10-31

    The traffic AAA-ATPase PilF is essential for pilus biogenesis and natural transformation of Thermus thermophilus HB27. Recently, we showed that PilF forms hexameric complexes containing six zinc atoms coordinated by conserved tetracysteine motifs. Here we report that zinc binding is essential for complex stability. However, zinc binding is neither required for pilus biogenesis nor natural transformation. A number of the mutants did not exhibit any pili during growth at 64 °C but still were transformable. This leads to the conclusion that type 4 pili and the DNA translocator are distinct systems. At lower growth temperatures (55 °C) the zinc-depleted multiple cysteine mutants were hyperpiliated but defective in pilus-mediated twitching motility. This provides evidence that zinc binding is essential for the role of PilF in pilus dynamics. Moreover, we found that zinc binding is essential for complex stability but dispensable for ATPase activity. In contrast to many polymerization ATPases from mesophilic bacteria, ATP binding is not required for PilF complex formation; however, it significantly increases complex stability. These data suggest that zinc and ATP binding increase complex stability that is important for functionality of PilF under extreme environmental conditions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. 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

    2015-09-18

    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 of 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, ABCG1 and SR-B1 but not involving PI3K and Akt. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Handling of Drugs, Metabolites, and Uremic Toxins by Kidney Proximal Tubule Drug Transporters.

    Science.gov (United States)

    Nigam, Sanjay K; Wu, Wei; Bush, Kevin T; Hoenig, Melanie P; Blantz, Roland C; Bhatnagar, Vibha

    2015-11-06

    The proximal tubule of the kidney plays a crucial role in the renal handling of drugs (e.g., diuretics), uremic toxins (e.g., indoxyl sulfate), environmental toxins (e.g., mercury, aristolochic acid), metabolites (e.g., uric acid), dietary compounds, and signaling molecules. This process is dependent on many multispecific transporters of the solute carrier (SLC) superfamily, including organic anion transporter (OAT) and organic cation transporter (OCT) subfamilies, and the ATP-binding cassette (ABC) superfamily. We review the basic physiology of these SLC and ABC transporters, many of which are often called drug transporters. With an emphasis on OAT1 (SLC22A6), the closely related OAT3 (SLC22A8), and OCT2 (SLC22A2), we explore the implications of recent in vitro, in vivo, and clinical data pertinent to the kidney. The analysis of murine knockouts has revealed a key role for these transporters in the renal handling not only of drugs and toxins but also of gut microbiome products, as well as liver-derived phase 1 and phase 2 metabolites, including putative uremic toxins (among other molecules of metabolic and clinical importance). Functional activity of these transporters (and polymorphisms affecting it) plays a key role in drug handling and nephrotoxicity. These transporters may also play a role in remote sensing and signaling, as part of a versatile small molecule communication network operative throughout the body in normal and diseased states, such as AKI and CKD. Copyright © 2015 by the American Society of Nephrology.

  10. The ABC transporter Rv1272c of Mycobacterium tuberculosis enhances the import of long-chain fatty acids in Escherichia coli.

    Science.gov (United States)

    Martin, Audrey; Daniel, Jaiyanth

    2018-02-05

    Mycobacterium tuberculosis (Mtb), which causes tuberculosis, is capable of accumulating triacylglycerol (TAG) by utilizing fatty acids from host cells. ATP-binding cassette (ABC) transporters are involved in transport processes in all organisms. Among the classical ABC transporters in Mtb none have been implicated in fatty acid import. Since the transport of fatty acids from the host cell is important for dormancy-associated TAG synthesis in the pathogen, mycobacterial ABC transporter(s) could potentially be involved in this process. Based on sequence identities with a bacterial ABC transporter that mediates fatty acid import for TAG synthesis, we identified Rv1272c, a hitherto uncharacterized ABC-transporter in Mtb that also shows sequence identities with a plant ABC transporter involved in fatty acid transport. We expressed Rv1272c in E. coli and show that it enhances the import of radiolabeled fatty acids. We also show that Rv1272c causes a significant increase in the metabolic incorporation of radiolabeled long-chain fatty acids into cardiolipin, a tetra-acylated phospholipid, and phosphatidylglycerol in E. coli. This is the first report on the function of Rv1272c showing that it displays a long-chain fatty acid transport function. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. 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).

  12. 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.

  13. Probing allocrite preferences of 2 naturally occurring variants of the wheat LR34 ABC transporter.

    Science.gov (United States)

    Rajagopalan, Nandhakishore; Halasz, Annamaria; Lu, Yuping; Liu, Enwu; Monteil-Rivera, Fanny; Loewen, Michele C

    2016-10-01

    For almost a century, the wheat Lr34 gene has conferred durable resistance against fungal rust diseases. While sequence homology predicts a putative ATP binding cassette transporter, the molecules that are transported (allocrites) by the encoded LR34 variants, and any associated mechanism of resistance, remain enigmatic. Here, the in vitro transport characteristics of 2 naturally occurring Lr34 variants (that differ in their ability to mediate disease resistance; Lr34sus and Lr34res) are investigated. Initially, a method to express and purify recombinant LR34Sus and LR34Res pseudo half-molecules from Saccharomyces cerevisiae, is described. Subsequently, a semi-targeted chlorophyll catabolite (CC) extraction from Lr34res-expressing wheat plants was performed based on previous reports highlighting increased accumulation of CCs in Lr34res-expressing flag leaves. Following partial biochemical characterization, this extract was applied to an LR34 in vitro proteoliposome transport assay. Mass spectroscopic analyses of transported metabolites revealed that LR34Sus imported a wheat metabolite of 618 Da and that the LR34Res transporter did not. While the identity of the LR34Sus transported metabolite remains to be confirmed and any allocrites of LR34Res remain to be detected, this work demonstrates that these variants have different allocrite preferences, a finding that may be relevant to the mechanism of disease resistance.

  14. Flavonoids as modulators of metabolic enzymes and drug transporters.

    Science.gov (United States)

    Miron, Anca; Aprotosoaie, Ana Clara; Trifan, Adriana; Xiao, Jianbo

    2017-06-01

    Flavonoids, natural compounds found in plants and in plant-derived foods and beverages, have been extensively studied with regard to their capacity to modulate metabolic enzymes and drug transporters. In vitro, flavonoids predominantly inhibit the major phase I drug-metabolizing enzyme CYP450 3A4 and the enzymes responsible for the bioactivation of procarcinogens (CYP1 enzymes) and upregulate the enzymes involved in carcinogen detoxification (UDP-glucuronosyltransferases, glutathione S-transferases (GSTs)). Flavonoids have been reported to inhibit ATP-binding cassette (ABC) transporters (multidrug resistance (MDR)-associated proteins, breast cancer-resistance protein) that contribute to the development of MDR. P-glycoprotein, an ABC transporter that limits drug bioavailability and also induces MDR, was differently modulated by flavonoids. Flavonoids and their phase II metabolites (sulfates, glucuronides) inhibit organic anion transporters involved in the tubular uptake of nephrotoxic compounds. In vivo studies have partially confirmed in vitro findings, suggesting that the mechanisms underlying the modulatory effects of flavonoids are complex and difficult to predict in vivo. Data summarized in this review strongly support the view that flavonoids are promising candidates for the enhancement of oral drug bioavailability, chemoprevention, and reversal of MDR. © 2017 New York Academy of Sciences.

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

    Directory of Open Access Journals (Sweden)

    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

  16. Protein Kinases C-Mediated Regulations of Drug Transporter Activity, Localization and Expression

    Science.gov (United States)

    Mayati, Abdullah; Moreau, Amélie; Le Vée, Marc; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Fardel, Olivier

    2017-01-01

    Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug–drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs) in transporter regulations are summarized and discussed. Both solute carrier (SLC) and ATP-binding cassette (ABC) drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug–drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders. PMID:28375174

  17. MFS multidrug transporters in pathogenic fungi: do they have real clinical impact?

    Science.gov (United States)

    Costa, Catarina; Dias, Paulo J; Sá-Correia, Isabel; Teixeira, Miguel C

    2014-01-01

    Infections caused by opportunistic fungal pathogens have reached concerning numbers due to the increase of the immunocrompromised human population and to the development of antifungal resistance. This resistance is often attributed to the action of multidrug efflux pumps, belonging to the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS). Although many studies have focused on the role of ABC multidrug efflux transporters, little is still known on the part played by the Drug:H(+) Antiporter (DHA) family of the MFS in this context. This review summarizes current knowledge on the role in antifungal drug resistance, mode of action and phylogenetic relations of DHA transporters, from the model yeast S. cerevisiae to pathogenic yeasts and filamentous fungi. Through the compilation of the predicted DHA transporters in the medically relevant Candida albicans, C. glabrata, C. parapsilosis, C. lusitaniae, C. tropicalis, C. guilliermondii, Cryptococcus neoformans, and Aspergillus fumigatus species, the fact that only 5% of the DHA transporters from these organisms have been characterized so far is evidenced. The role of these transporters in antifungal drug resistance and in pathogen-host interaction is described and their clinical relevance discussed. Given the knowledge gathered for these few DHA transporters, the need to carry out a systematic characterization of the DHA multidrug efflux pumps in fungal pathogens, with emphasis on their clinical relevance, is highlighted.

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

    Directory of Open Access Journals (Sweden)

    Saroj Velamakanni

    2009-07-01

    Full Text Available 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.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.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.

  19. In vitro characterization and endocrine regulation of cholesterol and phospholipid transport in the mammary gland.

    Science.gov (United States)

    Ontsouka, Corneille Edgar; Huang, Xiao; Aliyev, Eldar; Albrecht, Christiane

    2017-01-05

    Cell-based studies previously showed that the ATP-binding cassette transporter A1 (ABCA1) transfers cholesterol across mammary epithelial cells (MEC). Data for phospholipid transport are lacking, and it is unclear from which cellular source the transported cholesterol stems, whether this transport activates signaling pathways, and how lactogenic hormones regulate it. To clarify these aspects, lipid transport and expressional analyses were performed in bovine primary (bMEC) and/or immortalized (MAC-T) MEC cultures. Lipid efflux and ABCA1, ABCG1 and liver X receptorα mRNA levels were higher in MAC-T than bMEC. In MAC-T, the transported cholesterol originated mainly from the plasma membrane. ABCA1 dependent cholesterol efflux was higher than phosphatidylcholine efflux, was suppressed by probucol (ABCA1 inhibitor), AG490 (janus kinase-2 inhibitor), PD98059 (mitogen activated protein kinase kinase inhibitor) and pretreatment with β-cyclodextrin (lowering membrane cholesterol). Insulin was the only hormone significantly increasing cholesterol efflux. In conclusion, this study gives novel mechanistic and regulatory insights into the transport of cholesterol and phospholipids in MEC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. OsABCB14 functions in auxin transport and iron homeostasis in rice (Oryza sativa L.).

    Science.gov (United States)

    Xu, Yanxia; Zhang, Saina; Guo, Haipeng; Wang, Suikang; Xu, Ligen; Li, Chuanyou; Qian, Qian; Chen, Fan; Geisler, Markus; Qi, Yanhua; Jiang, De An

    2014-07-01

    Members of the ATP Binding Cassette B/Multidrug-Resistance/P-glyco-protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock-down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock-down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (-Fe). Their Fe concentration is higher and upregulation of Fe deficiency-responsive genes is lower in osabcb14 mutants than in wild-type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  1. 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. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Leishmania amazonensis: metabolic adaptations induced by resistance to an ABC transporter blocker.

    Science.gov (United States)

    Machuca, Claudia; Rodríguez, Adriana; Herrera, Marymar; Silva, Sonia; Ponte-Sucre, Alicia

    2006-09-01

    We compared growth rate, cell glucose turnover and expression of ATP-binding-cassette (ABC) transporters in Leishmania amazonensis (LTB0016; LTB) versus LTB(160) selected for resistance against the ABC transporter blocker glibenclamide. Additionally, we evaluated the influence of drug-resistance on Leishmania sensitivity against 2-mercaptoacetate and 2-deoxyglucose. Our data demonstrate that (1) LTB(160) and LTB constitutively express ABC transporters for neutral substrates, (2) glibenclamide resistance induces the expression of organic anion ABC transporters, members of the drug resistance associated transporters subfamily, (3) LTB(160) parasites use less glucose as energy substrate and exhibit a slower glucose uptake than LTB cells, and (4) LTB(160) parasites are less sensitive to 2-mercaptoacetate and 2-deoxyglucose than the glibenclamide-sensitive Leishmania LTB. Together these and previous results indicate that the metabolic adaptations expressed in drug-resistant LTB(160) differ from those described for mammalian drug resistant cells and constitute general mechanisms that underlie drug resistance in Leishmania and may be helpful for identifying alternative strategies to circumvent drug resistance in leishmaniasis.

  3. Hepatic ABC transporters and triglyceride metabolism.

    Science.gov (United States)

    Parks, John S; Chung, Soonkyu; Shelness, Gregory S

    2012-06-01

    Elevated plasma triglyceride and reduced HDL concentrations are prominent features of metabolic syndrome and type 2 diabetes. Individuals with Tangier disease also have elevated plasma triglyceride concentrations and very low HDL, resulting from mutations in ATP-binding cassette transporter A1 (ABCA1), an integral membrane protein that facilitates nascent HDL particle assembly. Past studies attributed the inverse relationship between plasma HDL and triglyceride to intravascular lipid exchange and catabolic events. However, recent studies also suggest that hepatic signaling and lipid mobilization and secretion may explain how HDL affects plasma triglyceride concentrations. Hepatocyte-specific ABCA1 knockout mice have markedly reduced plasma HDL and a two-fold increase in triglyceride due to failure to assemble nascent HDL particles by hepatocytes, causing increased catabolism of HDL apolipoprotein A-I and increased hepatic production of triglyceride-enriched VLDL. In-vitro studies suggest that nascent HDL particles may induce signaling to decrease triglyceride secretion. Inhibition of microRNA 33 expression in nonhuman primates augments hepatic ABCA1, genes involved in fatty acid oxidation, and decreases expression of lipogenic genes, causing increased plasma HDL and decreased triglyceride levels. New evidence suggests potential mechanisms by which hepatic ABCA1-mediated nascent HDL formation regulates VLDL-triglyceride production and contributes to the inverse relationship between plasma HDL and triglyceride.

  4. Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences.

    Science.gov (United States)

    Haider, Ameena J; Cox, Megan H; Jones, Natalie; Goode, Alice J; Bridge, Katherine S; Wong, Kelvin; Briggs, Deborah; Kerr, Ian D

    2015-07-17

    ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be 'rescued' by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. © 2015 Authors.

  5. The ABCG family of membrane-associated transporters: you don't have to be big to be mighty.

    Science.gov (United States)

    Kerr, Ian D; Haider, Ameena J; Gelissen, Ingrid C

    2011-12-01

    Along with many other mammalian ATP-binding cassette (ABC) transporters, members of the ABCG group are involved in the regulated transport of hydrophobic compounds across cellular membranes. In humans, five ABCG family members have been identified, encoding proteins ranging from 638 to 678 amino acids in length. All five have been the subject of intensive investigation to better understand their physiological roles, expression patterns, interactions with substrates and inhibitors, and regulation at both the transcript and protein level. The principal substrates for at least four of the ABCG proteins are endogenous and dietary lipids, with ABCG1 implicated in particular in the export of cholesterol, and ABCG5 and G8 forming a functional heterodimer responsible for plant sterol elimination from the body. ABCG2 has a much broader substrate specificity and its ability to transport numerous diverse pharmaceuticals has implications for the absorption, distribution, metabolism, excretion and toxicity (ADMETOx) profile of these compounds. ABCG2 is one of at least three so-called multidrug resistant ABC transporters expressed in humans, and its activity is associated with decreased efficacy of anti-cancer agents in several carcinomas. In addition to its role in cancer, ABCG2 also plays a role in the normal physiological transport of urate and haem, the implications of which are described. We summarize here data on all five human ABCG transporters and provide a current perspective on their roles in human health and disease. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  6. Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

    Science.gov (United States)

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-07-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters.

  7. Vacuolar Transport of the Medicinal Alkaloids from Catharanthus roseus Is Mediated by a Proton-Driven Antiport1[W

    Science.gov (United States)

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-01-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3′,4′-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4+ and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H+ gradient preestablished across the tonoplast by either vacuolar H+-ATPase or vacuolar H+-pyrophosphatase. The initial rates of H+ gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H+ antiport system and not by an ion-trap mechanism or ABC transporters. PMID:23686419

  8. Polymorphism in ABC transporter genes of Dirofilaria immitis

    Directory of Open Access Journals (Sweden)

    Thangadurai Mani

    2017-08-01

    Full Text Available Dirofilaria immitis, a filarial nematode, causes dirofilariasis in dogs, cats and occasionally in humans. Prevention of the disease has been mainly by monthly use of the macrocyclic lactone (ML endectocides during the mosquito transmission season. Recently, ML resistance has been confirmed in D. immitis and therefore, there is a need to find new classes of anthelmintics. One of the mechanisms associated with ML resistance in nematodes has been the possible role of ATP binding cassette (ABC transporters in reducing drug concentrations at receptor sites. ABC transporters, mainly from sub-families B, C and G, may contribute to multidrug resistance (MDR by active efflux of drugs out of the cell. Gene products of ABC transporters may thus serve as the targets for agents that may modulate susceptibility to drugs, by inhibiting drug transport. ABC transporters are believed to be involved in a variety of physiological functions critical to the parasite, such as sterol transport, and therefore may also serve as the target for drugs that can act as anthelmintics on their own. Knowledge of polymorphism in these ABC transporter genes in nematode parasites could provide useful information for the process of drug design. We have identified 15 ABC transporter genes from sub-families A, B, C and G, in D. immitis, by comparative genomic approaches and analyzed them for polymorphism. Whole genome sequencing data from four ML susceptible (SUS and four loss of efficacy (LOE pooled populations were used for single nucleotide polymorphism (SNP genotyping. Out of 231 SNPs identified in those 15 ABC transporter genes, 89 and 75 of them were specific to the SUS or LOE populations, respectively. A few of the SNPs identified may affect gene expression, protein function, substrate specificity or resistance development and may be useful for transporter inhibitor/anthelmintic drug design, or in order to anticipate resistance development.

  9. Polymorphism in ABC transporter genes of Dirofilaria immitis.

    Science.gov (United States)

    Mani, Thangadurai; Bourguinat, Catherine; Prichard, Roger K

    2017-08-01

    Dirofilaria immitis, a filarial nematode, causes dirofilariasis in dogs, cats and occasionally in humans. Prevention of the disease has been mainly by monthly use of the macrocyclic lactone (ML) endectocides during the mosquito transmission season. Recently, ML resistance has been confirmed in D. immitis and therefore, there is a need to find new classes of anthelmintics. One of the mechanisms associated with ML resistance in nematodes has been the possible role of ATP binding cassette (ABC) transporters in reducing drug concentrations at receptor sites. ABC transporters, mainly from sub-families B, C and G, may contribute to multidrug resistance (MDR) by active efflux of drugs out of the cell. Gene products of ABC transporters may thus serve as the targets for agents that may modulate susceptibility to drugs, by inhibiting drug transport. ABC transporters are believed to be involved in a variety of physiological functions critical to the parasite, such as sterol transport, and therefore may also serve as the target for drugs that can act as anthelmintics on their own. Knowledge of polymorphism in these ABC transporter genes in nematode parasites could provide useful information for the process of drug design. We have identified 15 ABC transporter genes from sub-families A, B, C and G, in D. immitis, by comparative genomic approaches and analyzed them for polymorphism. Whole genome sequencing data from four ML susceptible (SUS) and four loss of efficacy (LOE) pooled populations were used for single nucleotide polymorphism (SNP) genotyping. Out of 231 SNPs identified in those 15 ABC transporter genes, 89 and 75 of them were specific to the SUS or LOE populations, respectively. A few of the SNPs identified may affect gene expression, protein function, substrate specificity or resistance development and may be useful for transporter inhibitor/anthelmintic drug design, or in order to anticipate resistance development. Copyright © 2017 The Authors. Published by

  10. ABC Transporter Subfamily D: Distinct Differences in Behavior between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease

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    Kosuke Kawaguchi

    2016-01-01

    Full Text Available ATP-binding cassette (ABC transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1–3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease. Recently, it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B12 deficiency, respectively. In this review, the targeting mechanism and physiological functions of the ABCD transporters are summarized along with the related disease.

  11. ABC Transporter Subfamily D: Distinct Differences in Behavior between ABCD1–3 and ABCD4 in Subcellular Localization, Function, and Human Disease

    Science.gov (United States)

    2016-01-01

    ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1–3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA) or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease. Recently, it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B12 deficiency, respectively. In this review, the targeting mechanism and physiological functions of the ABCD transporters are summarized along with the related disease. PMID:27766264

  12. ABC transporters in fish species: a review

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    Marta eFerreira

    2014-07-01

    Full Text Available ATP-binding cassette (ABC proteins were first recognized for their role in multidrug resistance (MDR in chemotherapeutic treatments, which is a major impediment for the successful treatment of many forms of malignant tumors in humans. These proteins, highly conserved throughout vertebrate species, were later related to cellular detoxification and accounted as responsible for protecting aquatic organisms from xenobiotic insults in the so-called multixenobiotic resistance mechanism (MXR. In recent years, research on these proteins in aquatic species has highlighted their importance in the detoxification mechanisms in fish thus it is of extreme added value to continue these studies. Several transporters have been pointed out as relevant in the ecotoxicological context associated to the transport of xenobiotics, such as P-glycoproteins (Pgps, multidrug-resistance-associated proteins (MRPs 1-5 and breast resistance associated protein (BCRP. In mammals, several nuclear receptors have been identified as mediators of phase I and II metabolizing enzymes and ABC transporters. In aquatic species, knowledge on co-regulation of detoxification mechanism is scarce and needs to be addressed. The interaction of emergent contaminants, with chemosensitizer potential, with ABC transporters in aquatic organisms can compromise detoxification processes and have population effects and should be studied in more detail. This review intends to summarize the recent advances in research on MXR mechanisms in fish species, focusing in 1 regulation and functioning of ABC proteins; 2 cooperation with phase I and II biotransformation enzymes; and 3 ecotoxicological relevance and information on emergent pollutants with ability to modulate ABC transporters expression and activity. Several lines of evidence are clear suggesting the important role of these transporters in detoxification mechanisms and must be further investigated in fish.

  13. Kinetics of MDR transport in tumor-initiating cells.

    Science.gov (United States)

    Koshkin, Vasilij; Yang, Burton B; Krylov, Sergey N

    2013-01-01

    Multidrug resistance (MDR) driven by ABC (ATP binding cassette) membrane transporters is one of the major causes of treatment failure in human malignancy. MDR capacity is thought to be unevenly distributed among tumor cells, with higher capacity residing in tumor-initiating cells (TIC) (though opposite finding are occasionally reported). Functional evidence for enhanced MDR of TICs was previously provided using a "side population" assay. This assay estimates MDR capacity by a single parameter - cell's ability to retain fluorescent MDR substrate, so that cells with high MDR capacity ("side population") demonstrate low substrate retention. In the present work MDR in TICs was investigated in greater detail using a kinetic approach, which monitors MDR efflux from single cells. Analysis of kinetic traces obtained allowed for the estimation of both the velocity (V max) and affinity (K M) of MDR transport in single cells. In this way it was shown that activation of MDR in TICs occurs in two ways: through the increase of V max in one fraction of cells, and through decrease of K M in another fraction. In addition, kinetic data showed that heterogeneity of MDR parameters in TICs significantly exceeds that of bulk cells. Potential consequences of these findings for chemotherapy are discussed.

  14. ORF Sequence: NC_001136 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available and arsenite; also transports unconjugated bilirubin; similar to human cystic fibrosis protein CFTR; Ycf1p [... transporter of the ATP-binding cassette family, has a role in detoxifying metals such as cadmium, mercury,

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

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    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

  16. Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation.

    Science.gov (United States)

    Ziegler, Jörg; Schmidt, Stephan; Strehmel, Nadine; Scheel, Dierk; Abel, Steffen

    2017-06-16

    The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins.

  17. Effects of chronic kidney disease and uremia on hepatic drug metabolism and transport.

    Science.gov (United States)

    Yeung, Catherine K; Shen, Danny D; Thummel, Kenneth E; Himmelfarb, Jonathan

    2014-03-01

    The pharmacokinetics of non-renally cleared drugs in patients with chronic kidney disease is often unpredictable. Some of this variability may be due to alterations in the expression and activity of extra renal drug-metabolizing enzymes and transporters, primarily localized in the liver and intestine. Studies conducted in rodent models of renal failure have shown decreased mRNA and protein expression of many members of the cytochrome P450 enzyme (CYP) gene family and the ATP-binding cassette (ABC) and solute carrier (SLC) gene families of drug transporters. Uremic toxins interfere with transcriptional activation, cause downregulation of gene expression mediated by proinflammatory cytokines, and directly inhibit the activity of the cytochrome P450s and drug transporters. While much has been learned about the effects of kidney disease on non-renal drug disposition, important questions remain regarding the mechanisms of these effects, as well as the interplay between drug-metabolizing enzymes and drug transporters in the uremic milieu. In this review, we have highlighted the existing gaps in our knowledge and understanding of the impact of chronic kidney disease on non-renal drug clearance, and identified areas of opportunity for future research.

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

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    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.

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

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    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.

  20. Rad51 ATP binding but not hydrolysis is required to recruit Rad10 in synthesis-dependent strand annealing sites in S. cerevisiae.

    Science.gov (United States)

    Karlin, Justin; Fischhaber, Paula L

    2013-06-01

    Several modes of eukaryotic of DNA double strand break repair (DSBR) depend on synapsis of complementary DNA. The Rad51 ATPase, the S. cerevisiae homolog of E. coli RecA, plays a key role in this process by catalyzing homology searching and strand exchange between an invading DNA strand and a repair template (e.g. sister chromatid or homologous chromosome). Synthesis dependent strand annealing (SDSA), a mode of DSBR, requires Rad51. Another repair enzyme, the Rad1-Rad10 endonuclease, acts in the final stages of SDSA, hydrolyzing 3' overhanging single-stranded DNA. Here we show in vivo by fluorescence microscopy that the ATP binding function of yeast Rad51 is required to recruit Rad10 SDSA sites indicating that Rad51 pre-synaptic filament formation must occur prior to the recruitment of Rad1-Rad10. Our data also show that Rad51 ATPase activity, an important step in Rad51 filament disassembly, is not absolutely required in order to recruit Rad1-Rad10 to DSB sites.

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

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    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

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

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    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.

  3. Arsenic-glutathione conjugate transport by the human multidrug resistance proteins (MRPs/ABCCs).

    Science.gov (United States)

    Leslie, Elaine M

    2012-03-01

    Millions of people world-wide are chronically exposed to inorganic forms of the environmental toxicant arsenic in drinking water. This has led to a public health crisis because arsenic is a human carcinogen, and causes a myriad of other adverse health effects. In order to prevent and treat arsenic-induced toxicity it is critical to understand the cellular handling of this metalloid. A large body of literature describes the importance of the cellular tripeptide glutathione (γ-Glu-Cys-Gly,GSH/GS) in the excretion of arsenic. The triglutathione conjugate of arsenite [As(III)(GS)(3)] and the diglutathione conjugate of monomethylarsonous acid [MMA(III)(GS)(2)] have been isolated from rat bile and mouse urine, and account for the majority of excreted arsenic, suggesting these are important transportable forms. The ATP-binding cassette (ABC) transporter proteins, multidrug resistance protein 1 (MRP1/ABCC1) and the related protein MRP2 (ABCC2), are thought to play an important role in arsenic detoxification through the cellular efflux of arsenic-GSH conjugates. Current knowledge on the cellular handling of arsenic with a special emphasis on the transport pathways of the arsenic-GSH conjugates As(III)(GS)(3), MMA(III)(GS)(2), and dimethylarsenic glutathione DMA(III)(GS), as well as, the seleno-bis(S-glutathionyl) arsinium ion [(GS)(2)AsSe](-) are reviewed. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

    Science.gov (United States)

    Maruyama, Yukie; Itoh, Takafumi; Kaneko, Ai; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2015-09-01

    The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Transporters for the Intestinal Absorption of Cholesterol, Vitamin E, and Vitamin K.

    Science.gov (United States)

    Yamanashi, Yoshihide; Takada, Tappei; Kurauchi, Ryoya; Tanaka, Yusuke; Komine, Toko; Suzuki, Hiroshi

    2017-04-03

    Humans cannot synthesize fat-soluble vitamins such as vitamin E and vitamin K. For this reason, they must be obtained from the diet via intestinal absorption. As the deficiency or excess of these vitamins has been reported to cause several types of diseases and disorders in humans, the intestinal absorption of these nutrients must be properly regulated to ensure good health. However, the mechanism of their intestinal absorption remains poorly understood. Recent studies on cholesterol using genome-edited mice, genome-wide association approaches, gene mutation analyses, and the development of cholesterol absorption inhibitors have revealed that several membrane proteins play crucial roles in the intestinal absorption of cholesterol. Surprisingly, detailed analyses of these cholesterol transporters have revealed that they can also transport vitamin E and vitamin K, providing clues to uncover the molecular mechanisms underlying the intestinal absorption of these fat-soluble vitamins. In this review, we focus on the membrane proteins (Niemann-Pick C1 like 1, scavenger receptor class B type I, cluster of differentiation 36, and ATP-binding cassette transporter A1) that are (potentially) involved in the intestinal absorption of cholesterol, vitamin E, and vitamin K and discuss their physiological and pharmacological importance. We also discuss the related uncertainties that need to be explored in future studies.

  6. Family business: the multidrug-resistance related protein (MRP) ABC transporter genes in Arabidopsis thaliana.

    Science.gov (United States)

    Kolukisaoglu, H Uner; Bovet, Lucien; Klein, Markus; Eggmann, Thomas; Geisler, Markus; Wanke, Dierk; Martinoia, Enrico; Schulz, Burkhard

    2002-11-01

    Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein ( MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. Identification and characterization of the gltK gene encoding a membrane-associated glucose transport protein of pseudomonas aeruginosa.

    Science.gov (United States)

    Adewoye, L O; Worobec, E A

    2000-08-08

    The Pseudomonas aeruginosa oprB gene encodes the carbohydrate-selective OprB porin, which translocates substrate molecules across the outer membrane to the periplasmic glucose-binding protein. We identified and cloned two open reading frames (ORFs) flanking the oprB gene but are not in operonic arrangement with the oprB gene. The downstream ORF encodes a putative polypeptide homologous to members of a family of transcriptional repressors, whereas the oprB gene is preceded by an ORF encoding a putative product, which exhibits strong homology to several carbohydrate transport ATP-binding cassette (ABC) proteins. The genomic copy of the upstream ORF was mutagenized by homologous recombination. Analysis of the deletion mutant in comparison with the wild type revealed a significant reduction in [14C] glucose transport activity in the mutant strain, suggesting that this ORF likely encodes the inner membrane component of the glucose ABC transporter. It is thus designated gltK gene to reflect its homology to the Pseudomona fluorescens mtlK and its involvement in the high-affinity glucose transport system. Multiple alignment analysis revealed that the P. aeruginosa gltK gene product is a member of the MalK subfamily of ABC proteins.

  9. Inhibition of multidrug/xenobiotic resistance transporter by MK571 improves dye (Fura 2) accumulation in crustacean tissues from lobster, shrimp, and isopod.

    Science.gov (United States)

    Lüders, Ann-Katrin; Saborowski, Reinhard; Bickmeyer, Ulf

    2009-09-01

    Multidrug/xenobiotic resistance transporters are present in living organisms as a first line defence system against small, potentially harmful molecules from the environment or from internal metabolic reactions. Multidrug resistance associated proteins (MRP) are one type of ATP-Binding-Cassette (ABC) transporters, which also transport dyes such as Fura 2, a calcium chelating fluorescence indicator. The specific MRP inhibitor MK571 was used to investigate the fluorescence intensity of cells in tissues of the brain and the midgut gland of the crustaceans Homarus gammarus (lobster), Crangon crangon (brown shrimp) and Idotea emarginata (isopod) during incubation with Fura 2AM (1 microM). In the presence of the inhibitor MK571 (50 microM), the fluorescence of brain tissue significantly increased in all of the three species. The midgut gland of H. gammarus showed a significant increase of fluorescence, whereas there was no effect in the midgut glands of C. crangon and I. baltica. The half maximal concentration of MK571 was 50 microM as measured in the midgut gland of H. gammarus. In conclusion, MRP transporters are present in the three investigated crustacean nervous systems. Using the midgut glands of the three species, only in H. gammarus MK571 inhibited dye extrusion, indicating species-specific differences of transporter systems, their specificity, or tissue specific expression.

  10. Function of prokaryotic and eukaryotic ABC proteins in lipid transport.

    Science.gov (United States)

    Pohl, Antje; Devaux, Philippe F; Herrmann, Andreas

    2005-03-21

    ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry.

  11. Comparative effect of fish oil feeding and other dietary fatty acids on plasma lipoproteins, biliary lipids, and hepatic expression of proteins involved in reverse cholesterol transport in the rat.

    Science.gov (United States)

    Morgado, Nora; Rigotti, Attilio; Valenzuela, Alfonso

    2005-01-01

    While elevated plasma high-density lipoprotein (HDL) levels has been associated to a reduction in cardiovascular risk, dietary fish oils rich in omega-3 polyunsaturated fatty acids (PUFAs) may protect against this disease. The protective effect of HDL is associated to its participation in the reverse cholesterol transport pathway. On the other hand, omega-3 PUFAs decrease plasma HDL levels compared to other fatty acids, which may suggest an effect on reverse cholesterol transport. In this work, the effect of dietary fish oil on the fatty acid composition of hepatic membranes, plasma lipoprotein cholesterol profile, biliary lipids, and the expression of proteins involved in reverse cholesterol transport, was compared to other dietary oils having a different degree of fatty acid unsaturation. Male rats were fed a semi synthetic diet containing fish oil (omega-3), sunflower oil (omega-6), olive oil (omega-9) or coconut oil (saturated). Hepatic membrane fatty acid composition, plasma cholesterol levels, lipoprotein cholesterol profile, biliary lipids, hepatic mRNA levels for lecithin cholesterol acyltransferase, hepatic lipase, apo E, and apo A-I, and hepatic protein levels of the scavenger receptor class B type I, caveolin-1, and the ATP binding cassette transporter A1 were analyzed. Plasma apo A-I and apo E protein levels were also evaluated. Compared to the other diets, omega-3 PUFAs significantly changed omega-3/omega-6 fatty acid ratio of hepatic membranes, caused a reduction of plasma total and HDL cholesterol, and selectively increased biliary cholesterol secretion. No modification in the expression levels of lecithin cholesterol acyltransferase, hepatic lipase, apo A-I and apo E mRNA was observed. Hepatic scavenger receptor class B type I, caveolin-1, and the ATP binding cassette transporter A1 protein levels were also not affected. Plasma apo A-I, but not apo E, was reduced. These results show that dietary omega-3 PUFAs reduce plasma HDL cholesterol and

  12. 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.

  13. 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.

  14. Impaired Insulin Secretion in Four Tangier Disease Patients with ABCA1 Mutations

    National Research Council Canada - National Science Library

    Koseki, Masahiro; Matsuyama, Akifumi; Nakatani, Kazuhiro; Inagaki, Miwako; Nakaoka, Hajime; Kawase, Ryota; Yuasa-Kawase, Miyako; Tsubakio-Yamamoto, Kazumi; Masuda, Daisaku; C. Sandoval, Jose; Ohama, Tohru; Nakagawa-Toyama, Yumiko; Matsuura, Fumihiko; Nishida, Makoto; Ishigami, Masato; Hirano, Ken-ichi; Sakane, Naoki; Kumon, Yoshitaka; Suehiro, Tadashi; Nakamura, Tadashi; Shimomura, Iichiro; Yamashita, Shizuya

    2009-01-01

    Aim: Tangier disease (TD), caused by deficiency of ATP-binding cassette transporter A1, is characterized by the absence of high density lipoprotein and the accumulation of cholesteryl esters in many tissues...

  15. Reference: 342 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available lls and initiation of hyphae by B. g. hordei, suggesting that PEN3 contributes to defenses at the cell wall ...vate the salicylic acid pathway. Arabidopsis PEN3/PDR8, an ATP binding cassette transporter, contribute

  16. A Major Facilitator Superfamily Transporter Plays a Dual Role in Polar Auxin Transport and Drought Stress Tolerance in Arabidopsis[W

    Science.gov (United States)

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

    2013-01-01

    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. PMID:23524662

  17. Breast cancer resistance protein (BCRP/ABCG2) transports fluoroquinolone antibiotics and affects their oral availability, pharmacokinetics, and milk secretion.

    Science.gov (United States)

    Merino, Gracia; Alvarez, Ana I; Pulido, Mivis M; Molina, Antonio J; Schinkel, Alfred H; Prieto, Julio G

    2006-04-01

    The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells in intestine, liver, mammary gland, and other organs, affecting the pharmacological and toxicological behavior of many compounds, including their secretion into the milk. The purpose of this study was to determine whether three widely used fluoroquinolone antibiotics (ciprofloxacin, ofloxacin, and norfloxacin) are substrates of Bcrp1/BCRP and to investigate the possible role of this transporter in the in vivo pharmacokinetic profile of these compounds and their secretion into the milk. Using polarized cell lines, we found that ciprofloxacin, ofloxacin, and norfloxacin are transported by mouse Bcrp1 and human BCRP. In vivo pharmacokinetic studies showed that the ciprofloxacin plasma concentration was more than 2-fold increased in Bcrp1(-/-) compared with wild-type mice (1.77 +/- 0.73 versus 0.85 +/- 0.39 microg/ml, p milk concentration and milk/plasma ratio of ciprofloxacin were 2-fold higher in wild-type than in Bcrp1(-/-) lactating mice. We conclude that Bcrp1 is one of the determinants for the bioavailability of fluoroquinolones and their secretion into the milk.

  18. 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.

  19. Genome-Edited Human Pluripotent Stem Cell-Derived Macrophages as a Model of Reverse Cholesterol Transport--Brief Report.

    Science.gov (United States)

    Gupta, Rajat M; Meissner, Torsten B; Cowan, Chad A; Musunuru, Kiran

    2016-01-01

    To create isogenic human pluripotent stem cell-derived macrophages with and without ABCA1 expression as a model for reverse cholesterol transport. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) genome-editing system was used to introduce frameshift mutations into the coding sequence of ATP-binding cassette, subfamily A, member 1. Individual human pluripotent stem cell clones with deleterious mutations were identified, expanded, and differentiated into mature macrophages with a cytokine-based, feeder-free differentiation protocol. Wild-type cells demonstrated effective cholesterol efflux to apoAI acceptor, whereas ABCA1(-/-) cells displayed significantly reduced efflux ability and increased expression of proinflammatory cytokines. Human pluripotent stem cell-derived macrophages capable of reverse cholesterol transport can be rapidly generated and genetically edited with CRISPR/Cas9. Introduction of homozygous frameshift mutations results in loss of ABCA1 expression in differentiated macrophages and subsequent reduction of cholesterol efflux capability. This facile genome-editing approach and differentiation protocol pave the way for future studies of the molecular determinants of reverse cholesterol transport and other macrophage properties. © 2015 American Heart Association, Inc.

  20. ATP Hydrolysis Induced Conformational Changes in the Vitamin B12 Transporter BtuCD Revealed by MD Simulations.

    Science.gov (United States)

    Pan, Chao; Weng, Jingwei; Wang, Wenning

    2016-01-01

    ATP binding cassette (ABC) transporters utilize the energy of ATP hydrolysis to uni-directionally transport substrates across cell membrane. ATP hydrolysis occurs at the nucleotide-binding domain (NBD) dimer interface of ABC transporters, whereas substrate translocation takes place at the translocation pathway between the transmembrane domains (TMDs), which is more than 30 angstroms away from the NBD dimer interface. This raises the question of how the hydrolysis energy released at NBDs is "transmitted" to trigger the conformational changes at TMDs. Using molecular dynamics (MD) simulations, we studied the post-hydrolysis state of the vitamin B12 importer BtuCD. Totally 3-μs MD trajectories demonstrate a predominantly asymmetric arrangement of the NBD dimer interface, with the ADP-bound site disrupted and the ATP-bound site preserved in most of the trajectories. TMDs response to ATP hydrolysis by separation of the L-loops and opening of the cytoplasmic gate II, indicating that hydrolysis of one ATP could facilitate substrate translocation by opening the cytoplasmic end of translocation pathway. It was also found that motions of the L-loops and the cytoplasmic gate II are coupled with each other through a contiguous interaction network involving a conserved Asn83 on the extended stretch preceding TM3 helix plus the cytoplasmic end of TM2/6/7 helix bundle. These findings entail a TMD-NBD communication mechanism for type II ABC importers.

  1. Two Medicago truncatula Half-ABC Transporters Are Essential for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis[W

    Science.gov (United States)

    Zhang, Quan; Blaylock, Laura A.; Harrison, Maria J.

    2010-01-01

    In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungal symbiont resides in the root cortical cells where it delivers mineral nutrients to its plant host through branched hyphae called arbuscules. Here, we report a Medicago truncatula mutant, stunted arbuscule (str), in which arbuscule development is impaired and AM symbiosis fails. In contrast with legume symbiosis mutants reported previously, str shows a wild-type nodulation phenotype. STR was identified by positional cloning and encodes a half-size ATP binding cassette (ABC) transporter of a subfamily (ABCG) whose roles in plants are largely unknown. STR is a representative of a novel clade in the ABCG subfamily, and its orthologs are highly conserved throughout the vascular plants but absent from Arabidopsis thaliana. The STR clade is unusual in that it lacks the taxon-specific diversification that is typical of the ABCG gene family. This distinct phylogenetic profile enabled the identification of a second AM symbiosis-induced half-transporter, STR2. Silencing of STR2 by RNA interference results in a stunted arbuscule phenotype identical to that of str. STR and STR2 are coexpressed constitutively in the vascular tissue, and expression is induced in cortical cells containing arbuscules. STR heterodimerizes with STR2, and the resulting transporter is located in the peri-arbuscular membrane where its activity is required for arbuscule development and consequently a functional AM symbiosis. PMID:20453115

  2. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. E17110 promotes reverse cholesterol transport with liver X receptor β agonist activity in vitro

    Directory of Open Access Journals (Sweden)

    Ni Li

    2016-05-01

    Full Text Available Liver X receptor (LXR plays an important role in reverse cholesterol transport (RCT, and activation of LXR could reduce atherosclerosis. In the present study we used a cell-based screening method to identify new potential LXRβ agonists. A novel benzofuran-2-carboxylate derivative was identified with LXRβ agonist activity: E17110 showed a significant activation effect on LXRβ with an EC50 value of 0.72 μmol/L. E17110 also increased the expression of ATP-binding cassette transporter A1 (ABCA1 and G1 (ABCG1 in RAW264.7 macrophages. Moreover, E17110 significantly reduced cellular lipid accumulation and promoted cholesterol efflux in RAW264.7 macrophages. Interestingly, we found that the key amino acids in the LXRβ ligand-binding domain had distinct interactions with E17110 as compared to TO901317. These results suggest that E17110 was identified as a novel compound with LXRβ agonist activity in vitro via screening, and could be developed as a potential anti-atherosclerotic lead compound.

  4. 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.

  5. 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

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

    DEFF Research Database (Denmark)

    Frikke-Schmidt, Ruth; Nordestgaard, BG; 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.......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....

  7. 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

  8. 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

  9. 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.

  10. 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.

    2014-01-01

    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. PMID:24247241

  11. LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania

    Science.gov (United States)

    González-Rey, Elena; Delgado, Mario; Castanys, Santiago; Pérez-Victoria, José M.; Gamarro, Francisco

    2013-01-01

    Leishmaniasis is a neglected disease produced by the intracellular protozoan parasite Leishmania. In the present study, we show that LABCG2, a new ATP-binding cassette half-transporter (ABCG subfamily) from Leishmania, is involved in parasite virulence. Down-regulation of LABCG2 function upon expression of an inactive mutant version of this half-transporter (LABCG2K/M) is shown to reduce the translocation of short-chain analogues of phosphatidylserine (PS). This dominant-negative phenotype is specific for the headgroup of the phospholipid, as the movement of phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine or sphingomyelin is not affected. In addition, promastigotes expressing LABCG2K/M expose less endogenous PS in the stationary phase than control parasites. Transient exposure of PS at the outer leaflet of the plasma membrane is known to be one of the mechanisms used by Leishmania to infect macrophages and to silence their immune response. Stationary phase/metacyclic promastigotes expressing LABCG2K/M are less infective for macrophages and show decreased pathogenesis in a mouse model of cutaneous leishmaniasis. Thus, mice infected with parasites expressing LABCG2K/M did not develop any lesion and showed significantly lower inflammation and parasite burden than mice infected with control parasites. Our results indicate that LABCG2 function is required for the externalization of PS in Leishmania promastigotes, a process that is involved in the virulence of the parasite. PMID:23638200

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Scavenger receptor BI and ABCG5/G8 differentially impact biliary sterol secretion and reverse cholesterol transport in mice.

    Science.gov (United States)

    Dikkers, Arne; Freak de Boer, Jan; Annema, Wijtske; Groen, Albert K; Tietge, Uwe J F

    2013-07-01

    Biliary lipid secretion plays an important role in gallstone disease and reverse cholesterol transport (RCT). Using Sr-bI/Abcg5 double knockout mice (dko), the present study investigated the differential contribution of two of the most relevant transporters: adenosine triphosphate (ATP)-binding cassette subfamily G member 5 and 8 (ABCG5/G8) and scavenger receptor class B type I (SR-BI) to sterol metabolism and RCT. Plasma cholesterol levels increased in the following order, mainly due to differences in high density lipoprotein (HDL): Abcg5 ko Sr-bI ko (-16%) > Abcg5 ko (-75%) > Sr-bI/Abcg5 dko (-94%), all at least P G8-independent biliary cholesterol secretion under basal conditions; (2) biliary cholesterol mass secretion under maximal bile salt-stimulated conditions is fully dependent on ABCG5/G8; and (3) Sr-bI contributes to macrophage-to-feces RCT independent of Abcg5/g8. Copyright © 2013 American Association for the Study of Liver Diseases.

  14. 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

  15. Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

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    Rinne, Petteri; Rami, Martina; Nuutinen, Salla; Santovito, Donato; van der Vorst, Emiel P C; Guillamat-Prats, Raquel; Lyytikäinen, Leo-Pekka; Raitoharju, Emma; Oksala, Niku; Ring, Larisa; Cai, Minying; Hruby, Victor J; Lehtimäki, Terho; Weber, Christian; Steffens, Sabine

    2017-07-04

    The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse

  16. The molecular basis of glycogen breakdown and transport in Streptococcus pneumoniae.

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    Abbott, D Wade; Higgins, Melanie A; Hyrnuik, Susanne; Pluvinage, Benjamin; Lammerts van Bueren, Alicia; Boraston, Alisdair B

    2010-07-01

    The genome of Streptococcus pneumoniae strains, as typified by the TIGR4 strain, contain several genes encoding proteins putatively involved in alpha-glucan degradation, modification and synthesis. The extracellular components comprise an ATP binding cassette-transporter with its solute binding protein, MalX, and the hydrolytic enzyme SpuA. We show that of the commonly occurring exogenous alpha-glucans, S. pneumoniae TIGR4 is only able to grow on glycogen in a MalX- and SpuA-dependent manner. SpuA is able to degrade glycogen into a ladder of alpha-1,4-glucooligosaccharides while the high-affinity interaction (K(a) approximately 10(6) M(-1)) of MalX with maltooligosaccharides plays a key role in promoting the selective uptake of the glycogen degradation products that are produced by SpuA. The X-ray crystallographic analyses of apo- and complexed MalX illuminate the protein's specificity for the degradation products of glycogen and its striking ability to recognize the helical structure of the ligand. Overall, the results of this work provide new structural and functional insight into streptococcal alpha-glucan metabolism while supplying biochemical support for the hypothesis that the substrate of the S. pneumoniaealpha-glucan metabolizing machinery is glycogen, which in a human host is abundant in lung epithelial cells, a common target for invasive S. pneumoniae.

  17. Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure

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    Sager Georg

    2009-09-01

    Full Text Available Abstract The human ATP-binding cassette (ABC transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published cross-linking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5, Ile340 (TMH6, Phe343 (TMH6, Phe728 (TMH7, and Val982 (TMH12, form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the site-directed mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.

  18. Evaluation of para-Aminosalicylic Acid as a Substrate of Multiple Solute Carrier Uptake Transporters and Possible Drug Interactions with Nonsteroidal Anti-inflammatory Drugs In Vitro

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    Parvez, M. Masud; Shin, Ho Jung; Jung, Jin Ah

    2017-01-01

    ABSTRACT para-Aminosalicylic acid (PAS) is a second-line antituberculosis drug that has been used to treat multidrug-resistant and extensively drug-resistant tuberculosis for more than 60 years. Renal secretion and glomerular filtration are the major pathways for the elimination of PAS. We comprehensively studied PAS transport by using cell lines that overexpressed various transporters and found that PAS acts as a novel substrate of an organic anionic polypeptide (OATP1B1), organic cationic transporters (OCT1 and OCT2), and organic anion transporters (OAT1 and OAT3) but is not a substrate of any ATP-binding cassette (ABC) transporters. Net PAS uptake was measured, and the transport affinities (Km values) for OATP1B1, OCT1, OCT2, OAT1, and OAT3 were found to be 50.0, 20.3, 28.7, 78.1, and 100.1 μM, respectively. The net uptake rates suggested that renal OAT1 and OAT3 play relatively major roles in PAS elimination. The representative inhibitors rifampin for OATP1B1, probenecid for OAT1 and OAT3, and verapamil for OCT1 and OCT2 greatly inhibited PAS uptake, suggesting that PAS is dependent on multiple transporters for uptake. We also evaluated nonsteroidal anti-inflammatory drugs (NSAIDs), proton pump inhibitors (PPIs), and metformin for the inhibition of PAS uptake via these transporters. Half-maximal (50%) inhibitory concentrations (IC50s) were kinetically determined and used to predict the drug-drug interactions (DDIs) affecting these transporters' activity toward PAS. We found that rifampin, probenecid, ibuprofen, naproxen, cimetidine, and quinidine each exhibited a significant potential for in vivo DDIs with PAS. In this study, PAS was found to be a novel substrate of several transporters, and drugs that inhibit these transporters can reduce PAS elimination. PMID:28223391

  19. Assessment of ABCG2-mediated transport of pesticides across the rabbit placenta barrier using a novel MDCKII in vitro model.

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    Halwachs, Sandra; Schäfer, Ingo; Kneuer, Carsten; Seibel, Peter; Honscha, Walther

    2016-08-15

    In humans, the ATP-binding cassette efflux transporter ABCG2 contributes to the fetoprotective barrier function of the placenta, potentially limiting the toxicity of transporter substrates to the fetus. During testing of chemicals including pesticides, developmental toxicity studies are performed in rabbit. Despite its toxicological relevance, ABCG2-mediated transport of pesticides in rabbit placenta has not been yet elucidated. We therefore generated polarized MDCK II cells expressing the ABCG2 transporter from rabbit placenta (rbABCG2) and evaluated interaction of the efflux transporter with selected insecticides, fungicides, and herbicides. The Hoechst H33342 accumulation assay indicated that 13 widely used pesticidal active substances including azoxystrobin, carbendazim, chlorpyrifos, chlormequat, diflufenican, dimethoate, dimethomorph, dithianon, ioxynil, methiocarb, propamocarb, rimsulfuron and toclofos-methyl may be rbABCG2 inhibitors and/or substrates. No such evidence was obtained for chlorpyrifos-methyl, epoxiconazole, glyphosate, imazalil and thiacloprid. Moreover, chlorpyrifos (CPF), dimethomorph, tolclofos-methyl and rimsulfuron showed concentration-dependent inhibition of H33342 excretion in rbABCG2-transduced MDCKII cells. To further evaluate the role of rbABCG2 in pesticide transport across the placenta barrier, we generated polarized MDCKII-rbABCG2 monolayers. Confocal microscopy confirmed correct localization of rbABCG2 protein in the apical plasma membrane. In transepithelial flux studies, we showed the time-dependent preferential basolateral to apical (B>A) directed transport of [(14)C] CPF across polarized MDCKII-rbABCG2 monolayers which was significantly inhibited by the ABCG2 inhibitor fumitremorgin C (FTC). Using this novel in vitro cell culture model, we altogether showed functional secretory activity of the ABCG2 transporter from rabbit placenta and identified several pesticides like the insecticide CPF as potential rbABCG2 substrates

  20. Selective Increase of Two ABC Drug Efflux Transporters at the Blood-Spinal Cord Barrier Suggests Induced Pharmacoresistance in ALS

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    Jablonski, Michael R.; Jacob, Dena A.; Campos, Christopher; Miller, David S.; Maragakis, Nicholas J.; Pasinelli, Piera; Trotti, Davide

    2012-01-01

    ATP-binding cassette (ABC) drug efflux transporters in the CNS are predominantly localized to the luminal surface of endothelial cells in capillaries to impede CNS accumulation of xenobiotics. Inflammatory mediators and cellular stressors regulate their activity. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of upper and lower motor neurons characterized by extensive neuroinflammation. Here we tested the hypothesis that disease-driven changes in ABC transporter expression and function occur in ALS. Given the multitude of ABC transporters with their widespread substrate recognition, we began by examining expression levels of several ABC transporters. We found a selective increase in only two transporters; P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) both at mRNA and protein levels, in the SOD1-G93A mouse model of ALS, specifically in disease-affected CNS regions. Detailed analysis revealed a similar disease-driven increase in P-gp and BCRP levels in spinal cord microvessels, indicating that their altered expression occurs at the blood spinal cord barrier. Transport activity of P-gp and BCRP increased with disease progression in spinal cord and cerebral cortex capillaries. Finally, P-gp and BCRP protein expression also increased in spinal cords of ALS patients. Preclinical drug trials in the mouse model of ALS have failed to decisively slow or arrest disease progression; pharmacoresistance imparted by ABC transporters is one possible explanation for these failures. Our observations have large implications for ALS therapeutics in humans and suggest that the obstacle provided by these transporters to drug treatments must be overcome to develop effective ALS pharmacotherapies. PMID:22521463

  1. 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.

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    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Expression pattern of the human ABC transporters in pluripotent embryonic stem cells and in their derivatives.

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    Erdei, Zsuzsa; Lőrincz, Réka; Szebényi, Kornélia; Péntek, Adrienn; Varga, Nóra; Likó, István; Várady, György; Szakács, Gergely; Orbán, Tamás I; Sarkadi, Balázs; Apáti, Agota

    2014-09-01

    ATP-binding cassette (ABC) transporters have key roles in various physiological functions as well as providing chemical defense and stress tolerance in human tissues. In this study, we have examined the expression pattern of all ABC proteins in pluripotent human embryonic stem cells (hESCs) and in their differentiated progenies. We paid special attention to the cellular expression and localization of multidrug transporter ABC proteins. Stem cell differentiation was carried out without chemical induction or cell sorting, and specialized cell types were separated mechanically. Cellular features regarding pluripotency and tissue identity, as well as ABC transporter expression were studied by flow cytomtery, immuno-microscopy, and qPCR-based low-density arrays. Pluripotent hESCs and differentiated cell types (cardiomyocytes, neuronal cells, and mesenchymal stem cells) were distinguished by morphology, immunostaining markers, and selected mRNA expression patterns. We found that the mRNA expression levels of the 48 human ABC proteins also clearly distinguished the pluripotent and the respective differentiated cell types. When multidrug and lipid transporter ABC protein expression was examined by using well characterized specific antibodies by flow cytometry and confocal microscopy, the protein expression data corresponded well to the mRNA expression results. Moreover, the cellular localization of these important human ABC transporter proteins could be established in the pluripotent and differentiated hESC derived samples. These studies provide valuable information regarding ABC protein expression in human stem cells and their differentiated offspring. The results may also help to obtain further information concerning the specialized cellular functions of selected ABC transporters. © 2014 Clinical Cytometry Society.

  3. 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.

  4. Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP 4 and breast cancer resistance protein (BCRP

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    Rhiannon N. Hardwick

    2016-12-01

    Full Text Available Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI and aurora (AKI kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC transporters such as P-glycoprotein and breast cancer resistance protein (BCRP, which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs, remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of

  5. Quercetin improves macrophage reverse cholesterol transport in apolipoprotein E-deficient mice fed a high-fat diet.

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    Cui, Yingjie; Hou, Pengbo; Li, Fahui; Liu, Qinghua; Qin, Shucun; Zhou, Guanghai; Xu, Xuelian; Si, Yanhong; Guo, Shoudong

    2017-01-14

    Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to reduce hyperlipidaemia and atherosclerotic lesion formation. Reverse cholesterol transport (RCT) plays a crucial role in exporting cholesterol from peripheral cells, which is one mechanism utilized in the prevention and treatment of atherosclerosis. The aim of this study is to investigate whether quercetin reduces lipid accumulation by improving RCT in vivo. Apolipoprotein E-deficient mice fed a high-fat diet were used to investigate the effect of quercetin on RCT by an isotope tracing method, and the underlying mechanisms were clarified by molecular techniques. These novel results demonstrated that quercetin significantly improved [(3)H]-cholesterol transfer from [(3)H]-cholesterol-loaded macrophages to the plasma (approximately 34% increase), liver (30% increase), and bile (50% increase) and finally to the feces (approximately 40% increase) for excretion in apolipoprotein E-deficient mice fed a high-fat diet. Furthermore, quercetin markedly increased the cholesterol accepting ability of plasma and high-density lipoprotein (HDL) and dramatically decreased the content of malondialdehyde in plasma and oxidized phosphocholine carried by HDL. Therefore, the underlying mechanisms of quercetin in improving RCT may be partially due to the elevated cholesterol accepting ability of HDL, the increased expression levels of proteins related to RCT, such as ATP-binding cassettes (ABC) A1 and G1, and the improved antioxidant activity of HDL. Quercetin accelerates RCT in an atherosclerosis model, which is helpful in clarifying the lipid-lowering effect of quercetin.

  6. Crystallization and preliminary X-ray crystallographic analysis of BxlE, a xylobiose transporter from Streptomyces thermoviolaceus OPC-520

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    Seike, Kiho; Sato, Junji; Tomoo, Koji; Ishida, Toshimasa; Yamano, Akihito; Ikenishi, Sadao; Miyamoto, Katsushiro; Tsujibo, Hiroshi

    2007-01-01

    Together with the integral membrane proteins BxlF and BxlG, BxlE isolated from Streptomyces thermoviolaceus OPC-520 forms an ATP-binding cassette (ABC) transport system that mediates the uptake of xylan. To clarify the structural basis of sugar binding by BxlE at the atomic level, recombinant BxlE was crystallized using the hanging-drop vapour-diffusion method at 290 K. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 44.63, b = 63.27, c = 66.40 Å, β = 103.05°, and contained one 48 kDa molecule per asymmetric unit (V M = 1.96 Å3 Da−1). Diffraction data collected to a resolution of 1.65 Å using a rotating-anode X-ray source gave a data set with an overall R merge of 2.6% and a completeness of 91.3%. A data set from a platinum derivative is being used for phasing by the SAD method. PMID:17620710

  7. Cognition, learning behaviour and hippocampal synaptic plasticity are not disrupted in mice over-expressing the cholesterol transporter ABCG1.

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    Parkinson, Pamela F; Kannangara, Timal S; Eadie, Brennan D; Burgess, Braydon L; Wellington, Cheryl L; Christie, Brian R

    2009-02-24

    Cognitive deficits are a hallmark feature of both Down Syndrome (DS) and Alzheimer's Disease (AD). Extra copies of the genes on chromosome 21 may also play an important role in the accelerated onset of AD in DS individuals. Growing evidence suggests an important function for cholesterol in the pathogenesis of AD, particularly in APP metabolism and production of A beta peptides. The ATP-Binding Cassette-G1 (ABCG1) transporter is located on chromosome 21, and participates in the maintenance of tissue cholesterol homeostasis. To assess the role of ABCG1 in DS-related cognition, we evaluated the cognitive performance of mice selectively over-expressing the ABCG1 gene from its endogenous regulatory signals. Both wild-type and ABCG1 transgenic mice performed equivalently on several behavioral tests, including measures of anxiety, as well as on reference and working memory tasks. No deficits in hippocampal CA1 synaptic plasticity as determined with electrophysiological studies were apparent in mice over-expressing ABCG1. These findings indicate that although ABCG1 may play a role in maintaining cellular or tissue cholesterol homeostasis, it is unlikely that excess ABCG1 expression contributes to the cognitive deficits in DS individuals.

  8. Characterization of immortalized choroid plexus epithelial cell lines for studies of transport processes across the blood-cerebrospinal fluid barrier

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    Kläs Juliane

    2010-08-01

    Full Text Available Abstract Background Two rodent choroid plexus (CP epithelial cell lines, Z310 and TR-CSFB, were compared with primary rat CP epithelial cells and intact CP tissue with respect to transport protein expression, function and tight junction (TJ formation. Methods For expression profiles of transporters and TJ proteins, qPCR and western blot analysis were used. Uptake assays were performed to study the functional activity of transporters and TJ formation was measured by trans-epithelial electrical resistance (TEER and visualized by electron microscopy. Results The expression of known ATP-binding cassette (Abc transporter and solute carrier (Slc genes in CP was confirmed by qPCR. Primary cells and cell lines showed similar, but overall lower expression of Abc transporters and absent Slc expression when compared to intact tissue. Consistent with this Mrp1, Mrp4 and P-gp protein levels were higher in intact CP compared to cell lines. Functionality of P-gp and Mrp1 was confirmed by Calcein-AM and CMFDA uptake assays and studies using [3H]bis-POM-PMEA as a substrate indicated Mrp4 function. Cell lines showed low or absent TJ protein expression. After treatment of cell lines with corticosteroids, RNA expression of claudin1, 2 and 11 and occludin was elevated, as well as claudin1 and occludin protein expression. TJ formation was further investigated by freeze-fracture electron microscopy and only rarely observed. Increases in TJ particles with steroid treatment were not accompanied by an increase in transepithelial electrical resistance (TEER. Conclusion Taken together, immortalized cell lines may be a tool to study transport processes mediated by P-gp, Mrp1 or Mrp4, but overall expression of transport proteins and TJ formation do not reflect the situation in intact CP tissue.

  9. Tissue-specific transcript profiling for ABC transporters in the sequestering larvae of the phytophagous leaf beetle Chrysomela populi.

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    Anja S Strauss

    Full Text Available BACKGROUND: Insects evolved ingenious adaptations to use extraordinary food sources. Particularly, the diet of herbivores enriched with noxious plant secondary metabolites requires detoxification mechanisms. Sequestration, which involves the uptake, transfer, and concentration of occasionally modified phytochemicals into specialized tissues or hemolymph, is one of the most successful detoxification strategies found in most insect orders. Due to the ability of ATP-binding cassette (ABC carriers to transport a wide range of molecules including phytochemicals and xenobiotics, it is highly likely that they play a role in this sequestration process. To shed light on the role of ABC proteins in sequestration, we describe an inventory of putative ABC transporters in various tissues in the sequestering juvenile poplar leaf beetle, Chrysomela populi. RESULTS: In the transcriptome of C. populi, we predicted 65 ABC transporters. To link the proteins with a possible function, we performed comparative phylogenetic analyses with ABC transporters of other insects and of humans. While tissue-specific profiling of each ABC transporter subfamily suggests that ABCB, C and G influence the plant metabolite absorption in the gut, ABCC with 14 members is the preferred subfamily responsible for the excretion of these metabolites via Malpighian tubules. Moreover, salicin, which is sequestered from poplar plants, is translocated into the defensive glands for further deterrent production. In these glands and among all identified ABC transporters, an exceptionally high transcript level was observed only for Cpabc35 (Cpmrp. RNAi revealed the deficiency of other ABC pumps to compensate the function of CpABC35, demonstrating its key role during sequestration. CONCLUSION: We provide the first comprehensive phylogenetic study of the ABC family in a phytophagous beetle species. RNA-seq data from different larval tissues propose the importance of ABC pumps to achieve a

  10. Inactivation of the Ecs ABC transporter of Staphylococcus aureus attenuates virulence by altering composition and function of bacterial wall.

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    Ing-Marie Jonsson

    2010-12-01

    Full Text Available Ecs is an ATP-binding cassette (ABC transporter present in aerobic and facultative anaerobic gram-positive Firmicutes. Inactivation of Bacillus subtilis Ecs causes pleiotropic changes in the bacterial phenotype including inhibition of intramembrane proteolysis. The molecule(s transported by Ecs is (are still unknown.In this study we mutated the ecsAB operon in two Staphylococcus aureus strains, Newman and LS-1. Phenotypic and functional characterization of these Ecs deficient mutants revealed a defect in growth, increased autolysis and lysostaphin sensitivity, altered composition of cell wall proteins including the precursor form of staphylokinase and an altered bacterial surface texture. DNA microarray analysis indicated that the Ecs deficiency changed expression of the virulence factor regulator protein Rot accompanied by differential expression of membrane transport proteins, particularly ABC transporters and phosphate-specific transport systems, protein A, adhesins and capsular polysaccharide biosynthesis proteins. Virulence of the ecs mutants was studied in a mouse model of hematogenous S. aureus infection. Mice inoculated with the ecs mutant strains developed markedly milder infections than those inoculated with the wild-type strains and had consequently lower mortality, less weight loss, milder arthritis and decreased persistence of staphylococci in the kidneys. The ecs mutants had higher susceptibility to ribosomal antibiotics and plant alkaloids chelerythrine and sanguinarine.Our results show that Ecs is essential for staphylococcal virulence and antimicrobial resistance probably since the transport function of Ecs is essential for the normal structure and function of the cell wall. Thus targeting Ecs may be a new approach in combating staphylococcal infection.

  11. The ABC transporter gene family of Daphnia pulex

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    Cunningham Phil

    2009-04-01

    Full Text Available Abstract Background The large gene superfamily of ABC (ATP-binding cassette transporters encodes membrane proteins involved in trafficking processes across biological membranes and further essential cell biological functions. ABC transporters are evolutionary ancient and involved in the biochemical defence against toxicants. We report here a genome-wide survey of ABC proteins of Daphnia pulex, providing for the first time information on ABC proteins in crustacea, a primarily aquatic arthropod subphylum of high ecological and economical importance. Results We identified 64 ABC proteins in the Daphnia genome, which possesses members of all current ABC subfamilies A to H. To unravel phylogenetic relationships, ABC proteins of Daphnia were compared to those from yeast, worm, fruit fly and human. A high conservation of Daphnia of ABC transporters was observed for proteins involved in fundamental cellular processes, including the mitochondrial half transporters of the ABCB subfamily, which function in iron metabolism and transport of Fe/S protein precursors, and the members of subfamilies ABCD, ABCE and ABCF, which have roles in very long chain fatty acid transport, initiation of gene transcription and protein translation, respectively. A number of Daphnia proteins showed one-to-one orthologous relationships to Drosophila ABC proteins including the sulfonyl urea receptor (SUR, the ecdysone transporter ET23, and the eye pigment precursor transporter scarlet. As the fruit fly, Daphnia lacked homologues to the TAP protein, which plays a role in antigene processing, and the cystic fibrosis transmembrane conductance regulator (CFTR, which functions as a chloride channel. Daphnia showed two proteins homologous to MDR (multidrug resistance P-glycoproteins (ABCB subfamily and six proteins homologous to MRPs (multidrug resistance-associated proteins (ABCC subfamily. However, lineage specific gene duplications in the ABCB and ABCC subfamilies complicated the

  12. A new ABC half-transporter in Leishmania major is involved in resistance to antimony.

    Science.gov (United States)

    Manzano, J I; García-Hernández, R; Castanys, S; Gamarro, F

    2013-08-01

    The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

  13. Conserved ABC Transport System Regulated by the General Stress Response Pathways of Alpha- and Gammaproteobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Herrou, Julien; Willett, Jonathan W.; Czyz, Daniel M.; Babnigg, Gyorgy; Kim, Youngchang; Crosson, Sean

    2017-03-01

    Brucella abortus sigma(E1) is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon, bab1_0223-bab1_0226, is among the most highly activated gene sets in the sigma(E1) regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription of yehZYXW is activated by the general stress sigma factor sigma(S) in Enterobacteriaceae, which suggests a functional role for this transport system in bacterial stress response across the classes Alphaproteobacteria and Gammaproteobacteria. We present evidence that B. abortus YehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a sigma(E1)- null strain. The sole in vitro phenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li+ ion concentrations. A crystal structure of B. abortus YehZ revealed a class II periplasmic binding protein fold with significant structural homology to Archaeoglobus fulgidus ProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers

  14. Isolation and Characterization of the Colletotrichum acutatum ABC Transporter CaABC1

    Directory of Open Access Journals (Sweden)

    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.

  15. Isolation and Characterization of the Colletotrichum acutatum ABC Transporter CaABC1.

    Science.gov (United States)

    Kim, Suyoung; Park, Sook-Young; Kim, Hyejeong; Kim, Dongyoung; Lee, Seon-Woo; Kim, Heung Tae; Lee, Jong-Hwan; Choi, Woobong

    2014-12-01

    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.

  16. 27-Hydroxycholesterol regulates cholesterol synthesis and transport in C6 glioma cells.

    Science.gov (United States)

    An, Yu; Zhang, Dan-Di; Yu, Huan-Ling; Ma, Wei-Wei; Lu, Yan-Hui; Liu, Quan-Ri; Xiao, Rong

    2017-03-01

    The oxysterol 27-Hydroxycholesterol (27-OHC) is a major cholesterol metabolite that can cross the blood brain barrier (BBB) from peripheral circulation to the brain. Currently, the role of 27-OHC on cholesterol homeostasis in astrocytes and the underlying mechanisms are not defined. Since all brain cholesterol is essentially synthesized in brain itself and astrocytes as net producers of cholesterol are essential for normal brain function, here we investigated the effects of 27-OHC on cholesterol synthesis and transport in C6 glioma cells. C6 cells were treated with 5, 10 and 20μM 27-OHC for 24h and the cell viability and apoptosis, the cholesterol levels and metabolism-related mediators, genes and proteins were subsequently assessed using cell-counting kit (CCK)-8, Amplex red, ELISA, real-time PCR and Western blot, respectively. We found that 27-OHC decreased cholesterol levels by down-regulating the expression of sterol-regulated element binding protein-1 (SREBP-1a), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CR) and low density lipoprotein receptor (LDLR) and promoted cholesterol transport by up-regulating the expression of peroxisome proliferator-activated receptors-γ (PPAR-γ), liver X receptor-α (LXR-α), ATP-binding cassette transporter protein family member A1 (ABCA1) and apolipoprotein E (ApoE)genes. Our results suggested that 27-OHC may represent a sensitive modulator of cholesterol metabolism disorder by suppressing cholesterol synthesis and stimulating cholesterol transport in astrocytes. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Interactions of cyclin-dependent kinase inhibitors AT-7519, flavopiridol and SNS-032 with ABCB1, ABCG2 and ABCC1 transporters and their potential to overcome multidrug resistance in vitro.

    Science.gov (United States)

    Cihalova, Daniela; Staud, Frantisek; Ceckova, Martina

    2015-07-01

    ATP-binding cassette (ABC) transporters play an important role in multidrug resistance (MDR) toward anticancer drugs. Here, we evaluated interactions of cyclin-dependent kinase inhibitors (CDKi) AT-7519, flavopiridol and SNS-032 with the following ABC transporters in vitro: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated protein 1 (ABCC1). Inhibitory potency of studied CDKi to the transporters was evaluated by accumulation assays using fluorescent substrates and MDCKII cells overexpressing human ABCB1, ABCG2 or ABCC1. Resistance of transporter-expressing cells to the CDKi was evaluated by XTT proliferation assay. Observed interactions of CDKi were verified by ATPase assay in ABC transporter-expressing Sf9 membrane vesicles. Combination index analysis was additionally performed in ABC transporter-expressing cancer cell lines, HepG2 and T47D. Flavopiridol showed a significant inhibitory potency toward ABCG2 and ABCC1. SNS-032 also decreased ABCG2-mediated efflux, while AT-7519 failed to inhibit ABCB1, ABCG2 or ABCC1. Both flavopiridol and SNS-032 showed synergistic antiproliferative effects in combination with relevant ABC transporter substrates such as daunorubicin and topotecan in cancer cells. ABCB1 was found to confer significant resistance to AT-7519 and SNS-032, but not to flavopiridol. In contrast, ABCG2 and ABCC1 conferred resistance to flavopiridol, but not to AT-7519 and SNS-032. Our data provide detailed information on interactions of flavopiridol, SNS-032 and AT-7519 with ABC transporters, which may help elucidate the pharmacokinetic behavior and toxicity of these compounds. Moreover, we show the ability of flavopiridol and SNS-032, but not AT-7519, to overcome ABC transporter-mediated MDR.

  18. Phosphorylation at Ser²⁶ in the ATP-binding site of Ca²⁺/calmodulin-dependent kinase II as a mechanism for switching off the kinase activity.

    Science.gov (United States)

    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.

  19. CryoEM and Molecular Dynamics of the Circadian KaiB-KaiC Complex Indicates That KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. The ABCG2 Efflux Transporter in the Mammary Gland Mediates Veterinary Drug Secretion across the Blood-Milk Barrier into Milk of Dairy Cows.

    Science.gov (United States)

    Mahnke, Hanna; Ballent, Mariana; Baumann, Sven; Imperiale, Fernanda; von Bergen, Martin; Lanusse, Carlos; Lifschitz, Adrian L; Honscha, Walther; Halwachs, Sandra

    2016-05-01

    In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2 excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2 transport across polarized Madin-Darby canine kidney II cells-bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2(B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  1. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier.

    Science.gov (United States)

    Li, Yangfang; Wu, Qian; Li, Chen; Liu, Ling; Du, Kun; Shen, Jin; Wu, Yuqin; Zhao, Xiaofen; Zhao, Mei; Bao, Lingyun; Gao, Jin; Keep, Richard F; Xiang, Jianming

    2016-01-01

    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.

  2. Gene expression of ABC transporters in Cooperia oncophora after field and laboratory selection with macrocyclic lactones.

    Science.gov (United States)

    Tydén, Eva; Skarin, Moa; Höglund, Johan

    2014-12-01

    The most widespread helminth parasites of grazing cattle in northern Europe are the gastrointestinal nematodes Ostertagia ostertagi and Cooperia oncophora. Heavy reliance on the use of macrocyclic lactone (ML) in cattle has led to world-wide emergence of resistance to this drug class in C. oncophora. There is evidence that members of the ATP-binding cassette (ABC) transporter family, such as P-glycoproteins (P-gp) and multidrug-resistant proteins (MRP), play a role in resistance to ML. In this study gene expression of Con-pgp9, Con-pgp11, Con-pgp12, Con-pgp16 and Con-mrp1 was examined in two isolates of C. oncophora sharing the same genetic background but exposed to ML differently. For isolate one (Laboratory-selected), adult worms were recovered before and after treatment with ML in vivo. For isolate two (Field-selected), adult worms were collected from tracer animals that had never received anthelmintics themselves. One group grazed together with untreated animals and one group grazed with animals that received suppressive prophylactic treatment with ML at monthly intervals for up to two consecutive grazing seasons. Real-time PCR data demonstrated differences in gene expression after ML selection, with the highest constitutive expression levels for Con-pgp16 and Con-mrp1. Remarkably, the same pattern of increasing expression levels of the ABC transport genes was observed in both Laboratory- and Field-selected isolates, despite the Field-selected isolate not being directly exposed to ML. The higher expression levels of ABC transporters observed in the Field-selected isolate was thus not a response to direct exposure to ML, but rather appeared to reflect a genetic characteristic inherited from worms in the previous generation which had survived exposure to ML in the co-grazing treated animals. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. abc3+ encodes an iron-regulated vacuolar ABC-type transporter in Schizosaccharomyces pombe.

    Science.gov (United States)

    Pouliot, Benoît; Jbel, Mehdi; Mercier, Alexandre; Labbé, Simon

    2010-01-01

    Studies have shown the fundamental contribution of the yeast vacuole as a site for storage and detoxification of metals. Whereas the transmembrane proteins responsible for iron transport into and out of the vacuole have been identified in Saccharomyces cerevisiae, less information is available concerning the mobilization of vacuolar iron stores in Schizosaccharomyces pombe. In this study, we report the identification of a gene designated abc3(+) that encodes a protein which exhibits sequence homology with the ABCC subfamily of ATP-binding cassette transporters. The transcription of abc3(+) is induced by low concentrations of iron but repressed by high levels of iron. The iron-mediated repression of abc3(+) required a functional fep1(+) gene. Chromatin immunoprecipitation assays showed that Fep1 associates with the abc3(+) promoter in vivo, in an iron-dependent manner. Microscopic analyses revealed that a functional Abc3-green fluorescent protein localizes to the membrane vacuole when iron levels were low. Abc3 was required for growth in low-iron medium in the absence of the transport system mediated by Fio1 and Fip1. abc3Delta cells exhibited increased levels of expression of the frp1(+)-encoded ferric reductase, suggesting a loss of Fep1 repression and, consequently, the activation of Fep1-regulated genes. When abc3(+) was expressed using the nmt1(+) promoter system, its induction led to a reduced transcriptional activity of the frp1(+) gene. Because S. pombe does not possess vacuolar membrane-localized orthologs to S. cerevisiae Fth1, Fet5, and Smf3, our findings suggested that Abc3 may be responsible for mobilizing stored iron from the vacuole to the cytosol in response to iron deficiency.

  4. abc3+ Encodes an Iron-Regulated Vacuolar ABC-Type Transporter in Schizosaccharomyces pombe▿

    Science.gov (United States)

    Pouliot, Benoît; Jbel, Mehdi; Mercier, Alexandre; Labbé, Simon

    2010-01-01

    Studies have shown the fundamental contribution of the yeast vacuole as a site for storage and detoxification of metals. Whereas the transmembrane proteins responsible for iron transport into and out of the vacuole have been identified in Saccharomyces cerevisiae, less information is available concerning the mobilization of vacuolar iron stores in Schizosaccharomyces pombe. In this study, we report the identification of a gene designated abc3+ that encodes a protein which exhibits sequence homology with the ABCC subfamily of ATP-binding cassette transporters. The transcription of abc3+ is induced by low concentrations of iron but repressed by high levels of iron. The iron-mediated repression of abc3+ required a functional fep1+ gene. Chromatin immunoprecipitation assays showed that Fep1 associates with the abc3+ promoter in vivo, in an iron-dependent manner. Microscopic analyses revealed that a functional Abc3-green fluorescent protein localizes to the membrane vacuole when iron levels were low. Abc3 was required for growth in low-iron medium in the absence of the transport system mediated by Fio1 and Fip1. abc3Δ cells exhibited increased levels of expression of the frp1+-encoded ferric reductase, suggesting a loss of Fep1 repression and, consequently, the activation of Fep1-regulated genes. When abc3+ was expressed using the nmt1+ promoter system, its induction led to a reduced transcriptional activity of the frp1+ gene. Because S. pombe does not possess vacuolar membrane-localized orthologs to S. cerevisiae Fth1, Fet5, and Smf3, our findings suggested that Abc3 may be responsible for mobilizing stored iron from the vacuole to the cytosol in response to iron deficiency. PMID:19915076

  5. Attenuation of high sucrose diet-induced insulin resistance in ABC transporter deficient white mutant of Drosophila melanogaster.

    Science.gov (United States)

    Navrotskaya, Valeriya; Oxenkrug, Gregory; Vorobyova, Lyudmila; Summergrad, Paul

    2016-03-01

    Exposure to high sugar diet (HSD) is an experimental model of insulin resistance (IR) and type 2 diabetes (T2D) in mammals and insects. In Drosophila, HSD-induced IR delays emergence of pupae from larvae and eclosion of imago from pupae. Understanding of mechanisms of IR/T2D is essential for refining T2D prevention and treatment strategies. Dysregulation of tryptophan (Trp)-kynurenine (Kyn) pathway was suggested as one of the mechanisms of IR/T2D development. Rate-limiting enzyme of Trp-Kyn pathway in Drosophila is Trp 2,3-dioxygenase (TDO), an evolutionary conserved ortholog of human TDO. We previously reported attenuation of HSD-induced IR in vermilion mutants with inactive TDO. Conversion of Trp to Kyn is regulated not only by TDO activity but by intracellular Trp transport via ATP-binding cassette (ABC) transporter encoded by white gene in Drosophila. In order to evaluate the possible impact of deficient intracellular Trp transport on the inducement of IR by HSD, we compared the effect of HSD on pre-imago development in wild type flies, Canton-Special (C-S), and C-S flies containing white gene, white (C-S). Presence of white gene attenuated (by 50%) HSD-induced delay of pupae emergence from larvae and female and male imago eclosion from pupae. Present study together with our earlier report reveals that both decreased TDO activity (due to vermilion gene mutation) or deficient Trp transport into cell without affecting TDO levels (due to white gene mutation) attenuate HSD-induced development of IR in Drosophila model of T2D. Our data provide further support for hypothesis that dysregulation of Trp-Kyn pathway is one of the pathophysiological mechanisms and potential target for early diagnosis, prevention and treatment of IR/T2D.

  6. Gene expression modulation of ABC transporter genes in response to permethrin in adults of the mosquito malaria vector Anopheles stephensi.

    Science.gov (United States)

    Mastrantonio, Valentina; Ferrari, Marco; Epis, Sara; Negri, Agata; Scuccimarra, Giulia; Montagna, Matteo; Favia, Guido; Porretta, Daniele; Urbanelli, Sandra; Bandi, Claudio

    2017-07-01

    Living organisms have evolved an array of genes coding for detoxifying enzymes and efflux protein pumps, to cope with endogenous and xenobiotic toxic compounds. The study of the genes activated during toxic exposure is relevant to the area of arthropod vector control, since these genes are one of the targets upon which natural selection acts for the evolution of insecticide resistance. ATP-binding cassette (ABC) transporters participate to insecticide detoxification acting as efflux pumps, that reduce the intracellular concentration of toxic compounds, or of their metabolic derivatives. Here we analyzed the modulation of the expression of six genes coding for ABC transporters, after the exposure of adult females and males of the mosquito Anopheles stephensi, a major malaria vector in Asia, to permethrin. Male and female mosquitoes were exposed to insecticide for one hour, then the expression profiles of the ABC transporter genes AnstABCB2, AnstABCB3, AnstABCB4, AnstABCBmember6, AnstABCC11, and AnstABCG4 were analysed after one and 24h. Our results showed that three genes (AnstABCB2, AnstABCBmember6, AnstABCG4) were up-regulated in both sexes; two of these (AnstABCBmember6 and AnstABCG4) have previously been shown to be up-regulated also in larval stages of An. stephensi, supporting a role for these genes in permethrin defence in larvae as well as in adults. Finally, the same ABC transporter genes were activated both in females and males; however, the timing of gene induction was different, with a prompter induction in females than in males. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Finger-Actuated, Self-Contained Immunoassay Cassettes

    OpenAIRE

    Qiu, Xianbo; Thompson, Jason A.; Chen, Zongyuan; Liu, Changchun; Chen, Dafeng; Ramprasad, Sudhir; Mauk, Michael G; Ongagna, Serge; Barber, Cheryl; Abrams, William R.; Malamud, Daniel; Paul L A M Corstjens; Bau, Haim H.

    2009-01-01

    The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering ...

  8. Regulation of human hepatic drug transporter activity and expression by diesel exhaust particle extract.

    Directory of Open Access Journals (Sweden)

    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

  9. Fructus Gardenia Extract Ameliorates Oxonate-Induced Hyperuricemia with Renal Dysfunction in Mice by Regulating Organic Ion Transporters and mOIT3

    Directory of Open Access Journals (Sweden)

    Hui Ji

    2013-07-01

    Full Text Available The potent anti-hyperuricemia activities of Fructus Gardenia Extract (FGE have been well reported. The aim of this study was to evaluate the uricosuric and nephro-protective effects of FGE and explore its possible mechanisms of action in oxonate-induced hyperuricemic mice. FGE was orally administered to hyperuricemic and normal mice for 1 week. Serum and urinary levels of uric acid, creatinine and blood urea nitrogen (BUN, and fractional excretion of uric acid (FEUA were measured. The mRNA and protein levels of mouse urate transporter 1 (mURAT1, glucose transporter 9 (mGLUT9, ATP-binding cassette, subfamily G, 2 (mABCG2, organic anion transporter 1 (mOAT1, mOAT3, oncoprotein induced transcript 3 (mOIT3, organic cation/carnitine transporters in the kidney were analyzed. Simultaneously, Tamm-Horsfall glycoprotein (THP levels in urine and kidney were detected. FGE significantly reduced serum urate levels and increased urinary urate levels and FEUA in hyperuricemic mice. It could also effectively reverse oxonate-induced alterations in renal mURAT1, mGLUT9, mOAT1 and mOIT3 expressions, as well as THP levels, resulting in the enhancement of renal uric acid excretion. Moreover, FGE decreased serum creatinine and BUN levels, and up-regulated expression of organic cation/carnitine transporters, improving renal dysfunction in this model. Furthermore, FGE decreased renal mABCG2 expressions in hyperuricemic mice, contributing to its beneficial actions. However, further investigation is needed in clinical trials of FGE and its bioactive components.

  10. Gene expression analysis of ABC transporters in a resistant Cooperia oncophora isolate following in vivo and in vitro exposure to macrocyclic lactones.

    Science.gov (United States)

    De Graef, J; Demeler, J; Skuce, P; Mitreva, M; Von Samson-Himmelstjerna, G; Vercruysse, J; Claerebout, E; Geldhof, P

    2013-04-01

    Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

  11. Role of ABCG2 in transport of the mammalian lignan enterolactone and its secretion into milk in Abcg2 knockout mice.

    Science.gov (United States)

    Miguel, Verónica; Otero, Jon Andoni; García-Villalba, Rocío; Tomás-Barberán, Francisco; Espín, Juan Carlos; Merino, Gracia; Álvarez, Ana I

    2014-05-01

    Lignans are phytoestrogens that are metabolized by the gut microbiota to enterodiol and enterolactone, the main biologically active enterolignans. Substantial interindividual variation in plasma concentration and urinary excretion of enterolignans has been reported, this being determined, at least in part, by the intake of lignan precursors, the gut microbiota, and the host's phase 2 conjugating enzyme activity. However, the role of ATP-binding cassette (ABC) transporters in the transport and disposition of enterolactone has not been reported so far. Active transport assays using parental and Madin-Darby canine kidney epithelial cells transduced with murine and human ABCG2 showed a significant increase in apically directed translocation of enterolactone in transduced cells, which was confirmed by using the selective ABCG2 inhibitor Ko143. In addition, enterolactone also inhibited transport of the antineoplastic agent mitoxantrone as a model substrate, with inhibition percentages of almost 40% at 200 μM for human ABCG2. Furthermore, the endogenous levels in plasma and milk of enterolactone in wild-type and Abcg2((-/-)) knockout female mice were analyzed. The milk/plasma ratio decreased significantly in the Abcg2((-/-)) phenotype, as compared with the wild-type mouse group (0.4 ± 0.1 as against 6.4 ± 2.6). This paper is the first to report that enterolactone is a transported substrate and therefore most probably a competitive inhibitor of ABCG2, which suggests it has a role in the interindividual variations in the disposition of enterolactone and its secretion into milk. The inhibitory activity identified provides a solid basis for further investigation in possible food-drug interactions.

  12. Detergent screening and purification of the human liver ABC transporters BSEP (ABCB11 and MDR3 (ABCB4 expressed in the yeast Pichia pastoris.

    Directory of Open Access Journals (Sweden)

    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.

  13. ABC transporters are involved in defense against permethrin insecticide in the malaria vector Anopheles stephensi.

    Science.gov (United States)

    Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Comandatore, Francesco; Sassera, Davide; Rossi, Paolo; Cafarchia, Claudia; Otranto, Domenico; Favia, Guido; Genchi, Claudio; Bandi, Claudio; Urbanelli, Sandra

    2014-07-29

    Proteins from the ABC family (ATP-binding cassette) represent the largest known group of efflux pumps, responsible for transporting specific molecules across lipid membranes in both prokaryotic and eukaryotic organisms. In arthropods they have been shown to play a role in insecticide defense/resistance. The presence of ABC transporters and their possible association with insecticide transport have not yet been investigated in the mosquito Anopheles stephensi, the major vector of human malaria in the Middle East and South Asian regions. Here we investigated the presence and role of ABCs in transport of permethrin insecticide in a susceptible strain of this mosquito species. To identify ABC transporter genes we obtained a transcriptome from untreated larvae of An. stephensi and then compared it with the annotated transcriptome of Anopheles gambiae. To analyse the association between ABC transporters and permethrin we conducted bioassays with permethrin alone and in combination with an ABC inhibitor, and then we investigated expression profiles of the identified genes in larvae exposed to permethrin. Bioassays showed an increased mortality of mosquitoes when permethrin was used in combination with the ABC-transporter inhibitor. Genes for ABC transporters were detected in the transcriptome, and five were selected (AnstABCB2, AnstABCB3, AnstABCB4, AnstABCmember6 and AnstABCG4). An increased expression in one of them (AnstABCG4) was observed in larvae exposed to the LD50 dose of permethrin. Contrary to what was found in other insect species, no up-regulation was observed in the AnstABCB genes. Our results show for the first time the involvement of ABC transporters in larval defense against permethrin in An. stephensi and, more in general, confirm the role of ABC transporters in insecticide defense. The differences observed with previous studies highlight the need of further research as, despite the growing number of studies on ABC transporters in insects, the

  14. Fuel cell cassette with compliant seal

    Science.gov (United States)

    Karl, Haltiner, Jr. J.; Anthony, Derose J.; Klotzbach, Darasack C.; Schneider, Jonathan R.

    2017-11-07

    A fuel cell cassette for forming a fuel cell stack along a fuel cell axis includes a cell retainer, a plate positioned axially to the cell retainer and defining a space axially with the cell retainer, and a fuel cell having an anode layer and a cathode layer separated by an electrolyte layer. The outer perimeter of the fuel cell is positioned in the space between the plate and the cell retainer, thereby retaining the fuel cell and defining a cavity between the cell retainer, the fuel cell, and the plate. The fuel cell cassette also includes a seal disposed within the cavity for sealing the edge of the fuel cell. The seal is compliant at operational temperatures of the fuel cell, thereby allowing lateral expansion and contraction of the fuel cell within the cavity while maintaining sealing at the edge of the fuel cell.

  15. The role of the iron transporter ABCB7 in refractory anemia with ring sideroblasts.

    Directory of Open Access Journals (Sweden)

    Jacqueline Boultwood

    2008-04-01

    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.

  16. 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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. The role of sphingosine-1-phosphate transporter Spns2 in immune system function.

    Science.gov (United States)

    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.

  18. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    Science.gov (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  19. A novel two mode-acting inhibitor of ABCG2-mediated multidrug transport and resistance in cancer chemotherapy.

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2009-05-01

    Full Text Available Multidrug resistance (MDR is a major problem in successful treatment of cancers. Human ABCG2, a member of the ATP-binding cassette transporter superfamily, plays a key role in MDR and an important role in protecting cancer stem cells. Knockout of ABCG2 had no apparent adverse effect on the mice. Thus, ABCG2 is an ideal target for development of chemo-sensitizing agents for better treatment of drug resistant cancers and helping eradicate cancer stem cells.Using rational screening of representatives from a chemical compound library, we found a novel inhibitor of ABCG2, PZ-39 (N-(4-chlorophenyl-2-[(6-{[4,6-di(4-morpholinyl-1,3,5-triazin-2-yl]amino}-1,3-benzothiazol-2-ylsulfanyl]acetamide, that has two modes of actions by inhibiting ABCG2 activity and by accelerating its lysosome-dependent degradation. PZ-39 has no effect on ABCB1 and ABCC1-mediated drug efflux, resistance, and their expression, indicating that it may be specific to ABCG2. Analyses of its analogue compounds showed that the pharmacophore of PZ-39 is benzothiazole linked to a triazine ring backbone.Unlike any previously known ABCG2 transporter inhibitors, PZ-39 has a novel two-mode action by inhibiting ABCG2 activity, an acute effect, and by accelerating lysosome-dependent degradation, a chronic effect. PZ-39 is potentially a valuable probe for structure-function studies of ABCG2 and a lead compound for developing therapeutics targeting ABCG2-mediated MDR in combinational cancer chemotherapy.

  20. Disinfection Effect of Film Cassettes by Ultraviolet Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Kweon, Dae Cheol; Park, Peom [Ajou Univ., Suwon (Korea, Republic of)

    2001-12-15

    A bacteria infection on film cassette contact surface was examined at the diagnostic radiology department. Studies have demonstrated a bactericidal effect of ultraviolet irradiation, and to assess the contamination level on film cassette contact surface as a predictor of patient prevent from nosocomial infection. The study showed that the laboratory result was identified non-pathologic and pathologic bacterial in the five different cassette size of the contact surface. Film cassettes were exposed to ultraviolet light for 1, 2 and 3 minutes. Ultraviolet light disinfection practices suitable for bacteria. The study concludes that presence of a bacterial infection will prevent a using antiseptic technique on film cassette contact surface. In conclusion, ultraviolet irradiate on film cassette over the surface more than 2 minutes. Ultraviolet dose of 1565 {mu}W {center_dot} s/cm{sup 2}Win in 30 second relative to ultraviolet dose in time.

  1. 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.

  2. The multidrug ABC transporter BmrC/BmrD of Bacillus subtilis is regulated via a ribosome-mediated transcriptional attenuation mechanism.

    Science.gov (United States)

    Reilman, Ewoud; Mars, Ruben A T; van Dijl, Jan Maarten; Denham, Emma L

    2014-10-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Inhibition of ABCG2/BCRP transporter by soy isoflavones genistein and daidzein: effect on plasma and milk levels of danofloxacin in sheep.

    Science.gov (United States)

    Perez, Miriam; Otero, Jon A; Barrera, Borja; Prieto, Julio G; Merino, Gracia; Alvarez, Ana I

    2013-05-01

    Danofloxacin is a synthetic fluoroquinolone antibacterial agent and a substrate for ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). This protein actively extrudes drugs from cells in the intestine, liver, kidney, and other organs, such as the mammary gland. The purpose of this study was to determine whether genistein and daidzein, isoflavones present in soy and known inhibitors of ABCG2, could diminish danofloxacin secretion into milk. The results obtained from BCRP-transduced MDCK-II cells (Mardin-Darby canine kidney) showed that both isoflavones efficiently inhibited the in vitro transport of the drug. In addition, danofloxacin transport into milk was studied in Assaf sheep. The experimental design with ewes (n = 18) included ewes fed with standard forage, soy-enriched forage for 15 days prior to the experiment or standard forage paired with orally administered exogenous genistein and daidzein. The danofloxacin levels in the milk of ewes in the soy-enriched diet group were decreased. The area under concentration-time curve AUC (0-24 h) was 9.3 ± 4.6 vs. 16.58 ± 4.44 μgh/mL in the standard forage or control group. The plasma levels of danofloxacin were unmodified. The AUC (0-24 h) milk/plasma ratio decreased by over 50% in the soy-enriched diet group, compared to the control group (4.90 ± 2.65 vs. 9.58 ± 2.17). Exogenous administration of isoflavones did not modify danofloxacin secretion into milk. This study showed that milk excretion of a specific substrate of BCRP, such as danofloxacin, can be diminished by the presence of isoflavones in the diet. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Heteronuclear multidimensional NMR and homology modelling studies of the C-terminal nucleotide-binding domain of the human mitochondrial ABC transporter ABCB6

    Energy Technology Data Exchange (ETDEWEB)

    Kurashima-Ito, Kaori [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Ikeya, Teppei [National Institute of Advanced Industrial Science and Technology (AIST), (Japan); Senbongi, Hiroshi [Mitochondrial Diseases Group, MRC Dunn Human NutritionUnit (United Kingdom); Tochio, Hidehito [International Graduate School of Arts and Sciences, Supramolecular Biology, Yokohama City University, Molecular Biophysics Laboratory (Japan); Mikawa, Tsutomu [RIKEN, Cellular and Molecular Biology Laboratory (Japan); Shibata, Takehiko [RIKEN, Shibata Distinguished Senior Scientist Laboratory (Japan); Ito, Yutaka [RIKEN, Cellular and Molecular Biology Laboratory (Japan)], E-mail: ito-yutaka@center.tmu.ac.jp

    2006-05-15

    Human ATP-binding cassette, sub-family B, member 6 (ABCB6) is a mitochondrial ABC transporter, and presumably contributes to iron homeostasis. Aimed at understanding the structural basis for the conformational changes accompanying the substrate-transportation cycle, we have studied the C-terminal nucleotide-binding domain of ABCB6 (ABCB6-C) in both the nucleotide-free and ADP-bound states by heteronuclear multidimensional NMR and homology modelling. A non-linear sampling scheme was utilised for indirectly acquired {sup 13}C and {sup 15}N dimensions of all 3D triple-resonance NMR experiments, in order to overcome the instability and the low solubility of ABCB6-C. The backbone resonances for approximately 25% of non-proline residues, which are mostly distributed around the functionally important loops and in the Helical domain, were not observed for nucleotide-free form of ABCB6-C. From the pH, temperature and magnetic field strength dependencies of the resonance intensities, we concluded that this incompleteness in the assignments is mainly due to the exchange between multiple conformations at an intermediate rate on the NMR timescale. These localised conformational dynamics remained in ADP-bound ABCB6-C except for the loops responsible for adenine base and {alpha}/{beta}-phosphate binding. These results revealed that the localised dynamic cooperativity, which was recently proposed for a prokaryotic ABC MJ1267, also exists in a higher eukaryotic ABC, and is presumably shared by all members of the ABC family. Since the Helical domain is the putative interface to the transmembrane domain, this cooperativity may explain the coupled functions between domains in the substrate-transportation cycle.

  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. Mutational analysis of Cvab, an ABC transporter involved in the secretion of active colicin V.

    Directory of Open Access Journals (Sweden)

    Kai-Hui Wu

    Full Text Available CvaB is the central membrane transporter of the colicin V secretion system that belongs to an ATP-binding cassette superfamily. Previous data showed that the N-terminal and C-terminal domains of CvaB are essential for the function of CvaB. N-terminal domain of CvaB possesses Ca(2+-dependent cysteine proteolytic activity, and two critical residues, Cys32 and His105, have been identified. In this study, we also identify Asp121 as being the third residue of the putative catalytic triad within the active site of the enzyme. The Asp121 mutants lose both their colicin V secretion activity and N-terminal proteolytic activity. The adjacent residue Pro122 also appears to play a critical role in the colicin V secretion. However, the reversal of the two residues D121P - P122D results in loss of activity. Based on molecular modeling and protein sequence alignment, several residues adjacent to the critical residues, Cys32 and His105, were also examined and characterized. Site-directed mutagenesis of Trp101, Asp102, Val108, Leu76, Gly77, and Gln26 indicate that the neighboring residues around the catalytic triad affect colicin V secretion. Several mutated CvaB proteins with defective secretion were also tested, including Asp121 and Pro122, and were found to be structurally stable. These results indicate that the residues surrounding the identified catalytic triad are functionally involved in the secretion of biologically active colicin V.

  8. Stubborn contaminants: influence of detergents on the purity of the multidrug ABC transporter BmrA.

    Science.gov (United States)

    Wiseman, Benjamin; Kilburg, Arnaud; Chaptal, Vincent; Reyes-Mejia, Gina Catalina; Sarwan, Jonathan; Falson, Pierre; Jault, Jean-Michel

    2014-01-01

    Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.

  9. 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.

  10. A study on contamination and disinfection of film cassette

    Energy Technology Data Exchange (ETDEWEB)

    Kweon, Dae Cheol; Chung, Kyung Mo [Seoul National University Hospital, Seoul (Korea, Republic of); Choi, Ji Won [University of Sydney, Sydney (Australia)

    2000-04-15

    In July 2000, a bacteria infection on film cassette contact surface was examined at the diagnostic radiology department of the S. hospital. The objective of this study was to assess the contamination level on film cassette contact surface as a predictor of patient to prevent from nosocomial infection. The study showed that the laboratory result was identified non-pathologic bacterial in the four different cassette size of the contact surface. The study concludes that presence of a bacterial infection will prevent a using antiseptic technique on film cassette contact surface. Also the education of nosocomial infection for radiographer will be required.

  11. Finger-Actuated, Self-Contained Immunoassay Cassettes

    Science.gov (United States)

    Qiu, Xianbo; Thompson, Jason A.; Chen, Zongyuan; Liu, Changchun; Chen, Dafeng; Ramprasad, Sudhir; Mauk, Michael G.; Ongagna, Serge; Barber, Cheryl; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L.A.M.; Bau, Haim H.

    2010-01-01

    The building blocks for an inexpensive, disposable, luminescence-based microfluidic immunoassay cassette are described, and their integration in a point-of-care diagnostic system is demonstrated. Fluid motion in the cassette is driven by depressing finger-actuated pouches. All reagents needed for the immunoassay can be stored in the cassette in liquid form. Prior to use, the cassette consists of two separate parts. A top storage component contains pouches, sealed storage chambers, a metering chamber, and needle seats. The bottom processing component contains connection needles, a mixing chamber, and a detection chamber with immobilized proteins. Subsequent to sample introduction, the storage and processing components are mated. The needles form hydraulic connections between the two parts and, in some cases, close valves. The pouches are then actuated sequentially to induce flow of various reagents and facilitate process operations. The cassette is compatible with different detection modalities. Both a cassette with immunochromatographic-based detection and a cassette with microbead-based detection were constructed and evaluated. The immunochromatographic cassette was used to detect antibodies to HIV in saliva samples. The bead-based cassette was used to detect the proinflammatory chemokine IL-8. The experimental data demonstrates good repeatability and reasonable sensitivity. PMID:19597994

  12. Enterococcus faecalis Uses a Phosphotransferase System 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

    2017-05-01

    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, to our knowledge, 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, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically

  13. The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.

    Science.gov (United States)

    Holmes, Ann R; Keniya, Mikhail V; Ivnitski-Steele, Irena; Monk, Brian C; Lamping, Erwin; Sklar, Larry A; Cannon, Richard D

    2012-03-01

    Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.

  14. 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.

  15. Recombination between the dfrA12-orfF-aadA2 Cassette Array and an aadA1 Gene Cassette Creates a Hybrid Cassette, aadA8b

    Science.gov (United States)

    Gestal, Alicia M.; Stokes, H. W.; Partridge, Sally R.; Hall, Ruth M.

    2005-01-01

    Homologous recombination between closely related gene cassettes, such as aadA1 and aadA2, which are 89% identical, can create hybrid cassettes and hybrids of existing cassette arrays. A new cassette array, dfrA12-orfF-aadA8b, which was created by such a recombination event occurring within the aadA2 cassette in the dfrA12-orfF-aadA2 array, has been identified. PMID:16251327

  16. 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 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.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.The CT of Pxa2p is involved in its interaction with Pxa1p and in transporter function. This concept may be applied to human ALDP studies, helping to establish the pathological mechanism for CT-related X

  17. Associations between dru Types and SCCmec Cassettes

    DEFF Research Database (Denmark)

    Bartels, Mette D; Boye, Kit; Oliveira, Duarte C

    2013-01-01

    isolates with dru type variants indicating that dru typing is not useful as a first line epidemiological typing tool. However, MRSA isolates cultured from a single patient over a three year period exhibited a single dru type. The finding of dt10a in most SCCmec types suggests that dru and mecA originate......Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct...... repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring...

  18. 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...hese seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals...ype. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters. Al

  19. Increased expression of the yeast multidrug resistance ABC transporter Pdr18 leads to increased ethanol tolerance and ethanol production in high gravity alcoholic fermentation.

    Science.gov (United States)

    Teixeira, Miguel C; Godinho, Cláudia P; Cabrito, Tânia R; Mira, Nuno P; Sá-Correia, Isabel

    2012-07-27

    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. 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 ³H-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. 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 improve alcoholic fermentation performance for sustainable bio

  20. 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

  1. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Genome-wide transcriptome profiling of black poplar (Populus nigra L.) under boron toxicity revealed candidate genes responsible in boron uptake, transport and detoxification.

    Science.gov (United States)

    Yıldırım, Kubilay; Uylaş, Senem

    2016-12-01

    Boron (B) is an essential nutrient for normal growth of plants. Despite its low abundance in soils, it could be highly toxic to plants in especially arid and semi-arid environments. Poplars are known to be tolerant species to B toxicity and accumulation. However, physiological and gene regulation responses of these trees to B toxicity have not been investigated yet. Here, B accumulation and tolerance level of black poplar clones were firstly tested in the current study. Rooted cutting of these clones were treated with elevated B toxicity to select the most B accumulator and tolerant genotype. Then we carried out a microarray based transcriptome experiment on the leaves and roots of this genotype to find out transcriptional networks, genes and molecular mechanisms behind B toxicity tolerance. The results of the study indicated that black poplar is quite suitable for phytoremediation of B pollution. It could resist 15 ppm soil B content and >1500 ppm B accumulation in leaves, which are highly toxic concentrations for almost all agricultural plants. Transcriptomics results of study revealed totally 1625 and 1419 altered probe sets under 15 ppm B toxicity in leaf and root tissues, respectively. The highest induction were recorded for the probes sets annotated to tyrosine aminotransferase, ATP binding cassette transporters, glutathione S transferases and metallochaperone proteins. Strong up regulation of these genes attributed to internal excretion of B into the cell vacuole and existence of B detoxification processes in black poplar. Many other candidate genes functional in signalling, gene regulation, antioxidation, B uptake and transport processes were also identified in this hyper B accumulator plant for the first time with the current study. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. The reverse cholesterol transport pathway improves understanding of genetic networks for fat deposition and muscle growth in beef cattle.

    Directory of Open Access Journals (Sweden)

    Tyler F Daniels

    Full Text Available In the present study, thirteen genes involved in the reverse cholesterol transport (RCT pathway were investigated for their associations with three fat depositions, eight fatty acid compositions and two growth-related phenotypes in a Wagyu x Limousin reference population, including 6 F(1 bulls, 113 F(1 dams, and 246 F(2 progeny. A total of 37 amplicons were used to screen single nucleotide polymorphisms (SNPs on 6 F(1 bulls. Among 36 SNPs detected in 11 of these 13 genes, 19 were selected for genotyping by the Sequenom assay design on all F(2 progeny. Single-marker analysis revealed seven SNPs in ATP binding cassette A1, apolipoproteins A1, B and E, phospholipid transfer protein and paraoxinase 1 genes significantly associated with nine phenotypes (P<0.05. Previously, we reported genetic networks associated with 19 complex phenotypes based on a total of 138 genetic polymorphisms derived from 71 known functional genes. Therefore, after Bonferroni correction, these significant (adjusted P<0.05 and suggestive (adjusted P<0.10 associations were then used to identify genetic networks related to the RCT pathway. Multiple-marker analysis suggested possible genetic networks involving the RCT pathway for kidney-pelvic-heart fat percentage, rib-eye area, and subcutaneous fat depth phenotypes with markers derived from paraoxinase 1, apolipoproteins A1 and E, respectively. The present study confirmed that genes involved in cholesterol homeostasis are useful targets for investigating obesity in humans as well as for improving meat quality phenotypes in a livestock production.

  4. Strain- and sex-dependent circadian changes in abcc2 transporter expression: implications for irinotecan chronotolerance in mouse ileum.

    Directory of Open Access Journals (Sweden)

    Alper Okyar

    Full Text Available ATP-binding cassette transporter abcc2 is involved in the cellular efflux of irinotecan. The drug is toxic for mouse ileum, where abcc2 is highly expressed. Here, we investigate whether circadian changes in local abcc2 expression participate in the circadian rhythm of irinotecan toxicity for ileum mucosa, and further assess whether genetic background or sex modify this relation.Ileum mucosa was obtained every 3-4 h for 24 h in male and female B6D2F(1 and B6CBAF(1 mice synchronized with light from Zeitgeber Time (ZT0 to ZT12 alternating with 12 h of darkness. Irinotecan (50 mg/kg i.v. daily for 4 days was administered at the sex- and strain-specific times corresponding to least (ZT11-15 or largest drug-induced body weight loss (ZT23-03-07. Abcc2 expression was determined with qRT-PCR for mRNA and with immunohistochemistry and confocal microscopy for protein. Histopathologic lesions were graded in ileum tissues obtained 2, 4 or 6 days after treatment. Two- to six-fold circadian changes were demonstrated for mRNA and protein mean expressions of abcc2 in mouse ileum (p<0.05. ZT12 corresponded to high mRNA and protein expressions, with circadian waveforms differing according to genetic background and sex. The proportion of mice spared from ileum lesions varied three-fold according to irinotecan timing, with best tolerability at ZT11-15 (p = 0.00003. Irinotecan was also best tolerated in males (p = 0.05 and in B6CBAF(1 (p = 0.0006.Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan. This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.

  5. Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales.

    Science.gov (United States)

    Noll, Kenneth M; Lapierre, Pascal; Gogarten, J Peter; Nanavati, Dhaval M

    2008-01-15

    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. 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 have been horizontally acquired by a Thermotoga

  6. 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

  7. 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

  8. Multidrug resistance in lactic acid bacteria : molecular mechanisms and clinical relevance

    NARCIS (Netherlands)

    van Veen, HW; Margolles, A; Putman, M; 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. The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type

  9. 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

  10. Lack of ABCG2 shortens latency of BRCA1-deficient mammary tumors and this is not affected by genistein or resveratrol

    NARCIS (Netherlands)

    Zander, Serge A. L.; Kersbergen, Ariena; Sol, Wendy; Gonggrijp, Maaike; van de Wetering, Koen; Jonkers, Jos; Borst, Piet; Rottenberg, Sven

    2012-01-01

    In addition to their role in drug resistance, the ATP-binding cassette (ABC) transporters ABCG2 and ABCB1 have been suggested to protect cells from a broad range of substances that may foster tumorigenesis. Phytoestrogens or their metabolites are substrates of these transporters and the influence of

  11. ABCA7 and risk of dementia and vascular disease in the Danish population

    DEFF Research Database (Denmark)

    Kjeldsen, Emilie W.; Tybjærg-Hansen, Anne; Nordestgaard, Børge G.

    2018-01-01

    Objective: ATP-binding-cassette transporter A7(ABCA7) is suggested to be involved in lipid transport as well as in phagocytosis of amyloid-β in the brain. We tested the hypothesis that a common genetic variant in ABCA7 is associated with dementia, ischemic heart disease, ischemic cerebrovascular ...

  12. The effect of co-administered flavonoids on the metabolism of hesperetin and the disposition of its metabolites in Caco-2 cell monolayers

    NARCIS (Netherlands)

    Brand, W.; Padilla, B.; Bladeren, van P.J.; Williamson, G.; Rietjens, I.

    2010-01-01

    Metabolism by phase II enzymes and transport from intestinal cells back into the lumen by ATP binding cassette (ABC) transporters limits the bioavailability of the flavanone hesperetin, the aglycone of hesperidin. This study investigates to what extent other flavonoids modulate the metabolism and

  13. Molecular Properties of Drugs Interacting with SLC22 Transporters OAT1, OAT3, OCT1, and OCT2: A Machine-Learning Approach.

    Science.gov (United States)

    Liu, Henry C; Goldenberg, Anne; Chen, Yuchen; Lun, Christina; Wu, Wei; Bush, Kevin T; Balac, Natasha; Rodriguez, Paul; Abagyan, Ruben; Nigam, Sanjay K

    2016-10-01

    Statistical analysis was performed on physicochemical descriptors of ∼250 drugs known to interact with one or more SLC22 "drug" transporters (i.e., SLC22A6 or OAT1, SLC22A8 or OAT3, SLC22A1 or OCT1, and SLC22A2 or OCT2), followed by application of machine-learning methods and wet laboratory testing of novel predictions. In addition to molecular charge, organic anion transporters (OATs) were found to prefer interacting with planar structures, whereas organic cation transporters (OCTs) interact with more three-dimensional structures (i.e., greater SP3 character). Moreover, compared with OAT1 ligands, OAT3 ligands possess more acyclic tetravalent bonds and have a more zwitterionic/cationic character. In contrast, OCT1 and OCT2 ligands were not clearly distinquishable form one another by the methods employed. Multiple pharmacophore models were generated on the basis of the drugs and, consistent with the machine-learning analyses, one unique pharmacophore created from ligands of OAT3 possessed cationic properties similar to OCT ligands; this was confirmed by quantitative atomic property field analysis. Virtual screening with this pharmacophore, followed by transport assays, identified several cationic drugs that selectively interact with OAT3 but not OAT1. Although the present analysis may be somewhat limited by the need to rely largely on inhibition data for modeling, wet laboratory/in vitro transport studies, as well as analysis of drug/metabolite handling in Oat and Oct knockout animals, support the general validity of the approach-which can also be applied to other SLC and ATP binding cassette drug transporters. This may make it possible to predict the molecular properties of a drug or metabolite necessary for interaction with the transporter(s), thereby enabling better prediction of drug-drug interactions and drug-metabolite interactions. Furthermore, understanding the overlapping specificities of OATs and OCTs in the context of dynamic transporter tissue

  14. Exendin-4 Ameliorates Lipotoxicity-induced Glomerular Endothelial Cell Injury by Improving ABC Transporter A1-mediated Cholesterol Efflux in Diabetic apoE Knockout Mice.

    Science.gov (United States)

    Yin, Qing-Hua; Zhang, Rui; Li, Li; Wang, Yi-Ting; Liu, Jing-Ping; Zhang, Jie; Bai, Lin; Cheng, Jing-Qiu; Fu, Ping; Liu, Fang

    2016-12-16

    ATP-binding cassette transporter A1 (ABCA1), which promotes cholesterol efflux from cells and inhibits inflammatory responses, is highly expressed in the kidney. Research has shown that exendin-4, a glucagon-like peptide-1 receptor (GLP-1R) agonist, promotes ABCA1 expression in multiple tissues and organs; however, the mechanisms underlying exendin-4 induction of ABCA1 expression in glomerular endothelial cells are not fully understood. In this study we investigated the effect of exendin-4 on ABCA1 in glomerular endothelial cells of diabetic kidney disease (DKD) and the possible mechanism. We observed a marked increase in glomerular lipid deposits in tissues of patients with DKD and diabetic apolipoprotein E knock-out (apoE-/-) mice by Oil Red O staining and biochemical analysis of cholesterol. We found significantly decreased ABCA1 expression in glomerular endothelial cells of diabetic apoE-/- mice and increased renal lipid, cholesterol, and inflammatory cytokine levels. Exendin-4 decreased renal cholesterol accumulation and inflammation and increased cholesterol efflux by up-regulating ABCA1. In human glomerular endothelial cells, GLP-1R-mediated signaling pathways (e.g. Ca2+/calmodulin-dependent protein kinase, cAMP/PKA, PI3K/AKT, and ERK1/2) were involved in cholesterol efflux and inflammatory responses by regulating ABCA1 expression. We propose that exendin-4 increases ABCA1 expression in glomerular endothelial cells, which plays an important role in alleviating renal lipid accumulation, inflammation, and proteinuria in mice with type 2 diabetes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Predicting P-glycoprotein-mediated drug transport based on support vector machine and three-dimensional crystal structure of P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Zsolt Bikadi

    Full Text Available Human P-glycoprotein (P-gp is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com, which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening.

  16. Observing cassette culture: user interface implications for digital music libraries

    OpenAIRE

    Toal, Jason

    2007-01-01

    Many people keep their collections of music on cassette tape even if they rarely listen to them. Images of these collections can be found online on photo sharing websites. What can we learn from such collections and what might they tell us about designing interfaces for new digital music libraries? The author conducts an online ethnographic study of over two hundred cassette tape collections, and over sixty participants with the aim of guiding future design of music collections. The author pr...

  17. 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...

  18. 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.

  19. Protein Kinase C-Independent Inhibition of Organic Cation Transporter 1 Activity by the Bisindolylmaleimide Ro 31-8220.

    Directory of Open Access Journals (Sweden)

    Abdullah Mayati

    Full Text Available Ro 31-8220 is a potent protein kinase C (PKC inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs. Various PKC-independent effects of Ro 31-8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT 1, involved in uptake of marketed drugs in the liver, in a PKC-independent manner. Ro 31-8220, in contrast to other pan-PKC inhibitors such as staurosporine and chelerythrine, was thus shown to cis-inhibit uptake of the reference OCT1 substrate tetraethylammonium in OCT1-transfected HEK293 cells in a concentration-dependent manner (IC50 = 0.18 μM and without altering membrane expression of OCT1. This blockage of OCT1 was also observed in human hepatic HepaRG cells that constitutionally express OCT1. It likely occurred through a mixed mechanism of inhibition. Ro 31-8220 additionally trans-inhibited TEA uptake in OCT1-transfected HEK293 cells, which likely discards a transport of Ro 31-8220 by OCT1. Besides Ro 31-8220, 7 additional BIMs, including the PKC inhibitor LY 333531, inhibited OCT1 activity, whereas 4 other BIMs were without effect. In silico analysis of structure-activity relationships next revealed that various molecular descriptors, especially 3D-WHIM descriptors related to total size, correspond to key physico-chemical parameters for inhibition of OCT1 activity by BIMs. In addition to activity of OCT1, Ro 31-8220 inhibited those of other organic cation transporters such as multidrug and toxin extrusion protein (MATE 1 and MATE2-K, whereas, by contrast, it stimulated that of OCT2. Taken together, these data extend the nature of cellular off-targets of the BIM Ro 31-8220 to OCT1 and other organic cation transporters, which has likely to be kept in mind when using Ro 31-8220 and other BIMs as PKC inhibitors in experimental or

  20. 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

  1. Structures of OppA and PstS from Yersinia pestis indicate variability of interactions with transmembrane domains

    DEFF Research Database (Denmark)

    Tanabe, Mikio; Mirza, Osman; Bertrand, Thomas

    2007-01-01

    Bacterial ATP-binding cassette (ABC) transport systems couple ATP hydrolysis with the uptake and efflux of a wide range of substances across bacterial membranes. These systems are comprised of transmembrane domains, nucleotide binding domains and, in the case of uptake systems, periplasmic bindin...

  2. N-lactoyl-amino acids are ubiquitous metabolites that originate from CNDP2-mediated reverse proteolysis of lactate and amino acids

    NARCIS (Netherlands)

    Jansen, Robert S; Addie, Ruben; Merkx, Remco; Fish, Alexander; Mahakena, Sunny; Bleijerveld, Onno B|info:eu-repo/dai/nl/30483078X; Altelaar, Maarten|info:eu-repo/dai/nl/304833517; IJlst, Lodewijk; Wanders, Ronald J; Borst, P; van de Wetering, Koen

    2015-01-01

    Despite technological advances in metabolomics, large parts of the human metabolome are still unexplored. In an untargeted metabolomics screen aiming to identify substrates of the orphan transporter ATP-binding cassette subfamily C member 5 (ABCC5), we identified a class of mammalian metabolites,

  3. Plasma amyloid-β in patients with Tangier disease

    NARCIS (Netherlands)

    Shahim, Pashtun; Bochem, Andrea E.; Mattsson, Niklas; Lautner, Ronald; Blennow, Kaj; Hovingh, G. Kees; Motazacker, M. Mahdi; Zetterberg, Henrik

    2013-01-01

    Tangier disease (TD) is a rare genetic disorder caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene, which results in impaired cellular cholesterol efflux and high-density lipoprotein cholesterol deficiency. Animal and in vitro studies indicate that ABCA1 is involved in the

  4. Specific mutations in ABCA1 have discrete effects on ABCA1 function and lipid phenotypes both in vivo and in vitro

    NARCIS (Netherlands)

    Singaraja, Roshni R.; Visscher, Henk; James, Erick R.; Chroni, Angeliki; Coutinho, Jonathan M.; Brunham, Liam R.; Kang, Martin H.; Zannis, Vassilis I.; Chimini, Giovanna; Hayden, Michael R.

    2006-01-01

    Mutations in ATP-binding cassette transporter A1 (ABCA1) cause Tangier disease and familial hypoalphalipoproteinemia, resulting in low to absent plasma high-density lipoprotein cholesterol levels. However, wide variations in clinical lipid phenotypes are observed in patients with mutations in ABCA1.

  5. An Arabidopsis lipid flippase is required for timely recruitment of defenses to the host-pathogen interface at the plant cell surface

    Science.gov (United States)

    Deposition of cell wall-reinforcing papillae is an integral component of the plant immune response. The Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette (ABC) transporter plays a role in defense against numerous pathogens and is recruited to sites of pathogen detection where it accumulates with...

  6. Influence of ketoconazole on the fecal and urinary disposition of docetaxel

    NARCIS (Netherlands)

    Engels, Frederike K.; Loos, Walter J.; Mathot, Ron A. A.; van Schaik, Ron H. N.; Verweij, Jaap

    2007-01-01

    The anticancer drug docetaxel is extensively metabolized by cytochrome P450 (CYP) 3A isozymes. Furthermore, docetaxel is also a substrate for the transmembrane ATP-binding cassette efflux transporter protein ABCB1. CYP3A-inhibition significantly reduces docetaxel total systemic clearance, on average

  7. Sphingolipids in neuroblastoma : Their role in drug resistance mechanisms

    NARCIS (Netherlands)

    Sietsma, H; Dijkhuis, AJ; Kamps, W; Kok, JW

    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.,

  8. ABCG2 inhibition as a therapeutic approach for overcoming ...

    Indian Academy of Sciences (India)

    2016-02-16

    Feb 16, 2016 ... Breast cancer resistance protein (BCRP, ABCP or MXR)/ATP-binding cassette subfamily G member 2 (ABCG2) was characterized as a multidrug resistance efflux transporter in 1998. ABCG2 physiologically acts as a part of a self- defence mechanism for the organism; it enhances eliminating of toxic ...

  9. ABCG2 inhibition as a therapeutic approach for overcoming ...

    Indian Academy of Sciences (India)

    Breast cancer resistance protein (BCRP, ABCP or MXR) / ATP-binding cassette subfamily G member 2 (ABCG2) was characterized as a multidrug resistance efflux transporter in 1998. ABCG2 physiologically acts as a part of a self-defense mechanism for the organism; it enhances eliminating of toxic xenobiotic substances ...

  10. 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

  11. 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

  12. Data-driven homology modelling of P-glycoprotein in the ATP-bound state indicates flexibility of the transmembrane domains

    NARCIS (Netherlands)

    Stockner, T.; de Vries, S.J.|info:eu-repo/dai/nl/304837717; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Ecker, G.F.; Chiba, P.

    2009-01-01

    Human P-glycoprotein is an ATP-binding cassette transporter that plays an important role in the defence against potentially harmful molecules from the environment. It is involved in conferring resistance against cancer therapeutics and plays an important role for the pharmacokinetics of drugs. The

  13. Relative neurotoxicity of ivermectin and moxidectin in Mdr1ab channel activity

    National Research Council Canada - National Science Library

    Menez, Cecile; Sutra, Jean-Francois; Prichard, Roger; Lespine, Anne

    2012-01-01

    ... prevention of Dirofiilaria immitis infection in dogs. In general, MLs have a high margin of safety in mammals (Pulliam & Preston, 1989). Indeed, P-glycoprotein (P-gp, MDR1/ ABCB1), a plasma membrane efflux pump belonging to the ATP-binding cassette (ABC) transporters family, efficiently restricts their penetration in the brain at the blood-brain barr...

  14. Deficiency of either P-glycoprotein or breast cancer resistance protein protect against acute kidney injury.

    NARCIS (Netherlands)

    Huls, M.; Schoeber, J.P.H.; Verfaillie, C.M.; Luttun, A.; Ulloa-Montoya, F.; Menke, A.L.; Bolderen, L.R. van; Woestenenk, R.M.; Merkx, G.F.M.; Wetzels, J.F.M.; Russel, F.G.M.; Masereeuw, R.

    2010-01-01

    The kidney has a high capacity to regenerate after ischemic injury via several mechanisms, one of which involves bone marrow-derived (stem) cells. The ATP binding cassette transporters, P-glycoprotein and breast cancer resistance protein, are determinants for the enriched stem and progenitor cell

  15. High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

    NARCIS (Netherlands)

    Ros, Jenny E.; Libbrecht, Louis; Geuken, Mariska; Jansen, Peter L. M.; Roskams, Tania A. D.

    2003-01-01

    An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on

  16. 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

  17. 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...

  18. 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

  19. 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...

  20. [Integrons and resistance gene cassettes: structure and role against antimicrobials].

    Science.gov (United States)

    González, Gerardo; Mella, Sergio; Zemelman, Raúl; Bello, Helia; Domínguez, Mariana

    2004-05-01

    Bacteria have developed sophisticated and successful genetic mechanisms to evade the action of antimicrobials. Bacterial multiresistance has caused serious problems in the treatment of nosocomial infections. Integrons and gene cassettes are considered the main genetic elements in the evolution of plasmids and transposons that actively participate in the mobilization of genes, codifying different bacterial resistance mechanisms. This article reviews the historical and structural aspects of integrons and resistance gene cassettes and the presence of these structures in gram negative bacteria isolated from Chilean hospitals in the last ten years.

  1. Molecular Characterization of a High-Affinity Xylobiose Transporter of Streptomyces thermoviolaceus OPC-520 and Its Transcriptional Regulation

    Science.gov (United States)

    Tsujibo, Hiroshi; Kosaka, Mitsuo; Ikenishi, Sadao; Sato, Takaji; Miyamoto, Katsushiro; Inamori, Yoshihiko

    2004-01-01

    Streptomyces thermoviolaceus OPC-520 secretes two types of xylanases (StxI and StxII), an acetyl xylan esterase (StxIII), and an α-l-arabinofuranosidase (StxIV) in the presence of xylan. Xylan degradation products (mainly xylobiose) produced by the action of these enzymes entered the cell and were then degraded to xylose by an intracellular β-xylosidase (BxlA). A gene cluster involved in xylanolytic system of the strain was cloned and sequenced upstream of and including a BxlA-encoding gene (bxlA). The gene cluster consisted of four different open reading frames organized in the order bxlE, bxlF, bxlG, and bxlA. Reverse transcriptase PCR analysis revealed that the gene cluster is transcribed as polycistronic mRNA. The deduced gene products, comprising BxlE (a sugar-binding lipoprotein), BxlF (an integral membrane protein), and BxlG (an integral membrane protein), showed similarity to components of the bacterial ATP-binding cassette (ABC) transport system; however, the gene for the ATP binding protein was not linked to the bxl operon. The soluble recombinant BxlE protein was analyzed for its binding activity for xylooligosaccharides. The protein showed high-level affinity for xylobiose (Kd = 8.75 × 10−9 M) and for xylotriose (Kd = 8.42 × 10−8 M). Antibodies raised against the recombinant BxlE recognized the detergent-soluble BxlE isolated from S. thermoviolaceus membranes. The deduced BxlF and BxlG proteins are predicted to be integral membrane proteins. These proteins contained the conserved EAA loop (between the fourth and the fifth membrane-spanning segments) which is characteristic of membrane proteins from binding-protein-dependent ABC transporters. In addition, the bxlR gene located upstream of the bxl operon was cloned and expressed in Escherichia coli. The bxlR gene encoded a 343-residue polypeptide that is highly homologous to members of the GalR/LacI family of bacterial transcriptional regulators. The purified BxlR protein specifically bound to a 4

  2. 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.

  3. The Real World Spanish Cassette Program. Script Book.

    Science.gov (United States)

    Sternburg, Sheldon G.

    This dual cassette program, accompanied by a script book, is designed to give students listening practice in Spanish, particularly for regional differences of pronunciation and for variety in idiomatic construction. The program may be integrated with texts used in intermediate and advanced levels of instruction. The announcements, jingles, and…

  4. [Integrons and antimicrobial resistance gene cassettes in Shigella flexneri strains ].

    Science.gov (United States)

    Muñoz, Jeannette; Bello, Helia; Domínguez, Mariana; Mella, Sergio; Zemelman, Raúl; González, Gerardo

    2003-07-01

    The resistance of Shigella flexneri to antimicrobial agents can be associated to the presence of integrons that may contain and express antimicrobial resistance gene cassettes. To study antimicrobial resistance and the presence of integrons and antimicrobial gene cassettes in Shigella flexneri strains. In vitro susceptibility to 27 antimicrobials was studied in twenty four Shigella flexneri strains isolated from stools. The presence of integrons class 1, 2 and 3 and antimicrobial resistance gene cassettes was investigated by polymerase chain reaction (PCR) using specific primers for each gene. Most strains were resistant to one of the following antimicrobials: ampicillin, sulphonamide, trimethoprim, tetracycline, streptomycin, sulfamethoxazole-trimethoprim or chloramphenicol. Twenty nine percent were simultaneously resistant to all these antimicrobials. Integrons class 1 and 2 were found in 19 strains (79%). Class 3 integrons were not found. Gene cassettes dfrA1 and ant(3")I were associated to integrons class 2 in most strains (15/20, 75%). Genes cat, tetB and blarTEM were detected in 18/24 (75%), 7/24 (29%) and 4/24 (17%) of the strains, respectively and were not associated to any of the studied integrons. Genes that codify enzymes AAC(6')Ib and APH(3')VI were not detected in any strain. The high frequency of integrons found in the studied strains, could partly explain the increasing antimicrobial resistance of Shigella flexneri strains, isolated in Chile.

  5. 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.

  6. Co-assortment in integron-associated gene cassette assemblages in environmental DNA samples.

    Science.gov (United States)

    Michael, Carolyn A; Andrew, Nigel R

    2010-08-10

    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. 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. 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.

  7. Reaching Out: The Role of Audio Cassette Communication in Rural Development. Occasional Paper 19.

    Science.gov (United States)

    Adhikarya, Ronny; Colle, Royal D.

    This report describes the state-of-the-art of audio cassette technology (ACT) and reports findings from field tests, case studies, and pilot projects in several countries which demonstrate the potential of audio cassettes as a medium for communicating with rural people. Specific guidance is also offered on how a project can use cassettes as a…

  8. 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 only...... expressed in M cells and the gene product is responsible for the secretion of the mating pheromone. M-factor, a nonapeptide that is S-farnesylated and carboxy-methylated on its C-terminal cysteine residue. The predicted Mam1 protein is highly homologous to mammalian multiple drug-resistance proteins...... and to the Saccharomyces cerevisiae STE6 gene product, which mediates export of a-factor mating pheromone. We show that STE6 can also mediate secretion of M-factor in S. pombe....

  9. Ratiometric Matryoshka biosensors from a nested cassette of green- and orange-emitting fluorescent proteins.

    Science.gov (United States)

    Ast, Cindy; Foret, Jessica; Oltrogge, Luke M; De Michele, Roberto; Kleist, Thomas J; Ho, Cheng-Hsun; Frommer, Wolf B

    2017-09-05

    Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO- Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.

  10. 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

  11. 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

  12. Expression Patterns of ABC Transporter Genes in Fluconazole-Resistant Candida glabrata.

    Science.gov (United States)

    Gohar, Atefeh Abdollahi; Badali, Hamid; Shokohi, Tahereh; Nabili, Mojtaba; Amirrajab, Nasrin; Moazeni, Maryam

    2017-04-01

    Clinical management of fungal diseases is compromised by the emergence of antifungal drug resistance in fungi, which leads to elimination of available drug classes as treatment options. An understanding of antifungal resistance at molecular level is, therefore, essential for the development of strategies to combat the resistance. This study presents the assessment of molecular mechanisms associated with fluconazole resistance in clinical Candida glabrata isolates originated from Iran. Taking seven distinct fluconazole-resistant C. glabrata isolates, real-time PCRs were performed to evaluate the alternations in the regulation of the genes involved in drug efflux including CgCDR1, CgCDR2, CgSNQ2, and CgERG11. Gain-of-function (GOF) mutations in CgPDR1 alleles were determined by DNA sequencing. Cross-resistance to fluconazole, itraconazole, and voriconazole was observed in 2.5 % of the isolates. In the present study, six amino acid substitutions were identified in CgPdr1, among which W297R, T588A, and F575L were previously reported, whereas D243N, H576Y, and P915R are novel. CgCDR1 overexpression was observed in 57.1 % of resistant isolates. However, CgCDR2 was not co-expressed with CgCDR1. CgSNQ2 was upregulated in 71.4 % of the cases. CgERG11 overexpression does not seem to be associated with azole resistance, except for isolates that exhibited azole cross-resistance. The pattern of efflux pump gene upregulation was associated with GOF mutations observed in CgPDR1. These results showed that drug efflux mediated by adenosine-5-triphosphate (ATP)-binding cassette transporters, especially CgSNQ2 and CgCDR1, is the predominant mechanism of fluconazole resistance in Iranian isolates of C. glabrata. Since some novel GOF mutations were found here, this study also calls for research aimed at investigating other new GOF mutations to reveal the comprehensive understanding about efflux-mediated resistance to azole antifungal agents.

  13. Evolutionary Origin of the Staphylococcal Cassette Chromosome mec (SCCmec)

    DEFF Research Database (Denmark)

    Rolo, Joana; Worning, Peder; Nielsen, Jesper Boye

    2017-01-01

    Several lines of evidence indicate that the most primitive staphylococcal species, those of the Staphylococcus sciuri group, were involved in the first stages of evolution of the staphylococcal cassette chromosome mec (SCCmec), the genetic element carrying the β-lactam resistance gene mecA. Howev....... aureus clones) originated in S. sciuri possibly by a recombination event in a human host or a human-created environment and later was transferred to S. aureus....

  14. 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

  15. Impaired very long-chain acyl-CoA β-oxidation in human X-linked adrenoleukodystrophy fibroblasts is a direct consequence of ABCD1 transporter dysfunction.

    Science.gov (United States)

    Wiesinger, Christoph; Kunze, Markus; Regelsberger, Günther; Forss-Petter, Sonja; Berger, Johannes

    2013-06-28

    X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity.

  16. Impaired Very Long-chain Acyl-CoA β-Oxidation in Human X-linked Adrenoleukodystrophy Fibroblasts Is a Direct Consequence of ABCD1 Transporter Dysfunction*

    Science.gov (United States)

    Wiesinger, Christoph; Kunze, Markus; Regelsberger, Günther; Forss-Petter, Sonja; Berger, Johannes

    2013-01-01

    X-linked adrenoleukodystrophy (X-ALD), an inherited peroxisomal disorder, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (adrenoleukodystrophy protein, ALDP). Biochemically, X-ALD is characterized by an accumulation of very long-chain fatty acids and partially impaired peroxisomal β-oxidation. In this study, we used primary human fibroblasts from X-ALD and Zellweger syndrome patients to investigate the peroxisomal β-oxidation defect. Our results show that the degradation of C26:0-CoA esters is as severely impaired as degradation of unesterified very long-chain fatty acids in X-ALD and is abolished in Zellweger syndrome. Interestingly, the β-oxidation rates for both C26:0-CoA and C22:0-CoA were similarly affected, although C22:0 does not accumulate in patient fibroblasts. Furthermore, we show that the β-oxidation defect in X-ALD is directly caused by ABCD1 dysfunction as blocking ABCD1 function with a specific antibody reduced β-oxidation to levels observed in X-ALD fibroblasts. By quantification of mRNA and protein levels of the peroxisomal ABC transporters and by blocking with specific antibodies, we found that residual β-oxidation activity toward C26:0-CoA in X-ALD fibroblasts is mediated by ABCD3, although the efficacy of ABCD3 appeared to be much lower than that of ABCD1. Finally, using isolated peroxisomes, we show that β-oxidation of C26:0-CoA is independent of additional CoA but requires a cytosolic factor of >10-kDa molecular mass that is resistant to N-ethylmaleimide and heat inactivation. In conclusion, our findings in human cells suggest that, in contrast to yeast cells, very long-chain acyl-CoA esters are transported into peroxisomes by ABCD1 independently of additional synthetase activity. PMID:23671276

  17. 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...

  18. Is this charred material from a VHS video cassette?

    Science.gov (United States)

    Fruchtenicht, Tara; Blackledge, Robert D.; Williams, Teresa R.

    2010-06-01

    At his residence, a victim in a double homicide had installed a home-built video surveillance system. The suspects either knew of or discovered this system and removed it. In a backyard at a location associated with the suspects was a barrel used for burning trash. Could charred debris recovered from a metal bowl found among the contents of the barrel be the remains of a VHS video cassette? A positive answer to the question was obtained through a combination of optical microscopy, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and Energy Dispersive Spectroscopy (EDS).

  19. 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...

  20. Disinfection efficacy of an ultraviolet light on film cassettes for preventive of the nosocomial infection

    Energy Technology Data Exchange (ETDEWEB)

    Kweon, Dae Cheol [Seoul National Univ. Hospital, Seoul (Korea, Republic of); Jeon, Yong Woong; Cho, Am [Dongguk Univ., Seoul (Korea, Republic of)

    2001-06-01

    The bacterial infection on film cassette contact surface was examined at the diagnostic radiology department of the S. hospital. The objective of this study was to assess the contamination level on film cassette contact surface as a predictor of patient prevention from nosocomial infection and for improvement of the hospital environment. The laboratory result was identified non-pathologic bacterial in the five different cassette size of the contact surface. Film cassettes were exposed to ultraviolet light for 1, 2 and 3 minutes. Ultraviolet light disinfection is proven suitable for bacterial. The study concludes that presence of a bacterial infection will prevent a using antiseptic technique on film cassette contact surface. In addition education of nosocomial infection for radiographers will be required. In conclusion, ultraviolet is considered effective to irradiate bacterial. Additionally, two minutes are required to sterilize film cassettes.

  1. 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.

  2. Overcoming Multidrug Resistance in Human Cancer Cells by Natural Compounds

    OpenAIRE

    Nabekura, Tomohiro

    2010-01-01

    Multidrug resistance is a phenomenon whereby tumors become resistant to structurally unrelated anticancer drugs. P-glycoprotein belongs to the large ATP-binding cassette (ABC) transporter superfamily of membrane transport proteins. P-glycoprotein mediates resistance to various classes of anticancer drugs including vinblastine, daunorubicin, and paclitaxel, by actively extruding the drugs from the cells. The quest for inhibitors of anticancer drug efflux transporters has uncovered natural comp...

  3. 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

    2013-07-07

    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.

  4. 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

    2013-01-01

    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 achieve robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency though electrokinetic 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 electrokinetic for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future. PMID:23626988

  5. Emerging treatments in lung cancer – targeting the RLIP76 molecular transporter

    Directory of Open Access Journals (Sweden)

    Goldfinger LE

    2013-11-01

    Full Text Available Lawrence E Goldfinger,1,2 Seunghyung Lee1 1Department of Anatomy and Cell Biology, The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA, USA; 2Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, USA Abstract: Multidrug resistance in lung cancer cells is a significant obstacle in the treatment of lung cancer. Resistance to chemotherapeutic agents is often the result of efflux of the drugs from cancer cells, mediated by adenosine triphosphate (ATP-dependent drug transport across the plasma membrane. Thus, identifying molecular targets in the cancer cell transport machinery could be a key factor in successful combinatorial therapy, along with chemotherapeutic drugs. The transport protein Ral-interacting protein of 76 kDa (RLIP76, also known as Ral-binding protein 1 (RalBP1, is a highly promising target for lung cancer treatment. RLIP76 is an ATP-dependent non-ATP-binding cassette (ABC transporter, responsible for the major transport function in many cells, including many cancer cell lines, causing efflux of glutathione-electrophile conjugates of both endogenous metabolites and environmental toxins. RLIP76 is expressed in most human tissues, and is overexpressed in non-small-cell lung cancer cell lines and in many tumor types. The blockade of RLIP76 by various approaches has been shown to increase the sensitivity to radiation and chemotherapeutic drugs, and leads to apoptosis in cells. In xenograft tumor models in mice, RLIP76 blockade or depletion results in complete and sustained regression across many cancer cell types, including lung cancer cells. In addition to its transport function, RLIP76 has many other cellular and physiological functions based on its domain structure, which includes a unique Ral-binding domain and a Rho GTPase activating protein (RhoGAP-catalytic domain as well as docking sites for multiple signaling proteins. As a Ral effector, RhoGAP, and adapter protein, RLIP76

  6. Split tasks of asymmetric nucleotide-binding sites in the heterodimeric ABC exporter EfrCD.

    Science.gov (United States)

    Hürlimann, Lea M; Hohl, Michael; Seeger, Markus A

    2017-06-01

    Many heterodimeric ATP-binding cassette (ABC) exporters evolved asymmetric ATP-binding sites containing a degenerate site incapable of ATP hydrolysis due to noncanonical substitutions in conserved sequence motifs. Recent studies revealed that nucleotide binding to the degenerate site stabilizes contacts between the nucleotide-binding domains (NBDs) of the inward-facing transporter and regulates ATP hydrolysis at the consensus site via allosteric coupling mediated by the D-loops. However, it is unclear whether nucleotide binding to the degenerate site is strictly required for substrate transport. In this study, we examined the functional consequences of a systematic set of mutations introduced at the degenerate and consensus site of the multidrug efflux pump EfrCD of Enterococcus faecalis. Mutating motifs which differ among the two ATP-binding sites (Walker B, switch loop, and ABC signature) or which are involved in interdomain communication (D-loop and Q-loop) led to asymmetric results in the functional assays and were better tolerated at the degenerate site. This highlights the importance of the degenerate site to allosterically regulate the events at the consensus site. Mutating invariant motifs involved in ATP binding and NBD closure (A-loop and Walker A) resulted in equally reduced transport activities, regardless at which ATP-binding site they were introduced. In contrast to previously investigated heterodimeric ABC exporters, mutation of the degenerate site Walker A lysine completely inactivated ATPase activity and substrate transport, indicating that ATP binding to the degenerate site is essential for EfrCD. This study provides novel insights into the split tasks of asymmetric ATP-binding sites of heterodimeric ABC exporters. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  7. A survey of the radiographic cassettes disinfection of university hospitals in seoul

    Energy Technology Data Exchange (ETDEWEB)

    Kweon, Dae Cheol; Park, Peom [College of School, Ajou Univ., Suwon (Korea, Republic of); Kim, Moon Sun; Kim, Dong Sung [College of Medicine, Seoul National Univ., Seoul (Korea, Republic of)

    2001-04-01

    The purpose of this study is to prevent nosocomial infection in patients through contact of radiographic cassettes. Data were collected from radiographers working in 29 university hospitals in Seoul in February and March 2001. Radiographic cassettes were disinfected daily in 5 hospitals, weekly in 4 hospitals, monthly in 5 hospitals, bimonthly in 1 hospital and once every three months in another hospital. 12 other hospitals do not practice regular disinfections of radiographic cassettes. Gauze soaked in disinfectant solution is used in 7 hospitals while 11 hospitals used cotton and cloth soaked in disinfectant solution to clean the radiographic cassettes. 26 hospitals used 99% alcohol based disinfectant solutions while 3 hospitals used 75% alcohol based disinfectant, 26 hospitals use of intercourse cassettes outpatients and in patients. In 26 hospitals, all patients shared the same set of radiographic cassettes used in the hospitals, or in 26 hospitals, separate sets of radiographic cassettes are used for outpatients and inpatients. Separate sets of cassettes are used for ICU and inpatients in 6 others hospitals. 23 hospitals used the same sets of radiographic cassettes for all their patients. radiographic cassettes are cleaned in wash area in the study room of the radiographic department in 17 hospitals. 12 other hospitals do not have designated cleaning areas for the cassettes. All radiographers practiced hands washing with soap. All 29 hospitals surveyed have infection control committee. However, only 9 out of the 29 hospitals surveyed provided Infection {center_dot} disinfections control education to radiographers. Only 3 hospitals have radiographers sitting in the infection control committee. Infection management education is conducted in 63 hospitals annually, twice a year in 1 hospital and once every 3 months in 2 hospitals.

  8. 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

  9. Transportation

    Science.gov (United States)

    2006-01-01

    container. It now permits free transit of shipping containers from their western ports, if transported by rail directly to the U.S. ( Mireles , 2005, p...Transportation Industry Study Seminar. Mireles , Richard, Castillo. (2005, January). A Cure for West Coast Congestion. Logistics Today, Vol. 46, Issue 1. 1