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Sample records for background atp-binding cassette

  1. Two ATP Binding Cassette G Transporters, Rice ATP Binding Cassette G26 and ATP Binding Cassette G15, Collaboratively Regulate Rice Male Reproduction1[OPEN

    PubMed Central

    Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Xue, Feiyang; Luo, Qian; Zhu, Lu; Qu, Guorun; Chen, Mingjiao; Schreiber, Lukas; Zhang, Dabing

    2015-01-01

    Male reproduction in higher plants requires the support of various metabolites, including lipid molecules produced in the innermost anther wall layer (the tapetum), but how the molecules are allocated among different anther tissues remains largely unknown. Previously, rice (Oryza sativa) ATP binding cassette G15 (ABCG15) and its Arabidopsis (Arabidopsis thaliana) ortholog were shown to be required for pollen exine formation. Here, we report the significant role of OsABCG26 in regulating the development of anther cuticle and pollen exine together with OsABCG15 in rice. Cytological and chemical analyses indicate that osabcg26 shows reduced transport of lipidic molecules from tapetal cells for anther cuticle development. Supportively, the localization of OsABCG26 is on the plasma membrane of the anther wall layers. By contrast, OsABCG15 is polarly localized in tapetal plasma membrane facing anther locules. osabcg26 osabcg15 double mutant displays an almost complete absence of anther cuticle and pollen exine, similar to that of osabcg15 single mutant. Taken together, we propose that OsABCG26 and OsABCG15 collaboratively regulate rice male reproduction: OsABCG26 is mainly responsible for the transport of lipidic molecules from tapetal cells to anther wall layers, whereas OsABCG15 mainly is responsible for the export of lipidic molecules from the tapetal cells to anther locules for pollen exine development. PMID:26392263

  2. ATP binding cassette G transporters and plant male reproduction.

    PubMed

    Zhao, Guochao; Shi, Jianxin; Liang, Wanqi; Zhang, Dabing

    2016-01-01

    The function of ATP Binding Cassette G (ABCG) transporters in the regulation of plant vegetative organs development has been well characterized in various plant species. In contrast, their function in reproductive development particularly male reproductive development received considerably less attention till some ABCG transporters was reported to be associated with anther and pollen wall development in Arabidopsis thaliana and rice (Oryza sativa) during the past decade. This mini-review summarizes current knowledge of ABCG transporters regarding to their roles in male reproduction and underlying genetic and biochemical mechanisms, which makes it evident that ABCG transporters represent one of those conserved and divergent components closely related to male reproduction in plants. This mini-review also discusses the current challenges and future perspectives in this particular field. PMID:26906115

  3. Invited review: Architectures and mechanisms of ATP binding cassette proteins.

    PubMed

    Hopfner, Karl-Peter

    2016-08-01

    ATP binding cassette (ABC) ATPases form chemo-mechanical engines and switches that function in a broad range of biological processes. Most prominently, a very large family of integral membrane NTPases-ABC transporters-catalyzes the import or export of a diverse molecules across membranes. ABC proteins are also important components of the chromosome segregation, recombination, and DNA repair machineries and regulate or catalyze critical steps of ribosomal protein synthesis. Recent structural and mechanistic studies draw interesting architectural and mechanistic parallels between diverse ABC proteins. Here, I review this state of our understanding how NTP-dependent conformational changes of ABC proteins drive diverse biological processes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 492-504, 2016. PMID:27037766

  4. Evolutionary relationships of ATP-Binding Cassette (ABC) uptake porters

    PubMed Central

    2013-01-01

    Background The ATP-Binding Cassette (ABC) functional superfamily includes integral transmembrane exporters that have evolved three times independently, forming three families termed ABC1, ABC2 and ABC3, upon which monophyletic ATPases have been superimposed for energy-coupling purposes [e.g., J Membr Biol 231(1):1-10, 2009]. The goal of the work reported in this communication was to understand how the integral membrane constituents of ABC uptake transporters with different numbers of predicted or established transmembrane segments (TMSs) evolved. In a few cases, high resolution 3-dimensional structures were available, and in these cases, their structures plus primary sequence analyses allowed us to predict evolutionary pathways of origin. Results All of the 35 currently recognized families of ABC uptake proteins except for one (family 21) were shown to be homologous using quantitative statistical methods. These methods involved using established programs that compare native protein sequences with each other, after having compared each sequence with thousands of its own shuffled sequences, to gain evidence for homology. Topological analyses suggested that these porters contain numbers of TMSs ranging from four or five to twenty. Intragenic duplication events occurred multiple times during the evolution of these porters. They originated from a simple primordial protein containing 3 TMSs which duplicated to 6 TMSs, and then produced porters of the various topologies via insertions, deletions and further duplications. Except for family 21 which proved to be related to ABC1 exporters, they are all related to members of the previously identified ABC2 exporter family. Duplications that occurred in addition to the primordial 3 → 6 duplication included 5 → 10, 6 → 12 and 10 → 20 TMSs. In one case, protein topologies were uncertain as different programs gave discrepant predictions. It could not be concluded with certainty whether a 4 TMS ancestral

  5. Human ATP-binding cassette (ABC) transporter family

    PubMed Central

    2009-01-01

    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. PMID:19403462

  6. ATP-binding cassette transporters, atherosclerosis, and inflammation.

    PubMed

    Westerterp, Marit; Bochem, Andrea E; Yvan-Charvet, Laurent; Murphy, Andrew J; Wang, Nan; Tall, Alan R

    2014-01-01

    Although recent genome-wide association studies have called into question the causal relationship between high-density lipoprotein (HDL) cholesterol levels and cardiovascular disease, ongoing research in animals and cells has produced increasing evidence that cholesterol efflux pathways mediated by ATP-binding cassette (ABC) transporters and HDL suppress atherosclerosis. These differing perspectives may be reconciled by a modified HDL theory that emphasizes the antiatherogenic role of cholesterol flux pathways, initiated in cells by ABC transporters. ABCA1 and ABCG1 control the proliferation of hematopoietic stem and multipotential progenitor cells in the bone marrow and hematopoietic stem and multipotential progenitor cell mobilization and extramedullary hematopoiesis in the spleen. Thus, activation of cholesterol efflux pathways by HDL infusions or liver X receptor activation results in suppression of hematopoietic stem and multipotential progenitor cell mobilization and extramedullary hematopoiesis, leading to decreased production of monocytes and neutrophils and suppression of atherosclerosis. In addition, macrophage-specific knockout of transporters has confirmed their role in suppression of inflammatory responses in the arterial wall. Recent studies have also shown that ABCG4, a close relative of ABCG1, controls platelet production, atherosclerosis, and thrombosis. ABCG4 is highly expressed in megakaryocyte progenitors, where it promotes cholesterol efflux to HDL and controls the proliferative responses to thrombopoietin. Reconstituted HDL infusions act in an ABCG4-dependent fashion to limit hypercholesterolemia-driven excessive platelet production, thrombosis, and atherogenesis, as occurs in human myeloproliferative syndromes. Activation of ABC transporter-dependent cholesterol efflux pathways in macrophages, hematopoietic stem and multipotential progenitor cells, or platelet progenitors by reconstituted HDL infusion or liver X receptor activation remain

  7. Influence of ATP-binding cassette transporters in root exudation of phytoalexins, signals, and disease resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The roots of plants secrete compounds as a way to exchange information with organ-isms living in the soil. Here, we report the involvement of seven root-expressed ATP-binding cassette (ABC) transporters corresponding to both full and half-size molecules (Atabcg36, Atabcg37, Atabcc5, Atabcf1, Atabcf3...

  8. ATP-binding cassette (ABC) transporter expression and localization in sea urchin development

    PubMed Central

    Shipp, Lauren E.; Hamdoun, Amro

    2012-01-01

    Background ATP-binding cassette (ABC) transporters are membrane proteins that regulate intracellular concentrations of myriad compounds and ions. There are >100 ABC transporter predictions in the Strongylocentrotus purpuratus genome, including 40 annotated ABCB, ABCC, and ABCG “multidrug efflux” transporters. Despite the importance of multidrug transporters for protection and signaling, their expression patterns have not been characterized in deuterostome embryos. Results Sea urchin embryos expressed 20 ABCB, ABCC, and ABCG transporter genes in the first 58 hours of development, from unfertilized egg to early prism. We quantified transcripts of ABCB1a, ABCB4a, ABCC1, ABCC5a, ABCC9a, and ABCG2b, and found that ABCB1a mRNA was 10–100 times more abundant than other transporter mRNAs. In situ hybridization showed ABCB1a was expressed ubiquitously in embryos, while ABCC5a was restricted to secondary mesenchyme cells and their precursors. Fluorescent protein fusions showed localization of ABCB1a on apical cell surfaces, and ABCC5a on basolateral surfaces. Conclusions Embryos utilize many ABC transporters with predicted functions in cell signaling, lysosomal and mitochondrial homeostasis, potassium channel regulation, pigmentation, and xenobiotic efflux. Detailed characterization of ABCB1a and ABCC5a revealed that they have different temporal and spatial gene expression profiles and protein localization patterns that correlate to their predicted functions in protection and development, respectively. PMID:22473856

  9. New ATP-binding cassette A3 mutation causing surfactant metabolism dysfunction pulmonary type 3.

    PubMed

    Piersigilli, Fiammetta; Peca, Donatella; Campi, Francesca; Corsello, Mirta; Landolfo, Francesca; Boldrini, Renata; Danhaive, Olivier; Dotta, Andrea

    2015-10-01

    Respiratory distress syndrome (RDS) may occur in term and near-term infants because of mutations in surfactant-related genes. ATP-binding cassette A3 (ABCA3), a phospholipid carrier specifically expressed in the alveolar epithelium, is the most frequently involved protein. We report the case of a couple of late-preterm fraternal twin infants of opposite sex carrying the same compound heterozygous ABCA3 mutations, one of which has never been previously reported, with different disease severity, suggesting variable penetrance or sex-related differences. ABCA3 deficiency should be considered in term or near-term babies who develop unexplained RDS. PMID:26508177

  10. Phylogenetic and functional classification of ATP-binding cassette (ABC) systems.

    PubMed

    Bouige, Philippe; Laurent, David; Piloyan, Linda; Dassa, Elie

    2002-10-01

    ATP binding cassette (ABC) systems constitute one of the most abundant superfamilies of proteins. They are involved in the transport of a wide variety of substances, but also in many cellular processes and in their regulation. In this paper, we made a comparative analysis of the properties of ABC systems and we provide a phylogenetic and functional classification. This analysis will be helpful to accurately annotate ABC systems discovered during the sequencing of the genome of living organisms and to identify the partners of the ABC ATPases. PMID:12370001

  11. ATP-Binding Cassette Proteins: Towards a Computational View of Mechanism

    NASA Astrophysics Data System (ADS)

    Liao, Jielou

    2004-03-01

    Many large machine proteins can generate mechanical force and undergo large-scale conformational changes (LSCC) to perform varying biological tasks in living cells by utilizing ATP. Important examples include ATP-binding cassette (ABC) transporters. They are membrane proteins that couple ATP binding and hydrolysis to the translocation of substrates across membranes [1]. To interpret how the mechanical force generated by ATP binding and hydrolysis is propagated, a coarse-grained ATP-dependent harmonic network model (HNM) [2,3] is applied to the ABC protein, BtuCD. This protein machine transports vitamin B12 across membranes. The analysis shows that subunits of the protein move against each other in a concerted manner. The lowest-frequency modes of the BtuCD protein are found to link the functionally critical domains, and are suggested to be responsible for large-scale ATP-coupled conformational changes. [1] K. P. Locher, A. T. Lee and D. C. Rees. Science 296, 1091-1098 (2002). [2] Atilgan, A. R., S. R. Durell, R. L. Jernigan, M. C. Demirel, O. Keskin, and I. Bahar. Biophys. J. 80, 505-515(2002); M. M Tirion, Phys. Rev. Lett. 77, 1905-1908 (1996). [3] J. -L. Liao and D. N. Beratan, 2003, to be published.

  12. Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

    PubMed Central

    Davidson, Amy L.; Dassa, Elie; Orelle, Cedric; Chen, Jue

    2008-01-01

    Summary: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases. PMID:18535149

  13. ATP binding cassette transporter gene expression in rat liver progenitor cells

    PubMed Central

    Ros, J E; Roskams, T A D; Geuken, M; Havinga, R; Splinter, P L; Petersen, B E; LaRusso, N F; van der Kolk, D M; Kuipers, F; Faber, K N; Müller, M; Jansen, P L M

    2003-01-01

    Background and aim: Liver regeneration after severe liver damage depends in part on proliferation and differentiation of hepatic progenitor cells (HPCs). Under these conditions they must be able to withstand the toxic milieu of the damaged liver. ATP binding cassette (ABC) transporters are cytoprotective efflux pumps that may contribute to the preservation of these cells. The aim of this study was to determine the ABC transporter phenotype of HPCs. Methods: HPC activation was studied in rats treated with 2- acetylaminofluorene (2-AAF) followed by partial hepatectomy (PHx). ABC transporter gene expression was determined by real time detection reverse transcription-polymerase chain reaction in isolated HPCs, hepatocytes, cholangiocytes, and cultured progenitor cell-like RLF φ 13 cells and by immunohistochemistry of total liver samples. ABC transporter efflux activity was studied in RLF φ 13 cells by flow cytometry. Results: 2-AAF/PHx treated animals showed increased hepatic mRNA levels of the genes encoding multidrug resistance proteins Mdr1b, Mrp1, and Mrp3. Immunohistochemistry demonstrated expression of Mrp1 and Mrp3 proteins in periportal progenitor cells and of the Mdr1b protein in periportal hepatocytes. Freshly isolated Thy-1 positive cells and cultured RLF φ 13 progenitor cells highly expressed Mrp1 and Mrp3 mRNA while the hepatocyte specific transporters Mdr2, Bsep, Mrp2, and Mrp6 were only minimally expressed. Blocking Mrp activity by MK-571 resulted in accumulation of the Mrp specific substrate carboxyfluorescein in RLF φ 13 cells. Conclusion: HPCs express high levels of active Mrp1 and Mrp3. These may have a cytoprotective role in conditions of severe hepatotoxicity. PMID:12801967

  14. Formation of a Chloride-conducting State in the Maltose ATP-binding Cassette (ABC) Transporter.

    PubMed

    Carlson, Michael L; Bao, Huan; Duong, Franck

    2016-06-01

    ATP-binding cassette transporters use an alternating access mechanism to move substrates across cellular membranes. This mode of transport ensures the selective passage of molecules while preserving membrane impermeability. The crystal structures of MalFGK2, inward- and outward-facing, show that the transporter is sealed against ions and small molecules. It has yet to be determined whether membrane impermeability is maintained when MalFGK2 cycles between these two conformations. Through the use of a mutant that resides in intermediate conformations close to the transition state, we demonstrate that not only is chloride conductance occurring, but also to a degree large enough to compromise cell viability. Introduction of mutations in the periplasmic gate lead to the formation of a channel that is quasi-permanently open. MalFGK2 must therefore stay away from these ion-conducting conformations to preserve the membrane barrier; otherwise, a few mutations that increase access to the ion-conducting states are enough to convert an ATP-binding cassette transporter into a channel. PMID:27059961

  15. Selenodiglutathione uptake by the Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p.

    PubMed

    Lazard, Myriam; Ha-Duong, Nguyet-Thanh; Mounié, Stéphanie; Perrin, Romary; Plateau, Pierre; Blanquet, Sylvain

    2011-11-01

    The Saccharomyces cerevisiae vacuolar ATP-binding cassette transporter Ycf1p is involved in heavy metal detoxification by mediating the ATP-dependent transport of glutathione-metal conjugates to the vacuole. In the case of selenite toxicity, deletion of YCF1 was shown to confer increased resistance, rather than sensitivity, to selenite exposure [Pinson B, Sagot I & Daignan-Fornier B (2000) Mol Microbiol36, 679-687]. Here, we show that when Ycf1p is expressed from a multicopy plasmid, the toxicity of selenite is exacerbated. Using secretory vesicles isolated from a sec6-4 mutant transformed either with the plasmid harbouring YCF1 or the control plasmid, we establish that the glutathione-conjugate selenodigluthatione is a high-affinity substrate of this ATP-binding cassette transporter and that oxidized glutathione is also efficiently transported. Finally, we show that the presence of Ycf1p impairs the glutathione/oxidized glutathione ratio of cells subjected to a selenite stress. Possible mechanisms by which Ycf1p-mediated vacuolar uptake of selenodiglutathione and oxidized glutathione enhances selenite toxicity are discussed. PMID:21880115

  16. Microarray study of single nucleotide polymorphisms and expression of ATP-binding cassette genes in breast tumors

    NASA Astrophysics Data System (ADS)

    Tsyganov, M. M.; Ibragimova, M. K.; Karabut, I. V.; Freydin, M. B.; Choinzonov, E. L.; Litvyakov, N. V.

    2015-11-01

    Our previous research establishes that changes of expression of the ATP-binding cassette genes family is connected with the neoadjuvant chemotherapy effect. However, the mechanism of regulation of resistance gene expression remains unclear. As many researchers believe, single nucleotide polymorphisms can be involved in this process. Thereupon, microarray analysis is used to study polymorphisms in ATP-binding cassette genes. It is thus found that MDR gene expression is connected with 5 polymorphisms, i.e. rs241432, rs241429, rs241430, rs3784867, rs59409230, which participate in the regulation of expression of own genes.

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

    PubMed

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

    2013-11-01

    Currently, efficacious treatments for chemotherapy-induced alopecia (hair loss) are lacking, and incidences of permanent hair loss following high-dose chemotherapy are on the increase. In this article, we describe mechanisms by which the pharmacological defense status of the hair follicle might be enhanced, thereby reducing the accumulation of cytotoxic cancer drugs and preventing or reducing hair loss and damage. We believe this could be achieved via the selective increase in ATP-binding cassette (ABC) transporter expression within the hair follicle epithelium, following application of topical agonists for regulatory nuclear receptors. Clinical application would require the development of hair follicle-targeted formulations, potentially utilizing nanoparticle technology. This novel approach has the potential to yield entirely new therapeutic options for the treatment and management of chemotherapy-induced alopecia, providing significant psychological and physical benefit to cancer patients. PMID:24100054

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

    PubMed

    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. PMID:26517907

  19. Transport in technicolor: Mapping ATP-binding cassette transporters in sea urchin embryos

    PubMed Central

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

    2014-01-01

    One quarter of eukaryotic genes encode membrane proteins. These include nearly 1000 transporters that translocate nutrients, signaling molecules, and xenobiotics across membranes. While it is well appreciated that membrane transport is critical for development, the specific roles of many transporters have remained cryptic, in part because of their abundance and the diversity of their substrates. Multi-drug resistance ATP-binding cassette (ABC) efflux transporters are one example of cryptic membrane proteins. Although most organisms utilize these ABC transporters during embryonic development, many of these transporters have broad substrate specificity, and their developmental functions remain incompletely understood. Here, we review advances in our understanding of ABC transporters in sea urchin embryos, and methods developed to spatially and temporally map these proteins. These studies reveal that multifunctional transporters are required for signaling, homeostasis, and protection of the embryo, and shed light on how they are integrated into ancestral developmental pathways recapitulated in disease. PMID:25156004

  20. TSH increases synthesis of hepatic ATP-binding cassette subfamily A member 1 in hypercholesterolemia.

    PubMed

    Zhang, Tiantian; Zhou, Lingyan; Li, Cong Cong; Shi, Hong; Zhou, Xinli

    2016-07-22

    Epidemiological evidence suggests that thyrotropin (TSH) levels are closely correlated with the severity of hypercholesterolemia. Reverse cholesterol transfer (RCT) plays an important role in regulating bloodcholesterol. However, the molecular mechanism of hypercholesterolemia in subclinical hypothyroidism (SCH) has not been fully clarified. The SCH mouse model, which is characterized by elevated serum TSH but not thyroid hormone levels, demonstrated a significant increase in plasma cholesterol compared with controls. Interestingly, Tshr KO mice, with normal thyroid hormone levels after thyroid hormone supplementation, showed lower plasma cholesterol levels compared with their wild-type littermates. ATP binding cassette subfamily A member 1(ABCA1) is a member of the ABC superfamily, which induces transfer of intracellular cholesterol to extracellular apolipoprotein. TSH upregulated hepatic ABCA1 to promote the efflux of intercellular cumulative cholesterol, resulting in increased plasma cholesterol. These data might partially explain the pathogenesis of hypercholesterolemia in SCH. PMID:27179782

  1. Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers*

    PubMed Central

    Geillon, Flore; Gondcaille, Catherine; Charbonnier, Soëli; Van Roermund, Carlo W.; Lopez, Tatiana E.; Dias, Alexandre M. M.; Pais de Barros, Jean-Paul; Arnould, Christine; Wanders, Ronald J.; Trompier, Doriane; Savary, Stéphane

    2014-01-01

    ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2Δ yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters. PMID:25043761

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

    EPA Science Inventory

    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...

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

    PubMed

    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. PMID:27521922

  4. Masitinib antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance

    PubMed Central

    KATHAWALA, RISHIL J.; CHEN, JUN-JIANG; ZHANG, YUN-KAI; WANG, YI-JUN; PATEL, ATISH; WANG, DE-SHEN; TALELE, TANAJI T.; ASHBY, CHARLES R.; CHEN, ZHE-SHENG

    2014-01-01

    In this in vitro study, we determined whether masitinib could reverse multidrug resistance (MDR) in cells overexpressing the ATP binding cassette subfamily G member 2 (ABCG2) transporter. Masitinib (1.25 and 2.5 μM) significantly decreases the resistance to mitoxantrone (MX), SN38 and doxorubicin in HEK293 and H460 cells overexpressing the ABCG2 transporter. In addition, masitinib (2.5 μM) significantly increased the intracellular accumulation of [3H]-MX, a substrate for ABCG2, by inhibiting the function of ABCG2 and significantly decreased the efflux of [3H]-MX. However, masitinib (2.5 μM) did not significantly alter the expression of the ABCG2 protein. In addition, a docking model suggested that masitinib binds within the transmembrane region of a homology-modeled human ABCG2 transporter. Overall, our in vitro findings suggest that masitinib reverses MDR to various anti-neoplastic drugs in HEK293 and H460 cells overexpressing ABCG2 by inhibiting their transport activity as opposed to altering their levels of expression. PMID:24626598

  5. ATP-binding cassette transporters as pitfalls in selection of transgenic cells.

    PubMed

    Theile, Dirk; Staffen, Bianca; Weiss, Johanna

    2010-04-15

    Puromycin, hygromycin, and geneticin (G418) are antibiotics frequently used to select genetically engineered eukaryotic cells after transfection or transduction. Because intrinsic or acquired high expression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp/ABCB1) and multidrug resistance-associated proteins (MRP/ABCC1), can hamper efficient selection, it is important to know whether these antibiotics are substrates and/or inducers of efflux transporters. Therefore, we investigated the influence of these antibiotics on drug transporter expression by quantitative real-time polymerase chain reaction in the induction model cell line LS180. Moreover, we assessed whether ABC transporters influence the growth inhibitory effects of these antibiotics by proliferation assays using Madin-Darby canine kidney II (MDCKII) cells overexpressing the particular transporter. The results obtained indicate that puromycin and G418 are substrates of several ABC transporters, mainly Pgp/ABCB1. In contrast, hygromycin seems to be no good substrate for any of the ABC transporters investigated. Puromycin induced ABCC1/MRP1, whereas G418 suppressed ABCB1/Pgp, at the messenger RNA (mRNA) level. In contrast, hygromycin had no effect on ABC transporter mRNA expressions. In conclusion, this study emphasizes the significance of ABC transporters for the efficacy of selection processes. Consciousness of the results is supposed to guide the molecular biologist to the right choice of adequate experimental conditions for successful selection of genetically engineered eukaryotic cells. PMID:20018165

  6. Multidrug ATP-binding cassette transporters are essential for hepatic development of Plasmodium sporozoites.

    PubMed

    Rijpma, Sanna R; van der Velden, Maarten; González-Pons, Maria; Annoura, Takeshi; van Schaijk, Ben C L; van Gemert, Geert-Jan; van den Heuvel, Jeroen J M W; Ramesar, Jai; Chevalley-Maurel, Severine; Ploemen, Ivo H; Khan, Shahid M; Franetich, Jean-Francois; Mazier, Dominique; de Wilt, Johannes H W; Serrano, Adelfa E; Russel, Frans G M; Janse, Chris J; Sauerwein, Robert W; Koenderink, Jan B; Franke-Fayard, Blandine M

    2016-03-01

    Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development. PMID:26332724

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

    PubMed

    Sugiyama, Akifumi; Fukuda, Shoju; Takanashi, Kojiro; Yoshioka, Miki; Yoshioka, Hirofumi; Narusaka, Yoshihiro; Narusaka, Mari; Kojima, Mikiko; Sakakibara, Hitoshi; Shitan, Nobukazu; Sato, Shusei; Tabata, Satoshi; Kawaguchi, Masayoshi; Yazaki, Kazufumi

    2015-01-01

    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. PMID:26418593

  8. Tracing the structural evolution of eukaryotic ATP binding cassette transporter superfamily

    PubMed Central

    Xiong, Jie; Feng, Jinmei; Yuan, Dongxia; Zhou, Jun; Miao, Wei

    2015-01-01

    The ATP binding cassette (ABC) transporters superfamily is one of the largest classes of membrane proteins. The core of the ABC transporter protein is composed of transmembrane domains (TMDs) and nucleotide binding domains (NBD). Eukaryotes ABC transporters are classified into seven main families (ABCA to ABCG) based on sequence similarity and domain organizations. With different domain number and domain organizations, eukaryote ABC transporters show diverse structures: the single structure (NBD or TMD), the ABC2 structure (NBD-NBD), the half structure (TMD-NBD or NBD-TMD) and the full structure (TMD-NBD-TMD-NBD or NBD-TMD-NBD-TMD). However, studies on how various ABC transporter gene structures evolved is still absent. Therefore, in this study, we comprehensively investigated the structural evolution of eukaryotic ABC transporters. The seven eukaryote ABC transporter families (A to G) fell into three groups: A&G group, B,C&D group and E&F group. There were at least four times the number of NBD and TMD fusion events in the origin of the half structure transporter. Two fusion modes were found in the full and ABC2 structure origination. Based on these findings, we present a putative structural evolutionary path of eukaryote ABC transporters that will increase our understanding on their origin, divergence and function. PMID:26577702

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

    PubMed Central

    Sugiyama, Akifumi; Fukuda, Shoju; Takanashi, Kojiro; Yoshioka, Miki; Yoshioka, Hirofumi; Narusaka, Yoshihiro; Narusaka, Mari; Kojima, Mikiko; Sakakibara, Hitoshi; Shitan, Nobukazu; Sato, Shusei; Tabata, Satoshi; Kawaguchi, Masayoshi; Yazaki, Kazufumi

    2015-01-01

    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. PMID:26418593

  10. Akt isoform-dependent regulation of ATP-Binding cassette A1 expression by apolipoprotein E.

    PubMed

    Okoro, Emmanuel U; Guo, Zhongmao; Yang, Hong

    2016-08-12

    We previously reported that apolipoprotein E (apoE) upregulates ATP-binding cassette transporter A1 (ABCA1) transcription through phosphatidylinositol 3-kinase (PI3K). Here we demonstrate that treatment of murine macrophages with human apoE3 enhanced Akt phosphorylation, and upregulated ABCA1 protein and mRNA expression. Inhibition of PI3K weakened apoE3-induced Akt phosphorylation, and ABCA1 protein and mRNA increase. In contrast, inhibition of Akt only diminished apoE-induced ABCA1 protein but not the mRNA level. Suppression of protein synthesis did not erase the ability of apoE3 to increase ABCA1 protein level. Further, apoE3 increased the resistance of ABCA1 protein to calpain-mediated degradation without affecting calpain activity. Treatment of macrophages with apoE3 selectively enhanced the phosphorylation of Akt1 and Akt2, but not Akt3. Knockdown of Akt1 or Akt2 increased and decreased ABCA1 protein level, respectively; while overexpression of these Akt isoenzymes caused changes in ABCA1 protein level opposite to those induced by knockdown of the corresponding Akt. These data imply that apoE3 guards against calpain-mediated ABCA1 degradation through Akt2. PMID:27297104

  11. A novel ATP-binding cassette transporter, ABCG6 is involved in chemoresistance of Leishmania.

    PubMed

    BoseDasgupta, Somdeb; Ganguly, Agneyo; Roy, Amit; Mukherjee, Tanmoy; Majumder, Hemanta K

    2008-04-01

    ATP-binding cassette (ABC) transporters constitute the biggest family of membrane proteins involved in drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries and in many instances it is due to overexpressed ABC efflux pumps. Progressively adapted camptothecin (CPT)-resistant parasites show overexpression of a novel ABC transporter, which was classified as ABCG6. Transfection and overexpression of LdABCG6 in wild type parasites, shows its localization primarily in the plasma membrane and flagellar pocket region. Overexpressed LdABCG6 confers substantial CPT resistance to the parasites by rapid drug efflux. Various inhibitors have been tested for their ability to revert the CPT-resistant phenotype to specifically understand the inhibition of LdABCG6 transporter. Transport experiments using everted membrane vesicles were carried out to gain an insight into the kinetics of drug transport. This study provides further knowledge of specific membrane traffic ATPase and its involvement in the chemoresistance of Leishmania. PMID:18243364

  12. A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

    PubMed Central

    Chavan, Hemantkumar; Young, Susan; Ma, Xiaochao; Waller, Anna; Garcia, Matthew; Perez, Dominique; Chavez, Stephanie; Strouse, Jacob J.; Haynes, Mark K.; Bologa, Cristian G.; Oprea, Tudor I.; Tegos, George P.; Sklar, Larry A.; Krishnamurthy, Partha

    2012-01-01

    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. PMID:22808084

  13. Inflammatory Regulation of ATP Binding Cassette Efflux Transporter Expression and Function in Microglia

    PubMed Central

    Gibson, Christopher J.; Hossain, Muhammad M.; Richardson, Jason R.

    2012-01-01

    ATP-binding cassette (ABC) efflux transporters, including multidrug resistance protein 1 (Mdr1), breast cancer resistance protein (Bcrp), and multidrug resistance-associated proteins (Mrps) extrude chemicals from the brain. Although ABC transporters are critical for blood-brain barrier integrity, less attention has been placed on the regulation of these proteins in brain parenchymal cells such as microglia. Prior studies demonstrate that inflammation after lipopolysaccharide (LPS) treatment alters transporter expression in the livers of mice. Here, we sought to determine the effects of inflammation on the expression and function of transporters in microglia. To test this, the expression and function of ABC efflux transport proteins were quantified in mouse BV-2 microglial cells in response to activation with LPS. Intracellular retention of fluorescent rhodamine 123, Hoechst 33342, and calcein acetoxymethyl ester was increased in LPS-treated microglia, suggesting that the functions of Mdr1, Bcrp, and Mrps were decreased, respectively. LPS reduced Mdr1, Bcrp, and Mrp4 mRNA and protein expression between 40 and 70%. Conversely, LPS increased expression of Mrp1 and Mrp5 mRNA and protein. Immunofluorescent staining confirmed reduced Bcrp and Mrp4 and elevated Mrp1 and Mrp5 protein in activated microglia. Pharmacological inhibition of nuclear factor κB (NF-κB) transcriptional signaling attenuated down-regulation of Mdr1a mRNA and potentiated up-regulation of Mrp5 mRNA in LPS-treated cells. Together, these data suggest that LPS stimulates microglia and impairs efflux of prototypical ABC transporter substrates by altering mRNA and protein expression, in part through NF-κB signaling. Decreased transporter efflux function in microglia may lead to the retention of toxic chemicals and aberrant cell-cell communication during neuroinflammation. PMID:22942241

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

    PubMed Central

    van der Deen, Margaretha; de Vries, Elisabeth GE; Timens, Wim; Scheper, Rik J; Timmer-Bosscha, Hetty; Postma, Dirkje S

    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 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 (mal)function 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. PMID:15967026

  15. ATP-binding cassette transporter A7 (ABCA7) loss of function alters Alzheimer amyloid processing.

    PubMed

    Satoh, Kanayo; Abe-Dohmae, Sumiko; Yokoyama, Shinji; St George-Hyslop, Peter; Fraser, Paul E

    2015-10-01

    The ATP-binding cassette transporter A7 (ABCA7) has been identified as a susceptibility factor of late onset Alzheimer disease in genome-wide association studies. ABCA7 has been shown to mediate phagocytosis and affect membrane trafficking. The current study examined the impact of ABCA7 loss of function on amyloid precursor protein (APP) processing and generation of amyloid-β (Aβ). Suppression of endogenous ABCA7 in several different cell lines resulted in increased β-secretase cleavage and elevated Aβ. ABCA7 knock-out mice displayed an increased production of endogenous murine amyloid Aβ42 species. Crossing ABCA7-deficient animals to an APP transgenic model resulted in significant increases in the soluble Aβ as compared with mice expressing normal levels of ABCA7. Only modest changes in the amount of insoluble Aβ and amyloid plaque densities were observed once the amyloid pathology was well developed, whereas Aβ deposition was enhanced in younger animals. In vitro studies indicated a more rapid endocytosis of APP in ABCA7 knock-out cells that is mechanistically consistent with the increased Aβ production. These in vitro and in vivo findings indicate a direct role of ABCA7 in amyloid processing that may be associated with its primary biological function to regulate endocytic pathways. Several potential loss-of-function ABCA7 mutations and deletions linked to Alzheimer disease that in some instances have a greater impact than apoE allelic variants have recently been identified. A reduction in ABCA7 expression or loss of function would be predicted to increase amyloid production and that may be a contributing factor in the associated Alzheimer disease susceptibility. PMID:26260791

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

    PubMed

    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

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

    PubMed Central

    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

  18. Function and expression of ATP-binding cassette transporters in cultured human Y79 retinoblastoma cells.

    PubMed

    Ishikawa, Yuka; Nagai, Junya; Okada, Yumi; Sato, Koya; Yumoto, Ryoko; Takano, Mikihisa

    2010-01-01

    The aim of this study was to reveal the expression and function of P-glycoprotein and multidrug resistance-associated proteins (MRP), members of the ATP-binding cassette (ABC) superfamily of drug transporters, in cultured human Y79 retinoblastoma cells. ABC transporter mRNA expression was evaluated by conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analyses. Cellular accumulation of rhodamine 123 (P-glycoprotein substrate), calcein (MRP substrate), and doxorubicin (P-glycoprotein/MRP substrate) was analyzed by fluorometry. Conventional RT-PCR analysis showed the expression of multidrug resistance 1 (MDR1), MRP1, MRP2 and lung resistance-related protein (LRP) mRNAs. Real-time RT-PCR analysis revealed that the expression levels of the MDR1 and MRP2 genes in Y79 cells were much lower than those in human intestinal cell line Caco-2, while the expression level of MRP1 was higher than that in Caco-2 cells. The accumulation of rhodamine 123 was not enhanced by verapamil or reversin 205, inhibitors of P-glycoprotein, indicating no function of P-glycoprotein in Y79 cells. The accumulation of calcein was significantly increased by various MRP inhibitors including probenecid, indicating that MRP functions in Y79 cells. The accumulation of doxorubicin was increased in the presence of metabolic inhibitors (10 mM 2-deoxyglucose and 5 mM sodium azide). However, most MRP inhibitors such as probenecid and indomethacin did not affect doxorubicin accumulation, while cyclosporin A and taclorimus significantly increased doxorubicin accumulation. These results suggest that MRP, but not P-glycoprotein, functions in Y79 cells, and that the efflux of doxorubicin from Y79 cells may be due to an ATP-dependent transporter, which has not been identified yet. PMID:20190417

  19. Contributions of Aspergillus fumigatus ATP-Binding Cassette Transporter Proteins to Drug Resistance and Virulence

    PubMed Central

    Paul, Sanjoy; Diekema, Daniel

    2013-01-01

    In yeast cells such as those of Saccharomyces cerevisiae, expression of ATP-binding cassette (ABC) transporter proteins has been found to be increased and correlates with a concomitant elevation in azole drug resistance. In this study, we investigated the roles of two Aspergillus fumigatus proteins that share high sequence similarity with S. cerevisiae Pdr5, an ABC transporter protein that is commonly overproduced in azole-resistant isolates in this yeast. The two A. fumigatus genes encoding the ABC transporters sharing the highest sequence similarity to S. cerevisiae Pdr5 are called abcA and abcB here. We constructed deletion alleles of these two different ABC transporter-encoding genes in three different strains of A. fumigatus. Loss of abcB invariably elicited increased azole susceptibility, while abcA disruption alleles had variable phenotypes. Specific antibodies were raised to both AbcA and AbcB proteins. These antisera allowed detection of AbcB in wild-type cells, while AbcA could be visualized only when overproduced from the hspA promoter in A. fumigatus. Overproduction of AbcA also yielded increased azole resistance. Green fluorescent protein fusions were used to provide evidence that both AbcA and AbcB are localized to the plasma membrane in A. fumigatus. Promoter fusions to firefly luciferase suggested that expression of both ABC transporter-encoding genes is inducible by azole challenge. Virulence assays implicated AbcB as a possible factor required for normal pathogenesis. This work provides important new insights into the physiological roles of ABC transporters in this major fungal pathogen. PMID:24123268

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

    PubMed

    Xue, Shanshan; Wang, Jiaxing; Zhang, Xu; Shi, Ying; Li, Bochuan; Bao, Qiankun; Pang, Wei; Ai, Ding; Zhu, Yi; He, Jinlong

    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(-/-)) 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(-/-) mouse aortas, EC-ABCG1-Tg/Ldlr(-/-) aortas showed decreased early-stage lesions. Furthermore, the lesion area in the EC-ABCG1-Tg/Ldlr(-/-) 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. PMID:27297110

  1. Association of ATP-Binding Cassette Transporter A1 Gene Polymorphisms in Type 2 Diabetes Mellitus among Malaysians

    PubMed Central

    Haghvirdizadeh, Polin; Ramachandran, Vasudevan; Etemad, Ali; Heidari, Farzad; Ghodsian, Nooshin; Bin Ismail, Norzian; Ismail, Patimah

    2015-01-01

    Background. Type 2 diabetes mellitus (T2DM) is a complex polygenic disorder characterized by impaired insulin resistance, insulin secretion, and dysregulation of lipid and protein metabolism with environmental and genetic factors. ATP-binding cassette transporter A1 (ABCA1) gene polymorphisms are reported as the one of the genetic risk factors for T2DM in various populations with conflicting results. This study was conducted based on PCR-HRM to determine the frequency of ABCA1 gene by rs2230806 (R219K), rs1800977 (C69T), and rs9282541 (R230C) polymorphisms Malaysian subjects. Methods. A total of 164 T2DM and 165 controls were recruited and their genotypes for ABCA1 gene polymorphisms were determined based on the real time high resolution melting analysis. Results. There was a significant difference between the subjects in terms of age, BMI, FPG, HbA1c, HDL, LDL, and TG (P < 0.05). There was a significant association between HOM of R219K (P = 0.005), among Malaysian subjects; moreover, allele frequency revealed the significant difference in A allele of R219K (P = 0.003). But, there was no significant difference in genotypic and allelic frequencies of C69T and R230C polymorphism. Conclusion. R219K polymorphism of ABCA1 gene can be considered as a genetic risk factor for T2DM subjects among Malaysians. PMID:26451383

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

    SciTech Connect

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

    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

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

    PubMed Central

    2012-01-01

    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. PMID:22906146

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

    SciTech Connect

    Miao, Y.C.; Liu, C.

    2010-12-28

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

  5. The Allosteric Regulatory Mechanism of the Escherichia coli MetNI Methionine ATP Binding Cassette (ABC) Transporter*

    PubMed Central

    Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    The MetNI methionine importer of Escherichia coli, an ATP binding cassette (ABC) transporter, uses the energy of ATP binding and hydrolysis to catalyze the high affinity uptake of d- and l-methionine. Early in vivo studies showed that the uptake of external methionine is repressed by the level of the internal methionine pool, a phenomenon termed transinhibition. Our understanding of the MetNI mechanism has thus far been limited to a series of crystal structures in an inward-facing conformation. To understand the molecular mechanism of transinhibition, we studied the kinetics of ATP hydrolysis using detergent-solubilized MetNI. We find that transinhibition is due to noncompetitive inhibition by l-methionine, much like a negative feedback loop. Thermodynamic analyses revealed two allosteric methionine binding sites per transporter. This quantitative analysis of transinhibition, the first to our knowledge for a structurally defined transporter, builds upon the previously proposed structurally based model for regulation. This mechanism of regulation at the transporter activity level could be applicable to not only ABC transporters but other types of membrane transporters as well. PMID:25678706

  6. CpABC, a Cryptosporidium parvum ATP-binding cassette protein at the host–parasite boundary in intracellular stages

    PubMed Central

    Perkins, Margaret E.; Riojas, Ynolde A.; Wu, Teresa W.; Le Blancq, Sylvie M.

    1999-01-01

    The intracellular parasite Cryptosporidium parvum develops inside a vacuole at the apex of its epithelial host cell. The developing parasite is separated from the host cell cytoplasm by a zone of attachment that consists of an extensively folded membranous structure known as the feeder organelle. It has been proposed that the feeder organelle is the site of regulation of transport of nutrients and drugs into the parasite. In this report, we localize an ≈200-kDa integral membrane protein, CpABC, from Cryptosporidium parvum to the host–parasite boundary, possibly the feeder organelle. The predicted amino acid sequence of CpABC has significant structural similarity with the cystic fibrosis conductance regulator and the multidrug resistance protein subfamily of ATP-binding cassette proteins. This is an example of a parasite-encoded transport protein localized at the parasite–host interface of an intracellular protozoan. PMID:10318953

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

    PubMed Central

    Wang, Yi-Jun; Zhang, Yun-Kai; Kathawala, Rishil J.; Chen, Zhe-Sheng

    2014-01-01

    The phenomenon of multidrug resistance (MDR) has attenuated the efficacy of anticancer drugs and the possibility of successful cancer chemotherapy. ATP-binding cassette (ABC) transporters play an essential role in mediating MDR in cancer cells by increasing efflux of drugs from cancer cells, hence reducing the intracellular accumulation of chemotherapeutic drugs. Interestingly, small-molecule tyrosine kinase inhibitors (TKIs), such as AST1306, lapatinib, linsitinib, masitinib, motesanib, nilotinib, telatinib and WHI-P154, have been found to have the capability to overcome anticancer drug resistance by inhibiting ABC transporters in recent years. This review will focus on some of the latest and clinical developments with ABC transporters, TKIs and anticancer drug resistance. PMID:25268163

  8. In Vivo Bioluminescent Imaging of ATP-Binding Cassette Transporter-Mediated Efflux at the Blood-Brain Barrier.

    PubMed

    Bakhsheshian, Joshua; Wei, Bih-Rong; Hall, Matthew D; Simpson, R Mark; Gottesman, Michael M

    2016-01-01

    We provide a detailed protocol for imaging ATP-binding cassette subfamily G member 2 (ABCG2) function at the blood-brain barrier (BBB) of transgenic mice. D-Luciferin is specifically transported by ABCG2 found on the apical side of endothelial cells at the BBB. The luciferase-luciferin enzymatic reaction produces bioluminescence, which allows a direct measurement of ABCG2 function at the BBB. Therefore bioluminescence imaging (BLI) correlates with ABCG2 function at the BBB and this can be measured by administering luciferin in a mouse model that expresses luciferase in the brain parenchyma. BLI allows for a relatively low-cost alternative for studying transporter function in vivo compared to other strategies such as positron emission tomography. This method for imaging ABCG2 function at the BBB can be used to investigate pharmacokinetic inhibition of the transporter. PMID:27424909

  9. In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

    PubMed Central

    Clifton, Matthew C.; Simon, Michael J.; Erramilli, Satchal K.; Zhang, Huide; Zaitseva, Jelena; Hermodson, Mark A.; Stauffacher, Cynthia V.

    2015-01-01

    Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters. PMID:25533465

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

    PubMed Central

    2012-01-01

    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 < 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). 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 (...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Identification and expression analysis of ABC protein-encoding genes in Toxoplasma gondii. Toxoplasma gondii ATP-binding cassette superfamily.

    PubMed

    Sauvage, Virginie; Millot, Jean-Marc; Aubert, Dominique; Visneux, Vincent; Marle-Plistat, Maggy; Pinon, Jean-Michel; Villena, Isabelle

    2006-06-01

    The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii. PMID:16600400

  14. Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5.

    PubMed

    Matsuda, Shuichi; Takano, Sho; Sato, Moeko; Furukawa, Kaoru; Nagasawa, Hidetaka; Yoshikawa, Shoko; Kasuga, Jun; Tokuji, Yoshihiko; Yazaki, Kazufumi; Nakazono, Mikio; Takamure, Itsuro; Kato, Kiyoaki

    2016-03-01

    Water stress is one of the major environmental stresses that affect agricultural production worldwide. Water loss from plants occurs primarily through stomatal pores. Here, we report that an Oryza sativa half-size ATP-binding cassette (ABC) subfamily G protein, RCN1/OsABCG5, is involved in stomatal closure mediated by phytohormone abscisic acid (ABA) accumulation in guard cells. We found that the GFP-RCN1/OsABCG5-fusion protein was localized at the plasma membrane in guard cells. The percentage of guard cell pairs containing both ABA and GFP-RCN1/OsABCG5 increased after exogenous ABA treatment, whereas they were co-localized in guard cell pairs regardless of whether exogenous ABA was applied. ABA application resulted in a smaller increase in the percentage of guard cell pairs containing ABA in rcn1 mutant (A684P) and RCN1-RNAi than in wild-type plants. Furthermore, polyethylene glycol (drought stress)-inducible ABA accumulation in guard cells did not occur in rcn1 mutants. Stomata closure mediated by exogenous ABA application was strongly reduced in rcn1 mutants. Finally, rcn1 mutant plants had more rapid water loss from detached leaves than the wild-type plants. These results indicate that in response to drought stress, RCN1/OsABCG5 is involved in accumulation of ABA in guard cells, which is indispensable for stomatal closure. PMID:26708605

  15. Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters.

    PubMed

    Saurin, W; Hofnung, M; Dassa, E

    1999-01-01

    ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems. PMID:9873074

  16. HG-829 is a potent noncompetitive inhibitor of the ATP-binding cassette multidrug resistance transporter ABCB1.

    PubMed

    Caceres, Gisela; Robey, Robert W; Sokol, Lubomir; McGraw, Kathy L; Clark, Justine; Lawrence, Nicholas J; Sebti, Said M; Wiese, Michael; List, Alan F

    2012-08-15

    Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrug-resistant malignancies. PMID:22761337

  17. Genome-wide analysis of the ATP-binding cassette (ABC) transporter gene family in the silkworm, Bombyx mori.

    PubMed

    Xie, Xiaodong; Cheng, Tingcai; Wang, Genhong; Duan, Jun; Niu, Weihuan; Xia, Qingyou

    2012-07-01

    The ATP-binding cassette (ABC) superfamily is a larger protein family with diverse physiological functions in all kingdoms of life. We identified 53 ABC transporters in the silkworm genome, and classified them into eight subfamilies (A-H). Comparative genome analysis revealed that the silkworm has an expanded ABCC subfamily with more members than Drosophila melanogaster, Caenorhabditis elegans, or Homo sapiens. Phylogenetic analysis showed that the ABCE and ABCF genes were highly conserved in the silkworm, indicating possible involvement in fundamental biological processes. Five multidrug resistance-related genes in the ABCB subfamily and two multidrug resistance-associated-related genes in the ABCC subfamily indicated involvement in biochemical defense. Genetic variation analysis revealed four ABC genes that might be evolving under positive selection. Moreover, the silkworm ABCC4 gene might be important for silkworm domestication. Microarray analysis showed that the silkworm ABC genes had distinct expression patterns in different tissues on day 3 of the fifth instar. These results might provide new insights for further functional studies on the ABC genes in the silkworm genome. PMID:22311044

  18. Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver*

    PubMed Central

    Chavan, Hemantkumar; Li, Feng; Tessman, Robert; Mickey, Kristen; Dorko, Kenneth; Schmitt, Timothy; Kumer, Sean; Gunewardena, Sumedha; Gaikwad, Nilesh; Krishnamurthy, Partha

    2015-01-01

    Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s. PMID:25623066

  19. The ATP-binding cassette transporter OsABCG15 is required for anther development and pollen fertility in rice.

    PubMed

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

    2013-08-01

    Plant male reproductive development is a complex biological process, but the underlying mechanism is not well understood. Here, we characterized a rice (Oryza sativa L.) male sterile mutant. Based on map-based cloning and sequence analysis, we identified a 1,459-bp deletion in an adenosine triphosphate (ATP)-binding cassette (ABC) transporter gene, OsABCG15, causing abnormal anthers and male sterility. Therefore, we named this mutant osabcg15. Expression analysis showed that OsABCG15 is expressed specifically in developmental anthers from stage 8 (meiosis II stage) to stage 10 (late microspore stage). Two genes CYP704B2 and WDA1, involved in the biosynthesis of very-long-chain fatty acids for the establishment of the anther cuticle and pollen exine, were downregulated in osabcg15 mutant, suggesting that OsABCG15 may play a key function in the processes related to sporopollenin biosynthesis or sporopollenin transfer from tapetal cells to anther locules. Consistently, histological analysis showed that osabcg15 mutants developed obvious abnormality in postmeiotic tapetum degeneration, leading to rapid degredation of young microspores. The results suggest that OsABCG15 plays a critical role in exine formation and pollen development, similar to the homologous gene of AtABCG26 in Arabidopsis. This work is helpful to understand the regulatory network in rice anther development. PMID:23570336

  20. ABCC1, an ATP Binding Cassette Protein from Grape Berry, Transports Anthocyanidin 3-O-Glucosides[W][OA

    PubMed Central

    Francisco, Rita Maria; Regalado, Ana; Ageorges, Agnès; Burla, Bo J.; Bassin, Barbara; Eisenach, Cornelia; Zarrouk, Olfa; Vialet, Sandrine; Marlin, Thérèse; Chaves, Maria Manuela; Martinoia, Enrico; Nagy, Réka

    2013-01-01

    Accumulation of anthocyanins in the exocarp of red grapevine (Vitis vinifera) cultivars is one of several events that characterize the onset of grape berry ripening (véraison). Despite our thorough understanding of anthocyanin biosynthesis and regulation, little is known about the molecular aspects of their transport. The participation of ATP binding cassette (ABC) proteins in vacuolar anthocyanin transport has long been a matter of debate. Here, we present biochemical evidence that an ABC protein, ABCC1, localizes to the tonoplast and is involved in the transport of glucosylated anthocyanidins. ABCC1 is expressed in the exocarp throughout berry development and ripening, with a significant increase at véraison (i.e., the onset of ripening). Transport experiments using microsomes isolated from ABCC1-expressing yeast cells showed that ABCC1 transports malvidin 3-O-glucoside. The transport strictly depends on the presence of GSH, which is cotransported with the anthocyanins and is sensitive to inhibitors of ABC proteins. By exposing anthocyanin-producing grapevine root cultures to buthionine sulphoximine, which reduced GSH levels, a decrease in anthocyanin concentration is observed. In conclusion, we provide evidence that ABCC1 acts as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate. PMID:23723325

  1. Stickleback embryos use ATP-binding cassette transporters as a buffer against exposure to maternally derived cortisol.

    PubMed

    Paitz, Ryan T; Bukhari, Syed Abbas; Bell, Alison M

    2016-03-16

    Offspring from females that experience stressful conditions during reproduction often exhibit altered phenotypes and many of these effects are thought to arise owing to increased exposure to maternal glucocorticoids. While embryos of placental vertebrates are known to regulate exposure to maternal glucocorticoids via placental steroid metabolism, much less is known about how and whether egg-laying vertebrates can control their steroid environment during embryonic development. We tested the hypothesis that threespine stickleback (Gasterosteus aculeatus) embryos can regulate exposure to maternal steroids via active efflux of maternal steroids from the egg. Embryos rapidly (within 72 h) cleared intact steroids, but blocking ATP-binding cassette (ABC) transporters inhibited cortisol clearance. Remarkably, this efflux of cortisol was sufficient to prevent a transcriptional response of embryos to exogenous cortisol. Taken together, these findings suggest that, much like their placental counterparts, developing fish embryos can actively regulate their exposure to maternal cortisol. These findings highlight the fact that even in egg-laying vertebrates, the realized exposure to maternal steroids is mediated by both maternal and embryonic processes and this has important implications for understanding how maternal stress influences offspring development. PMID:26984623

  2. Whole-Genome Survey of the Putative ATP-Binding Cassette Transporter Family Genes in Vitis vinifera

    PubMed Central

    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. PMID:24244377

  3. Functional coupling of ATP-binding cassette transporter Abcb6 to cytochrome P450 expression and activity in liver.

    PubMed

    Chavan, Hemantkumar; Li, Feng; Tessman, Robert; Mickey, Kristen; Dorko, Kenneth; Schmitt, Timothy; Kumer, Sean; Gunewardena, Sumedha; Gaikwad, Nilesh; Krishnamurthy, Partha

    2015-03-20

    Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s. PMID:25623066

  4. Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter.

    PubMed Central

    Ortiz, D F; Kreppel, L; Speiser, D M; Scheel, G; McDonald, G; Ow, D W

    1992-01-01

    In response to heavy metal stress, plants and certain fungi, such as the fission yeast Schizosaccharomyces pombe, synthesize small metal-binding peptides known as phytochelatins. We have identified a cadmium sensitive S. pombe mutant deficient in the accumulation of a sulfide-containing phytochelatin-cadmium complex, and have isolated the gene, designated hmt1, that complements this mutant. The deduced protein sequence of the hmt1 gene product shares sequence identity with the family of ABC (ATP-binding cassette)-type transport proteins which includes the mammalian P-glycoproteins and CFTR, suggesting that the encoded product is an integral membrane protein. Analysis of fractionated fission yeast cell components indicates that the HMT1 polypeptide is associated with the vacuolar membrane. Additionally, fission yeast strains harboring an hmt1-expressing multicopy plasmid exhibit enhanced metal tolerance along with a higher intracellular level of cadmium, implying a relationship between HMT1 mediated transport and compartmentalization of heavy metals. This suggests that tissue-specific overproduction of a functional hmt1 product in transgenic plants might be a means to alter the tissue localization of these elements, such as for sequestering heavy metals away from consumable parts of crop plants. Images PMID:1396551

  5. Inventory and general analysis of the ATP-binding cassette (ABC) gene superfamily in maize (Zea mays L.).

    PubMed

    Pang, Kaiyuan; Li, Yanjiao; Liu, Menghan; Meng, Zhaodong; Yu, Yanli

    2013-09-10

    The metabolic functions of ATP-binding cassette (or ABC) proteins, one of the largest families of proteins presented in all organisms, have been investigated in many protozoan, animal and plant species. To facilitate more systematic and complicated studies on maize ABC proteins in the future, we present the first complete inventory of these proteins, including 130 open reading frames (ORFs), and provide general descriptions of their classifications, basic structures, typical functions, evolution track analysis and expression profiles. The 130 ORFs were assigned to eight subfamilies based on their structures and homological features. Five of these subfamilies consist of 109 proteins, containing transmembrane domains (TM) performing as transporters. The rest three subfamilies contain 21 soluble proteins involved in various functions other than molecular transport. A comparison of ABC proteins among nine selected species revealed either convergence or divergence in each of the ABC subfamilies. Generally, plant genomes contain far more ABC genes than animal genomes. The expression profiles and evolution track of each maize ABC gene were further investigated, the results of which could provide clues for analyzing their functions. Quantitative real-time polymerase chain reaction experiments (PCR) were conducted to detect induced expression in select ABC genes under several common stresses. This investigation provides valuable information for future research on stress tolerance in plants and potential strategies for enhancing maize production under stressful conditions. PMID:23747399

  6. Characterization of the ATP-binding cassette transporter gene expression profile in Y79: a retinoblastoma cell line.

    PubMed

    Hendig, Doris; Langmann, Thomas; Zarbock, Ralf; Schmitz, Gerd; Kleesiek, Knut; Götting, Christian

    2009-08-01

    Chemotherapy failure was reported in treatment of retinoblastoma suggesting a role for ATP-binding cassette (ABC) proteins. Little is known about the expression pattern of ABC proteins in this cancer type. We investigated the gene expression profile of 47 ABC proteins in the human retinoblastoma cell line Y79 by TaqMan low-density array. Analysis revealed 31 ABC transporter genes expressed in this tumor cell line. Y79 cells demonstrate high gene expression of ABCA7, ABCA12, ABCB7, ABCB10, ABCC1, ABCC4, ABCD3, ABCE1, ABCF1, ABCF2, and ABCF3 (more than twofold compared to pooled RNA from different tissues). Moreover, we show that Y79 cells exhibit an active calcein efflux pointing to multidrug resistance protein (MRP)-like transporter activity. In summary, we present for the first time an ABC transporter gene expression profile in cells derived from retinoblastoma. Most of the highly expressed ABC transporter genes are typical markers of cancer cells and might exhibit potential targets for medical treatment of retinoblastoma. PMID:19266166

  7. Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters

    PubMed Central

    Uto-Kondo, Harumi; Ayaori, Makoto; Nakaya, Kazuhiro; Takiguchi, Shunichi; Yakushiji, Emi; Ogura, Masatsune; Terao, Yoshio; Ozasa, Hideki; Sasaki, Makoto; Komatsu, Tomohiro; Sotherden, Grace Megumi; Hosoai, Tamaki; Sakurada, Masami; Ikewaki, Katsunori

    2014-01-01

    Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30–49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL. PMID:25120277

  8. ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs.

    PubMed

    Jansen, Robert S; Mahakena, Sunny; de Haas, Marcel; Borst, Piet; van de Wetering, Koen

    2015-12-18

    The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate was 2.4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs. PMID:26515061

  9. Gestational and pregnane X receptor-mediated regulation of placental ATP-binding cassette drug transporters in mice.

    PubMed

    Gahir, Sarabjit S; Piquette-Miller, Micheline

    2011-03-01

    The ATP-binding cassette (ABC) drug transporters in the placenta are involved in controlling the exchange of endogenous and exogenous moieties. Pregnane X receptor (PXR) is a nuclear receptor that regulates the hepatic expression of several key ABC transporters, but it is unclear whether PXR is involved in the regulation of these transporters in the placenta. This study explores the role of PXR in the regulation of placental drug transporters. The placental mRNA expression of Mdr1a, Bcrp, and Mrp1, 2, and 3 was examined in PXR knockout (-/-), heterozygote (+/-), and wild-type (+/+) mice by quantitative PCR. The impact of PXR activation was examined in pregnant pregnane-16α-carbonitrile (PCN)-treated mice. Compared with that in controls, the basal expression of Mdr1a, Bcrp, Mrp1, and Mrp2 was significantly higher in (+/-) and (-/-) mice. Alterations in the expression of mdr1a, bcrp, and mrp1, 2, and 3 between gestational day (GD) 10 and GD 17 was dissimilar between (+/+) and (-/-) mice. Although PCN treatment induced maternal and fetal hepatic expression of Cyp3a11; placental expression of transporters were not significantly changed. Overall, our results suggest a repressive role of PXR in the basal expression of several placental transporters and a tissue-specific induction of these target genes after PXR activation. PMID:21127142

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

    PubMed

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

    2010-02-01

    One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins. PMID:19961540

  11. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    PubMed Central

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

  12. ATP and AMP mutually influence their interaction with the ATP-binding cassette (ABC) adenylate kinase cystic fibrosis transmembrane conductance regulator (CFTR) at separate binding sites.

    PubMed

    Randak, Christoph O; Dong, Qian; Ver Heul, Amanda R; Elcock, Adrian H; Welsh, Michael J

    2013-09-20

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP &lrarr2; 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5'-triphosphate (8-N3-ATP) and 8-azidoadenosine 5'-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P(1),P(5)-di(adenosine-5') pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2. PMID:23921386

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

    PubMed

    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. PMID:20732973

  14. The COMATOSE ATP-Binding Cassette Transporter Is Required for Full Fertility in Arabidopsis1[W][OA

    PubMed Central

    Footitt, Steven; Dietrich, Daniela; Fait, Aaron; Fernie, Alisdair R.; Holdsworth, Michael J.; Baker, Alison; Theodoulou, Frederica L.

    2007-01-01

    COMATOSE (CTS) encodes a peroxisomal ATP-binding cassette transporter required not only for β-oxidation of storage lipids during germination and establishment, but also for biosynthesis of jasmonic acid and conversion of indole butyric acid to indole acetic acid. cts mutants exhibited reduced fertilization, which was rescued by genetic complementation, but not by exogenous application of jasmonic acid or indole acetic acid. Reduced fertilization was also observed in thiolase (kat2-1) and peroxisomal acyl-Coenzyme A synthetase mutants (lacs6-1,lacs7-1), indicating a general role for β-oxidation in fertility. Genetic analysis revealed reduced male transmission of cts alleles and both cts pollen germination and tube growth in vitro were impaired in the absence of an exogenous carbon source. Aniline blue staining of pollinated pistils demonstrated that pollen tube growth was affected only when both parents bore the cts mutation, indicating that expression of CTS in either male or female tissues was sufficient to support pollen tube growth in vivo. Accordingly, abundant peroxisomes were detected in a range of maternal tissues. Although γ-aminobutyric acid levels were reduced in flowers of cts mutants, they were unchanged in kat2-1, suggesting that alterations in γ-aminobutyric acid catabolism do not contribute to the reduced fertility phenotype through altered pollen tube targeting. Taken together, our data support an important role for β-oxidation in fertility in Arabidopsis (Arabidopsis thaliana) and suggest that this pathway could play a role in the mobilization of lipids in both pollen and female tissues. PMID:17468211

  15. Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-23

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway. PMID:22303012

  16. Alternating Access to the Transmembrane Domain of the ATP-binding Cassette Protein Cystic Fibrosis Transmembrane Conductance Regulator (ABCC7)*

    PubMed Central

    Wang, Wuyang; Linsdell, Paul

    2012-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between “outwardly facing” and “inwardly facing” conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)2− ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)2−, access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional (“vestigial”) gates within the CFTR permeation pathway. PMID:22303012

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

    PubMed Central

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

    2014-01-01

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

  18. Astaxanthin enhances ATP-binding cassette transporter A1/G1 expressions and cholesterol efflux from macrophages.

    PubMed

    Iizuka, Maki; Ayaori, Makoto; Uto-Kondo, Harumi; Yakushiji, Emi; Takiguchi, Shunichi; Nakaya, Kazuhiro; Hisada, Tetsuya; Sasaki, Makoto; Komatsu, Tomohiro; Yogo, Makiko; Kishimoto, Yoshimi; Kondo, Kazuo; Ikewaki, Katsunori

    2012-01-01

    ATP-binding cassette transporters (ABC) A1 and G1 are key molecules in cholesterol efflux from macrophages, which is an initial step of reverse cholesterol transport (RCT), a major anti-atherogenic property of high-density lipoprotein (HDL). Astaxanthin is one of the naturally occurring carotenoids responsible for the pink-red pigmentation in a variety of living organisms. Although astaxanthin is known to be a strong antioxidant, it remains unclear through what mechanism of action it affects cholesterol homeostasis in macrophages. We therefore investigated the effects of astaxanthin on cholesterol efflux and ABCA1/G1 expressions in macrophages. Astaxanthin enhanced both apolipoprotein (apo) A-I- and HDL-mediated cholesterol efflux from RAW264.7 cells. In supporting these enhanced cholesterol efflux mechanisms, astaxanthin promoted ABCA1/G1 expression in various macrophages. In contrast, peroxisome proliferator-activated receptor γ, liver X receptor (LXR) α and LXRβ levels remained unchanged by astaxanthin. An experiment using actinomycin D demonstrated that astaxanthin transcriptionally induced ABCA1/G1 expression, and oxysterol depletion caused by overexpression of cholesterol sulfotransferase further revealed that these inductions in ABCA1/G1 were independent of LXR-mediated pathways. Finally, we performed luciferase assays using human ABCA1/G1 promoter-reporter constructs to reveal that astaxanthin activated both promoters irrespective of the presence or absence of LXR-responsive elements, indicating LXR-independence of these activations. In conclusion, astaxanthin increased ABCA1/G1 expression, thereby enhancing apoA-I/HDL-mediated cholesterol efflux from the macrophages in an LXR-independent manner. In addition to the anti-oxidative properties, the potential cardioprotective properties of astaxanthin might therefore be associated with an enhanced anti-atherogenic function of HDL. PMID:22790567

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

    PubMed Central

    Heumann, Jan; Taggart, John B.; Gharbi, Karim; Bron, James E.; Bekaert, Michaël; Sturm, Armin

    2015-01-01

    Salmon lice, Lepeophtheirus salmonis (Krøyer, 1837), are fish ectoparasites causing significant economic damage in the mariculture of Atlantic salmon, Salmo salar Linnaeus, 1758. The control of L. salmonis at fish farms relies to a large extent on treatment with anti-parasitic drugs. A problem related to chemical control is the potential for development of resistance, which in L. salmonis is documented for a number of drug classes including organophosphates, pyrethroids and avermectins. The ATP-binding cassette (ABC) gene superfamily is found in all biota and includes a range of drug efflux transporters that can confer drug resistance to cancers and pathogens. Furthermore, some ABC transporters are recognised to be involved in conferral of insecticide resistance. While a number of studies have investigated ABC transporters in L. salmonis, no systematic analysis of the ABC gene family exists for this species. This study presents a genome-wide survey of ABC genes in L. salmonis for which, ABC superfamily members were identified through homology searching of the L. salmonis genome. In addition, ABC proteins were identified in a reference transcriptome of the parasite generated by high-throughput RNA sequencing (RNA-seq) of a multi-stage RNA library. Searches of both genome and transcriptome allowed the identification of a total of 33 genes / transcripts coding for ABC proteins, of which 3 were represented only in the genome and 4 only in the transcriptome. Eighteen sequences were assigned to ABC subfamilies known to contain drug transporters, i.e. subfamilies B (4 sequences), C (11) and G (2). The results suggest that the ABC gene family of L. salmonis possesses fewer members than recorded for other arthropods. The present survey of the L. salmonis ABC gene superfamily will provide the basis for further research into potential roles of ABC transporters in the toxicity of salmon delousing agents and as potential mechanisms of drug resistance. PMID:26418738

  20. ATP-Binding Cassette (ABC) Transporters of the Human Respiratory Tract Pathogen, Moraxella catarrhalis: Role in Virulence

    PubMed Central

    Murphy, Timothy F; Brauer, Aimee L.; Johnson, Antoinette; Kirkham, Charmaine

    2016-01-01

    Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract. PMID:27391026

  1. ATP-Binding Cassette (ABC) Transporters of the Human Respiratory Tract Pathogen, Moraxella catarrhalis: Role in Virulence.

    PubMed

    Murphy, Timothy F; Brauer, Aimee L; Johnson, Antoinette; Kirkham, Charmaine

    2016-01-01

    Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract. PMID:27391026

  2. ATP-Binding Membrane Cassette Transporter A1 (ABCA1): A Possible Link between Inflammation and Reverse Cholesterol Transport

    PubMed Central

    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

  3. Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters

    PubMed Central

    King Jordan, I.; Kota, Karthik C.; Cui, Guiying; Thompson, Christopher H.; McCarty, Nael A.

    2008-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein. PMID:19020075

  4. Evolutionary and functional divergence between the cystic fibrosis transmembrane conductance regulator and related ATP-binding cassette transporters.

    PubMed

    Jordan, I King; Kota, Karthik C; Cui, Guiying; Thompson, Christopher H; McCarty, Nael A

    2008-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, an ancient family of proteins found in all phyla. In nearly all cases, ABC proteins are transporters that couple the hydrolysis of ATP to the transmembrane movement of substrate via an alternating access mechanism. In contrast, CFTR is best known for its activity as an ATP-dependent chloride channel. We asked why CFTR, which shares the domain architecture of ABC proteins that function as transporters, exhibits functional divergence. We compared CFTR protein sequences to those of other ABC transporters, which identified the ABCC4 proteins as the closest mammalian paralogs, and used statistical analysis of the CFTR-ABCC4 multiple sequence alignment to identify the specific domains and residues most likely to be involved in the evolutionary transition from transporter to channel activity. Among the residues identified as being involved in CFTR functional divergence, by virtue of being both CFTR-specific and conserved among all CFTR orthologs, was R352 in the sixth transmembrane helix (TM6). Patch-clamp experiments show that R352 interacts with D993 in TM9 to stabilize the open-channel state; D993 is absolutely conserved between CFTRs and ABCC4s. These data suggest that CFTR channel activity evolved, at least in part, by converting the conformational changes associated with binding and hydrolysis of ATP, as are found in true ABC Transporters, into an open permeation pathway by means of intraprotein interactions that stabilize the open state. This analysis sets the stage for understanding the evolutionary and functional relationships that make CFTR a unique ABC transporter protein. PMID:19020075

  5. Osimertinib (AZD9291) Attenuates the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCB1 in Vitro.

    PubMed

    Hsiao, Sung-Han; Lu, Yu-Jen; Li, Yan-Qing; Huang, Yang-Hui; Hsieh, Chia-Hung; Wu, Chung-Pu

    2016-06-01

    The effectiveness of cancer chemotherapy is often circumvented by multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in human cancer cells. Furthermore, some TKIs are transported by ABCB1, which results in low oral bioavailability, reduced distribution, and the development of acquired resistance to these TKIs. In this study, we investigated the interaction between ABCB1 and osimertinib, a novel selective, irreversible third-generation EGFR TKI that has recently been approved by the U.S. Food and Drug Administration. We also evaluated the potential impact of ABCB1 on the efficacy of osimertinib in cancer cells, which can present a therapeutic challenge to clinicians in the future. We revealed that although osimertinib stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer resistance to osimertinib. Our results suggest that it is unlikely that the overexpression of ABCB1 can be a major contributor to the development of osimertinib resistance in cancer patients. More significantly, we revealed an additional action of osimertinib that directly inhibits the function of ABCB1 without affecting the expression level of ABCB1, enhances drug-induced apoptosis, and reverses the MDR phenotype in ABCB1-overexpressing cancer cells. Considering that osimertinib is a clinically approved third-generation EGFR TKI, our findings suggest that a combination therapy with osimertinib and conventional anticancer drugs may be beneficial to patients with MDR tumors. PMID:27169328

  6. Ablation of the ATP-binding cassette transporter, Abca2 modifies response to estrogen-based therapies.

    PubMed

    Mack, Jody T; Brown, Carol B; Garrett, Tracy E; Uys, Joachim D; Townsend, Danyelle M; Tew, Kenneth D

    2012-09-01

    The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12×100 mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100 mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism. PMID:22898081

  7. Ablation of the ATP-binding cassette transporter, Abca2 modifies response to estrogen-based therapies

    PubMed Central

    Mack, Jody T.; Brown, Carol B.; Garrett, Tracy E.; Uys, Joachim D.; Townsend, Danyelle M.; Tew, Kenneth D.

    2013-01-01

    The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein expressed in oligodendrocytes and Schwann cells, prostate, ovary and macrophages. In cell cultures, ABCA2 over-expression has been linked with resistance to the anticancer agent, estramustine phosphate (EMP; a nor-nitrogen mustard conjugate of estradiol). The present study shows that Abca2 knockout (KO) mice have greater sensitivity to a variety of side effects induced by EMP treatment. Chronic EMP (12 × 100 mg/kg body weight) produced mortality in 36% of KO mice, but only 7% of age-matched wild type (WT). Side effects of the drug were also more prevalent in the KO mouse. For example, during the first week of EMP treatments, 67% of KO males (compared to 6% of WT males) responded with episodic erectile events. In WT mice, ABCA2 protein localized within pene corpuscles, (which rely on modified Schwann cells for amplification of tactile signals) suggesting that the transporter may function in the erectile process. Endothelial nitric oxide synthase (eNOS; a source of nitric oxide during erectile response) levels were similar in WT and KO male penile tissue. Treatment with 100 mg/kg EMP (once daily for four days) elevated serum estradiol and estrone in both WT and KO. However, the circulating levels of these estrogens were higher in KO mice implying a reduced plasma clearance of estrogens as a consequence of ABCA2 ablation. Consistent with the pro-convulsant effects of estrogens, KO mice also displayed an increased incidence of seizures following EMP (14% vs. 0%). Taken together, these data indicate that ABCA2 deficiency renders mice more sensitive to EMP treatment-induced effects implying that the transporter has a role in regulating EMP transport and/or metabolism. PMID:22898081

  8. The Yersiniabactin-Associated ATP Binding Cassette Proteins YbtP and YbtQ Enhance Escherichia coli Fitness during High-Titer Cystitis.

    PubMed

    Koh, Eun-Ik; Hung, Chia S; Henderson, Jeffrey P

    2016-05-01

    The Yersinia high-pathogenicity island (HPI) is common to multiple virulence strategies used by Escherichia coli strains associated with urinary tract infection (UTI). Among the genes in this island are ybtP and ybtQ, encoding distinctive ATP binding cassette (ABC) proteins associated with iron(III)-yersiniabactin import in Yersinia pestis In this study, we compared the impact of ybtPQ on a model E. coli cystitis strain during in vitro culture and experimental murine infections. A ybtPQ-null mutant exhibited no growth defect under standard culture conditions, consistent with nonessentiality in this background. A growth defect phenotype was observed and genetically complemented in vitro during iron(III)-yersiniabactin-dependent growth. Following inoculation into the bladders of C3H/HEN and C3H/HeOuJ mice, this strain exhibited a profound, 10(6)-fold competitive infection defect in the subgroup of mice that progressed to high-titer bladder infections. These results identify a virulence role for YbtPQ in the highly inflammatory microenvironment characteristic of high-titer cystitis. The profound competitive defect may relate to the apparent selection of Yersinia HPI-positive E. coli in uncomplicated clinical UTIs. PMID:26883590

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

    PubMed

    Lima, Estelita Pereira; Goulart, Marília Oliveira Fonseca; Rolim Neto, Modesto Leite

    2014-11-01

    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. PMID:25411004

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

    PubMed Central

    Lima, Estelita Pereira; Goulart, Marília Oliveira Fonseca; Rolim, Modesto Leite

    2014-01-01

    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.

  11. ATP-Binding Cassette Transport System Involved in Regulation of Morphological Differentiation in Response to Glucose in Streptomyces griseus

    PubMed Central

    Seo, Jeong-Woo; Ohnishi, Yasuo; Hirata, Aiko; Horinouchi, Sueharu

    2002-01-01

    Streptomyces griseus NP4, which was derived by UV mutagenesis from strain IFO13350, showed a bald and wrinkled colony morphology in response to glucose. Mutant NP4 formed ectopic septa at intervals along substrate hyphae, and each of the compartments developed into a spore which was indistinguishable from an aerial spore in size, shape, and thickness of the spore wall and in susceptibility to lysozyme and heat. The ectopic spores of NP4 formed in liquid medium differed from “submerged spores” in lysozyme sensitivity. Shotgun cloning experiments with a library of the chromosomal DNA of the parental strain and mutant NP4 as the host gave rise to DNA fragments giving two different phenotypes; one complementing the bald phenotype of the host, and the other causing much severe wrinkled morphology in the host. Subcloning identified a gene (dasR) encoding a transcriptional repressor belonging to the GntR family that was responsible for the reversal of the bald phenotype and a gene (dasA) encoding a lipoprotein probably serving as a substrate-binding protein in an ATP-binding cassette (ABC) transport system that was responsible for the severe wrinkled morphology. These genes were adjacent but divergently encoded. Two genes, named dasB and dasC, encoding a membrane-spanning protein were present downstream of dasA, which suggested that dasRABC comprises a gene cluster for an ABC transporter, probably for sugar import. dasR was transcribed actively during vegetative growth, and dasA was transcribed just after commencement of aerial hypha formation and during sporulation, indicating that both were developmentally regulated. Transcriptional analysis and direct sequencing of dasRA in mutant NP4 suggested a defect of this mutant in the regulatory system to control the expression of these genes. Introduction of multicopies of dasA into the wild-type strain caused ectopic septation in very young substrate hyphae after only 1 day of growth and subsequent sporulation in response

  12. PDE5 inhibitors, sildenafil and vardenafil, reverse multidrug resistance by inhibiting the efflux function of multidrug resistance protein 7 (ATP-binding Cassette C10) transporter.

    PubMed

    Chen, Jun-Jiang; Sun, Yue-Li; Tiwari, Amit K; Xiao, Zhi-Jie; Sodani, Kamlesh; Yang, Dong-Hua; Vispute, Saraubh G; Jiang, Wen-Qi; Chen, Si-Dong; Chen, Zhe-Sheng

    2012-08-01

    Phosphodiesterase type 5 (PDE5) inhibitors are widely used in the treatment of male erectile dysfunction and pulmonary hypertension. Recently, several groups have evaluated the ability of PDE5 inhibitors for their anticancer activities. Previously, we had shown that sildenafil, vardenafil and tadalafil could reverse P-glycoprotein (ATP-binding cassette B1)-mediated MDR. In the present study, we determined whether these PDE5 inhibitors have the potential to reverse multidrug resistance protein 7 (MRP7; ATP-binding cassette C10)-mediated MDR. We found that sildenafil and vardenafil dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to paclitaxel, docetaxel and vinblastine, while tadalafil had only a minimal effect. Accumulation and efflux experiments demonstrated that sildenafil and vardenafil increased the intracellular accumulation of [(3)H]-paclitaxel by inhibiting the efflux of [(3 H]-paclitaxel in HEK/MRP7 cells. In addition, immunoblot and immunofluorescence analyses indicated that no significant alterations of MRP7 protein expression and localization in plasma membranes were found after treatment with sildenafil, vardenafil or tadalafil. These results demonstrate that sildenafil and vardenafil reverse MRP7-mediated a MDR through inhibition of the drug efflux function of MRP7. Our findings indicate a potentially novel use of PDE5 inhibitors as an adjuvant chemotherapeutic agent in clinical practice. PMID:22578167

  13. Cystic fibrosis-type mutational analysis in the ATP-binding cassette transporter signature of human P-glycoprotein MDR1.

    PubMed

    Hoof, T; Demmer, A; Hadam, M R; Riordan, J R; Tümmler, B

    1994-08-12

    Members of the ATP-binding cassette transporter superfamily such as the P-glycoproteins (MDR) and the cystic fibrosis transmembrane conductance regulator (CFTR) share conserved sequence motifs in their nucleotide binding fold that are the major targets for CFTR mutations in patients with cystic fibrosis. Cystic fibrosis-type mutations were introduced at analogous positions into the human MDR1 gene. Heterologous expression of wild-type or mutated MDR1 revealed similar mRNA transcript levels in Chinese hamster ovary K1 recipients, but the subsequent processing was defective for all mutations that give rise to severe cystic fibrosis in the case of CFTR. Functional multidrug transporter MDR1, however, was obtained when amino acid substitutions were introduced into a less conserved position of the ATP-binding cassette transporter signature (codon 536 in MDR1). The profile of cross-resistance and chemosensitization was modulated in these codon 536 variants, which suggests that this region is involved in the drug transport function of P-glycoprotein. PMID:7914197

  14. Endogenous mutagenesis by an insertion sequence element identifies Aeromonas salmonicida AbcA as an ATP-binding cassette transport protein required for biogenesis of smooth lipopolysaccharide.

    PubMed Central

    Chu, S; Noonan, B; Cavaignac, S; Trust, T J

    1995-01-01

    Analysis of an Aeromonas salmonicida A layer-deficient/O polysaccharide-deficient mutant carrying a Tn5 insertion in the structural gene for A protein (vapA) showed that the abcA gene immediately downstream of vapA had been interrupted by the endogenous insertion sequence element ISAS1. Immunoelectron microscopy showed that O polysaccharides did not accumulate at the inner membrane-cytoplasm interface of this mutant. abcA encodes an unusual protein; it carries both an amino-terminal ATP-binding cassette (ABC) domain showing high sequence similarity to ABC proteins implicated in the transport of certain capsular and O polysaccharides and a carboxyl-terminal potential DNA-binding domain, which distinguishes AbcA from other polysaccharide transport proteins in structural and evolutionary terms. The smooth lipopolysaccharide phenotype was restored by complementation with abcA but not by abcA carrying site-directed mutations in the sequence encoding the ATP-binding site of the protein. The genetic organization of the A. salmonicida ABC polysaccharide system differs from other bacteria. abcA also differs in apparently being required for both O-polysaccharide synthesis and in energizing the transport of O polysaccharides to the cell surface. Images Fig. 2 Fig. 3 Fig. 4 PMID:7777581

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

    PubMed Central

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

    2015-01-01

    ABCB1-mediated multidrug resistance (MDR) remains a major obstacle to successful chemotherapy in ovarian cancer. Herein, afatinib at nontoxic concentrations significantly reversed ABCB1-mediated MDR in ovarian cancer cells in vitro (p < 0.05). Combining paclitaxel and afatinib caused tumor regressions and tumor necrosis in A2780T xenografts in vivo. More interestingly, unlike reversible TKIs, afatinib had a distinctive dual-mode action. Afatinib not only inhibited the efflux function of ABCB1, but also attenuated its expression transcriptionally via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB. Furthermore, apart from a substrate binding domain, afatinib could also bind to an ATP binding domain of ABCB1 through forming hydrogen bonds with Gly533, Gly534, Lys536 and Ala560 sites. Importantly, mutations in these four binding sites of ABCB1 and the tyrosine kinase domain of EGFR were not correlated with the reversal activity of afatinib on MDR. Given that afatinib is a clinically approved drug, our results suggest combining afatinib with chemotherapeutic drugs in ovarian cancer. This study can facilitate the rediscovery of superior MDR reversal agents from molecular targeted drugs to provide a more effective and safer way of resensitizing MDR. PMID:26317651

  16. Demonstration of phosphoryl group transfer indicates that the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) exhibits adenylate kinase activity.

    PubMed

    Randak, Christoph O; Ver Heul, Amanda R; Welsh, Michael J

    2012-10-19

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP <==> 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase. PMID:22948143

  17. Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity*

    PubMed Central

    Randak, Christoph O.; Ver Heul, Amanda R.; Welsh, Michael J.

    2012-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5′-monophosphate (AMP), CFTR Cl− channel function is coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the 32P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase. PMID:22948143

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

    PubMed

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

    2014-01-01

    Prokaryotic solute binding protein-dependent ATP-binding cassette import systems are divided into type I and type II and mechanistic differences in the transport process going along with this classification are under intensive investigation. Little is known about the conformational dynamics during the catalytic cycle especially concerning the transmembrane domains. The type I transporter for positively charged amino acids from Salmonella enterica serovar Typhimurium (LAO-HisQMP2) was studied by limited proteolysis in detergent solution in the absence and presence of co-factors including ATP, ADP, LAO/arginine, and Mg(2+) ions. Stable peptide fragments could be obtained and differentially susceptible cleavage sites were determined by mass spectrometry as Lys-258 in the nucleotide-binding subunit, HisP, and Arg-217/Arg-218 in the transmembrane subunit, HisQ. In contrast, transmembrane subunit HisM was gradually degraded but no stable fragment could be detected. HisP and HisQ were equally resistant under pre- and post-hydrolysis conditions in the presence of arginine-loaded solute-binding protein LAO and ATP/ADP. Some protection was also observed with LAO/arginine alone, thus reflecting binding to the transporter in the apo-state and transmembrane signaling. Comparable digestion patterns were obtained with the transporter reconstituted into proteoliposomes and nanodiscs. Fluorescence lifetime spectroscopy confirmed the change of HisQ(R218) to a more apolar microenvironment upon ATP binding and hydrolysis. Limited proteolysis was subsequently used as a tool to study the consequences of mutations on the transport cycle. Together, our data suggest similar conformational changes during the transport cycle as described for the maltose ABC transporter of Escherichia coli, despite distinct structural differences between both systems. PMID:24021237

  19. Human ATP-Binding Cassette Transporter ABCB1 Confers Resistance to Volasertib (BI 6727), a Selective Inhibitor of Polo-like Kinase 1.

    PubMed

    Wu, Chung-Pu; Hsieh, Chia-Hung; Hsiao, Sung-Han; Luo, Shi-Yu; Su, Ching-Ya; Li, Yan-Qing; Huang, Yang-Hui; Huang, Chiun-Wei; Hsu, Sheng-Chieh

    2015-11-01

    The overexpression of the serine/threonine specific polo-like kinase 1 (Plk1) is associated with poor prognosis in many types of cancer. Consequently, Plk1 has emerged as a valid therapeutic target for anticancer drug design. Volasertib is a potent inhibitor of Plk1 that inhibits the proliferation of multiple human cancer cell lines by promoting cell cycle arrest at nanomolar concentrations. However, the risk of developing drug resistance, which is often associated with the overexpression of the ATP-binding cassette (ABC) transporter ABCB1 (P-glycoprotein), can present a therapeutic challenge for volasertib and many other therapeutic drugs. Although volasertib is highly effective against the proliferation of numerous cancer cell lines, we found that the overexpression of ABCB1 in cancer cells leads to cellular resistance to volasertib and reduces the level of volasertib-stimulated G2/M cell cycle arrest and subsequent onset of apoptosis. Furthermore, we demonstrate that volasertib competitively inhibits the function of ABCB1 and stimulates the basal ATPase activity of ABCB1 in a concentration-dependent manner, which is consistent with substrate transport by ABCB1. More importantly, we discovered that the coadministration of an inhibitor or drug substrate of ABCB1 restored the anticancer activity of volasertib in ABCB1-overexpressing cancer cells. In conclusion, the results of our study reveal that ABCB1 negatively affects the efficacy of volasertib and supports its combination with a modulator of ABCB1 to improve clinical responses. PMID:26412161

  20. Hernandezine, a Bisbenzylisoquinoline Alkaloid with Selective Inhibitory Activity against Multidrug-Resistance-Linked ATP-Binding Cassette Drug Transporter ABCB1.

    PubMed

    Hsiao, Sung-Han; Lu, Yu-Jen; Yang, Chun-Chiao; Tuo, Wei-Cherng; Li, Yan-Qing; Huang, Yang-Hui; Hsieh, Chia-Hung; Hung, Tai-Ho; Wu, Chung-Pu

    2016-08-26

    The overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (P-glycoprotein, MDR1) is the most studied mechanism of multidrug resistance (MDR), which remains a major obstacle in clinical cancer chemotherapy. Consequently, resensitizing MDR cancer cells by inhibiting the efflux function of ABCB1 has been considered as a potential strategy to overcome ABCB1-mediated MDR in cancer patients. However, the task of developing a suitable modulator of ABCB1 has been hindered mostly by the lack of selectivity and high intrinsic toxicity of candidate compounds. Considering the wide range of diversity and relatively nontoxic nature of natural products, developing a potential modulator of ABCB1 from natural sources is particularly valuable. Through screening of a large collection of purified bioactive natural products, hernandezine was identified as a potent and selective reversing agent for ABCB1-mediated MDR in cancer cells. Experimental data demonstrated that the bisbenzylisoquinoline alkaloid hernandezine is selective for ABCB1, effectively inhibits the transport function of ABCB1, and enhances drug-induced apoptosis in cancer cells. More importantly, hernandezine significantly resensitizes ABCB1-overexpressing cancer cells to multiple chemotherapeutic drugs at nontoxic, nanomolar concentrations. Collectively, these findings reveal that hernandezine has great potential to be further developed into a novel reversal agent for combination therapy in MDR cancer patients. PMID:27504669

  1. Novel Mutation in the ATP-Binding Cassette Transporter A3 (ABCA3) Encoding Gene Causes Respiratory Distress Syndrome in A Term Newborn in Southwest Iran

    PubMed Central

    Rezaei, Farideh; Shafiei, Mohammad; Shariati, Gholamreza; Dehdashtian, Ali; Mohebbi, Maryam; Galehdari, Hamid

    2016-01-01

    Introduction ABCA3 glycoprotein belongs to the ATP-binding cassette (ABC) superfamily of transporters, which utilize the energy derived from hydrolysis of ATP for the translocation of a wide variety of substrates across the plasma membrane. Mutations in the ABCA3 gene are knowingly causative for fatal surfactant deficiency, particularly respiratory distress syndrome (RDS) in term babies. Case Presentation In this study, Sanger sequencing of the whole ABCA3 gene (NCBI NM_001089) was performed in a neonatal boy with severe RDS. A homozygous mutation has been identified in the patient. Parents were heterozygous for the same missense mutation GGA > AGA at position 202 in exon 6 of the ABCA3 gene (c.604G > A; p.G202R). Furthermore, 70 normal individuals have been analyzed for the mentioned change with negative results. Conclusions Regarding Human Genome Mutation Database (HGMD) and other literature recherche, the detected change is a novel mutation and has not been reported before. Bioinformatics mutation predicting tools prefer it as pathogenic. PMID:27437095

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

    PubMed Central

    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

  3. Copy number variation in the ATP-binding cassette transporter ABCC6 gene and ABCC6 pseudogenes in patients with pseudoxanthoma elasticum

    PubMed Central

    Kringen, Marianne K; Stormo, Camilla; Berg, Jens Petter; Terry, Sharon F; Vocke, Christine M; Rizvi, Samar; Hendig, Doris; Piehler, Armin P

    2015-01-01

    Single mutations in the ATP-binding cassette transporter (ABCC6) gene (OMIM 603234) are known to cause the rare autosomal recessive disease pseudoxanthoma elasticum (PXE). Recently, we have found that copy number variations (CNVs) in pseudogenes of the ABCC6 gene are quite common. The aim of this study was to investigate the frequency and possible contribution of CNV in ABCC6 and its pseudogenes in PXE. Genomic DNA from 212 PXE individuals were examined for copy number by pyrosequencing and quantitative polymerase chain reaction (PCR) and compared with healthy individuals. The frequency of PXE individuals with any CNV was higher than in healthy individuals. The majority of variation comprised known and possibly new deletions in the ABCC6 gene and duplications of the ABCC6P1 and ABCC6P2 genes. ABCC6 deletions and ABCC6P2 duplications were not observed in 142 healthy individuals. In conclusion, by pyrosequencing and quantitative PCR, we were able to detect known and possibly new deletions in the ABCC6 gene that may have caused the PXE phenotype. Pyrosequencing may be used in PXE patients who have obtained incomplete genotype from conventional techniques. The frequency of ABCC6P2 pseudogene duplication was more common in PXE patients than healthy individuals and may affect the PXE phenotype. PMID:26029710

  4. Arabidopsis PEN3/PDR8, an ATP Binding Cassette Transporter, Contributes to Nonhost Resistance to Inappropriate Pathogens That Enter by Direct Penetration[W][OA

    PubMed Central

    Stein, Mónica; Dittgen, Jan; Sánchez-Rodríguez, Clara; Hou, Bi-Huei; Molina, Antonio; Schulze-Lefert, Paul; Lipka, Volker; Somerville, Shauna

    2006-01-01

    Arabidopsis thaliana is a host to the powdery mildew Erysiphe cichoracearum and nonhost to Blumeria graminis f. sp hordei, the powdery mildew pathogenic on barley (Hordeum vulgare). Screening for Arabidopsis mutants deficient in resistance to barley powdery mildew identified PENETRATION3 (PEN3). pen3 plants permitted both increased invasion into epidermal cells and initiation of hyphae by B. g. hordei, suggesting that PEN3 contributes to defenses at the cell wall and intracellularly. pen3 mutants were compromised in resistance to the necrotroph Plectosphaerella cucumerina and to two additional inappropriate biotrophs, pea powdery mildew (Erysiphe pisi) and potato late blight (Phytophthora infestans). Unexpectedly, pen3 mutants were resistant to E. cichoracearum. This resistance was salicylic acid–dependent and correlated with chlorotic patches. Consistent with this observation, salicylic acid pathway genes were hyperinduced in pen3 relative to the wild type. The phenotypes conferred by pen3 result from the loss of function of PLEIOTROPIC DRUG RESISTANCE8 (PDR8), a highly expressed putative ATP binding cassette transporter. PEN3/PDR8 tagged with green fluorescent protein localized to the plasma membrane in uninfected cells. In infected leaves, the protein concentrated at infection sites. PEN3/PDR8 may be involved in exporting toxic materials to attempted invasion sites, and intracellular accumulation of these toxins in pen3 may secondarily activate the salicylic acid pathway. PMID:16473969

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

    PubMed

    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

  6. The Role of Arabidopsis ABCG9 and ABCG31 ATP Binding Cassette Transporters in Pollen Fitness and the Deposition of Steryl Glycosides on the Pollen Coat[W

    PubMed Central

    Choi, Hyunju; Ohyama, Kiyoshi; Kim, Yu-Young; Jin, Jun-Young; Lee, Saet Buyl; Yamaoka, Yasuyo; Muranaka, Toshiya; Suh, Mi Chung; Fujioka, Shozo; Lee, Youngsook

    2014-01-01

    The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen. PMID:24474628

  7. The Arabidopsis PLEIOTROPIC DRUG RESISTANCE8/ABCG36 ATP Binding Cassette Transporter Modulates Sensitivity to the Auxin Precursor Indole-3-Butyric Acid[C][W

    PubMed Central

    Strader, Lucia C.; Bartel, Bonnie

    2009-01-01

    Plants have developed numerous mechanisms to store hormones in inactive but readily available states, enabling rapid responses to environmental changes. The phytohormone auxin has a number of storage precursors, including indole-3-butyric acid (IBA), which is apparently shortened to active indole-3-acetic acid (IAA) in peroxisomes by a process similar to fatty acid β-oxidation. Whereas metabolism of auxin precursors is beginning to be understood, the biological significance of the various precursors is virtually unknown. We identified an Arabidopsis thaliana mutant that specifically restores IBA, but not IAA, responsiveness to auxin signaling mutants. This mutant is defective in PLEIOTROPIC DRUG RESISTANCE8 (PDR8)/PENETRATION3/ABCG36, a plasma membrane–localized ATP binding cassette transporter that has established roles in pathogen responses and cadmium transport. We found that pdr8 mutants display defects in efflux of the auxin precursor IBA and developmental defects in root hair and cotyledon expansion that reveal previously unknown roles for IBA-derived IAA in plant growth and development. Our results are consistent with the possibility that limiting accumulation of the IAA precursor IBA via PDR8-promoted efflux contributes to auxin homeostasis. PMID:19648296

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

    PubMed Central

    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

    2011-01-01

    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. PMID:21737747

  9. Control of Mycosphaerella graminicola on Wheat Seedlings by Medical Drugs Known To Modulate the Activity of ATP-Binding Cassette Transporters▿

    PubMed Central

    Roohparvar, Ramin; Huser, Aurelie; Zwiers, Lute-Harm; De Waard, Maarten 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 to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents. PMID:17545327

  10. Vascular and extravascular distribution of the ATP-binding cassette transporters ABCB1 and ABCC1 in aged human brain and pituitary

    PubMed Central

    Bernstein, Hans-Gert; Hölzl, Gloria; Dobrowolny, Henrik; Hildebrandt, Jens; Trübner, Kurt; Krohn, Markus; Bogerts, Bernhard; Pahnke, Jens

    2014-01-01

    ATP-binding cassette (ABC) transporters play an increasing role in the understanding of pathologic peptide deposition in neurodegenerative diseases (NDs), such as Alzheimer’s and Parkinson’s. To describe the location of the most important ABC transporters for NDs in human brain tissue, we investigated ABCB1 and ABCC1 immunohistologically in the adult human brain and pituitary. Both transporters have similar but not identical expression patterns. In brain regions with an established blood-brain barrier (BBB), ABCB1 and ABCC1 were ubiquitously expressed in endothelial cells of the microvasculature and in a subset of larger blood vessels (mostly venules). Remarkably, both transporters were also found in fenestrated capillaries in circumventricular organs where the BBB is absent. Moreover, ABCB1 and ABCC1 were also expressed in various non-endothelia cells such as pericytes, astrocytes, choroid plexus epithelia, ventricle ependymal cells, and neurons. With regard to their neuronal expression it was shown that both transporters are located in specific nerve cell populations, which are also immunopositive for three putative cell markers of purinergic cell signalling, namely 5´-nucleotidase, adenosine deaminase and nucleoside triphosphate diphosphohydrolase-2. Therefore, we speculate that neuronal expression of ABCB1 and ABCC1 might be linked to adenosinergic/purinergic neuromodulation. Lastly, both transporters were observed in multiple adenohypophyseal cells. PMID:25218792

  11. The Klebsiella pneumoniae O12 ATP-binding Cassette (ABC) Transporter Recognizes the Terminal Residue of Its O-antigen Polysaccharide Substrate.

    PubMed

    Mann, Evan; Mallette, Evan; Clarke, Bradley R; Kimber, Matthew S; Whitfield, Chris

    2016-04-29

    Export of the Escherichia coli serotype O9a O-antigenic polysaccharides (O-PS) involves an ATP-binding cassette (ABC) transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC 33257). The O12 polysaccharide is terminated at the non-reducing end by a β-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM, and export is dependent on the presence of the terminal β-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype, but they share only weak sequence similarity, and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures. PMID:26934919

  12. Reversal of multidrug resistance by the inhibition of ATP-binding cassette pumps employing "Generally Recognized As Safe" (GRAS) nanopharmaceuticals: A review.

    PubMed

    Sosnik, Alejandro

    2013-11-01

    Pumps of the ATP-binding cassette superfamily (ABCs) regulate the access of drugs to the intracellular space. In this context, the overexpression of ABCs is a well-known mechanism of multidrug resistance (MDR) in cancer and infectious diseases (e.g., viral hepatitis and the human immunodeficiency virus) and is associated with therapeutic failure. Since their discovery, ABCs have emerged as attractive therapeutic targets and the search of compounds that inhibit their genetic expression and/or their functional activity has gained growing interest. Different generations of pharmacological ABC inhibitors have been explored over the last four decades to address resistance in cancer, though clinical results have been somehow disappointing. "Generally Recognized As Safe" (GRAS) is a U.S. Food and Drug Administration designation for substances that are accepted as safe for addition in food. Far from being "inert", some amphiphilic excipients used in the production of pharmaceutical products have been shown to inhibit the activity of ABCs in MDR tumors, emerging as a clinically translatable approach to overcome resistance. The present article initially overviews the classification, structure and function of the different ABCs, with emphasis on those pumps related to drug resistance. Then, the different attempts to capitalize on the activity of GRAS nanopharmaceuticals as ABC inhibitors are discussed. PMID:24055628

  13. Cuticular Defects in Oryza sativa ATP-binding Cassette Transporter G31 Mutant Plants Cause Dwarfism, Elevated Defense Responses and Pathogen Resistance.

    PubMed

    Garroum, Imène; Bidzinski, Przemyslaw; Daraspe, Jean; Mucciolo, Antonio; Humbel, Bruno M; Morel, Jean-Benoit; Nawrath, Christiane

    2016-06-01

    The cuticle covers the surface of the polysaccharide cell wall of leaf epidermal cells and forms an essential diffusion barrier between plant and environment. Homologs of the ATP-binding cassette (ABC) transporter AtABCG32/HvABCG31 clade are necessary for the formation of a functional cuticle in both monocots and dicots. Here we characterize the osabcg31 knockout mutant and hairpin RNA interference (RNAi)-down-regulated OsABCG31 plant lines having reduced plant growth and a permeable cuticle. The reduced content of cutin in leaves and structural alterations in the cuticle and at the cuticle-cell wall interface in plants compromised in OsABCG31 expression explain the cuticle permeability. Effects of modifications of the cuticle on plant-microbe interactions were evaluated. The cuticular alterations in OsABCG31-compromised plants did not cause deficiencies in germination of the spores or the formation of appressoria of Magnaporthe oryzae on the leaf surface, but a strong reduction of infection structures inside the plant. Genes involved in pathogen resistance were constitutively up-regulated in OsABCG31-compromised plants, thus being a possible cause of the resistance to M. oryzae and the dwarf growth phenotype. The findings show that in rice an abnormal cuticle formation may affect the signaling of plant growth and defense. PMID:27121976

  14. Corticotropin-Releasing Hormone (CRH) Promotes Macrophage Foam Cell Formation via Reduced Expression of ATP Binding Cassette Transporter-1 (ABCA1).

    PubMed

    Cho, Wonkyoung; Kang, Jihee Lee; Park, Young Mi

    2015-01-01

    Atherosclerosis, the major pathology of cardiovascular disease, is caused by multiple factors involving psychological stress. Corticotropin-releasing hormone (CRH), which is released by neurosecretory cells in the hypothalamus, peripheral nerve terminals and epithelial cells, regulates various stress-related responses. Our current study aimed to verify the role of CRH in macrophage foam cell formation, the initial critical stage of atherosclerosis. Our quantitative real-time reverse transcriptase PCR (qRT-PCR), semi-quantitative reverse transcriptase PCR, and Western blot results indicate that CRH down-regulates ATP-binding cassette transporter-1 (ABCA1) and liver X receptor (LXR)-α, a transcription factor for ABCA1, in murine peritoneal macrophages and human monocyte-derived macrophages. Oil-red O (ORO) staining and intracellular cholesterol measurement of macrophages treated with or without oxidized LDL (oxLDL) and with or without CRH (10 nM) in the presence of apolipoprotein A1 (apoA1) revealed that CRH treatment promotes macrophage foam cell formation. The boron-dipyrromethene (BODIPY)-conjugated cholesterol efflux assay showed that CRH treatment reduces macrophage cholesterol efflux. Western blot analysis showed that CRH-induced down-regulation of ABCA1 is dependent on phosphorylation of Akt (Ser473) induced by interaction between CRH and CRH receptor 1(CRHR1). We conclude that activation of this pathway by CRH accelerates macrophage foam cell formation and may promote stress-related atherosclerosis. PMID:26110874

  15. A 20(S)-protopanoxadiol derivative overcomes multi-drug resistance by antagonizing ATP-binding cassette subfamily B member 1 transporter function

    PubMed Central

    Chen, Wantao; Xu, Qin; Xiao, Meng; Hu, Lihong; Mao, Li; Wang, Xu

    2016-01-01

    In cancer cells, failure of chemotherapy is often caused by the ATP-binding cassette subfamily B member 1 (ABCB1), and few drugs have been successfully developed to overcome ABCB1-mediated multi-drug resistance (MDR). To suppress ABCB1 activity, we previously designed and synthesized a new series of derivatives based on 20(S)-protopanoxadiol (PPD). In the present study, we investigated the role of PPD derivatives in the function of ABC transporters. Non-toxic concentrations of the PPD derivative PPD12 sensitized ABCB1-overexpressing cells to their anti-cancer substrates better than either the parental PPD or inactive PPD11. PPD12 increased intracellular accumulation of adriamycin and rhodamine123 in resistant cancer cells. Although PPD12 did not suppress the expression of ABCB1 mRNA or protein, it stimulated the activity of ABCB1 ATPase. Because PPD12 is a competitive inhibitor, it was predicted to bind to the large hydrophobic cavity of homology-modeled human ABCB1. PPD12 also enhanced the efficacy of adriamycin against ABCB1-overexpressing KB/VCR xenografts in nude mice. In conclusion, PPD12 enhances the efficacy of substrate drugs in ABCB1-overexpressing cancer cells. These findings suggest that a combination therapy consisting of PPD12 with conventional chemotherapeutic agents may be an effective treatment for ABCB1-mediated MDR cancer patients. PMID:26824187

  16. Whole-Transcriptome Survey of the Putative ATP-Binding Cassette (ABC) Transporter Family Genes in the Latex-Producing Laticifers of Hevea brasiliensis

    PubMed Central

    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

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

    PubMed

    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. PMID:21737747

  18. Intracellular ATP-binding cassette transporter A3 is expressed in lung cancer cells and modulates susceptibility to cisplatin and paclitaxel.

    PubMed

    Overbeck, Tobias R; Hupfeld, Timo; Krause, Doris; Waldmann-Beushausen, Regina; Chapuy, Bjoern; Güldenzoph, Bjoern; Aung, Thiha; Inagaki, Nobuya; Schöndube, Friedrich A; Danner, Bernhard C; Truemper, Lorenz; Wulf, Gerald G

    2013-01-01

    Patients with advanced-stage bronchial cancer benefit from systemic cytostatic therapy, in particular from regimens integrating cisplatin and taxanes. However, eventual disease progression leads to a fatal outcome in most cases, originating from tumor cells resisting chemotherapy. We here show that the intracellular ATP-binding cassette transporter A3 (ABCA3), previously recognized as critical for the secretion of surfactant components from type 2 pneumocytes, is expressed in non-small-cell lung cancer (NSCLC) cells. With some heterogeneity in a given specimen, expression levels detected immunohistochemically in primary cancer tissue were highest in adenocarcinomas and lowest in small cell lung cancers. Genetic silencing of ABCA3 in the NSCLC cell line models A549, NCI-H1650 and NCI-H1975 significantly increased tumor cell susceptibility to the cytostatic effects of both cisplatin (in all cell lines) and paclitaxel (in two of three cell lines). Taken together, ABCA3 emerges as a modulator of NSCLC cell susceptibility to cytostatic therapy. PMID:23689165

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

    PubMed Central

    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

  20. Improvement of the cellular quality of cryopreserved bovine blastocysts accompanied by enhancement of the ATP-binding cassette sub-family B member 1 expression.

    PubMed

    Mori, Miyuki; Kasa, Shojiro; Isozaki, Yoshihiro; Kamori, Tsugumitsu; Yamaguchi, Shoichiro; Ueda, Shuji; Kuwano, Toshio; Eguchi, Minako; Isayama, Keishiro; Nishimura, Shotaro; Tabata, Shoji; Yamauchi, Nobuhiko; Hattori, Masa-aki

    2013-01-01

    The ATP-binding cassette sub-family B member 1 (ABCB1) plays a critical role in maintaining the metabolic capability of cells as an efflux transporter that pumps xenobiotics out of cells. We investigated the effects of highly expressed ABCB1 on the development and viability of cryopreserved bovine embryos. The ABCB1 level in cultured bovine embryos was decreased during development to blastocyst-stage compared to germinal vesicle- and second metaphase-stage oocytes. When bovine embryos were cultured with forskolin and/or rifampicin, the ABCB1 level was significantly increased in blastocysts but embryo development was not significantly improved. After embryo cryopreservation, highly ABCB1-expressed blastocysts exhibited significant increases in viability and hatching rates. The high viability of the cryopreserved blastocysts was accompanied by a significant increase in cell proliferation during culture for 48 h. Thus, ABCB1 is expressed in bovine oocytes and embryos, and the cellular quality of bovine blastocysts is improved by the enhancement of ABCB1 expression. PMID:23164983

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

    SciTech Connect

    Lee, Asaka; Asahina, Kota; Okamoto, Takumi; Kawaguchi, Kosuke; Kostsin, Dzmitry G.; Kashiwayama, Yoshinori; Takanashi, Kojiro; Yazaki, Kazufumi; Imanaka, Tsuneo; Morita, Masashi

    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.

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

    PubMed Central

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

    2003-01-01

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

  3. Rescuing Trafficking Mutants of the ATP-binding Cassette Protein, ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease.

    PubMed

    Sabirzhanova, Inna; Lopes Pacheco, Miquéias; Rapino, Daniele; Grover, Rahul; Handa, James T; Guggino, William B; Cebotaru, Liudmila

    2015-08-01

    Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment. PMID:26092729

  4. An ATP Binding Cassette Transporter Is Required for Cuticular Wax Deposition and Desiccation Tolerance in the Moss Physcomitrella patens[W

    PubMed Central

    Buda, Gregory J.; Barnes, William J.; Fich, Eric A.; Park, Sungjin; Yeats, Trevor H.; Zhao, Lingxia; Domozych, David S.; Rose, Jocelyn K.C.

    2013-01-01

    The plant cuticle is thought to be a critical evolutionary adaptation that allowed the first plants to colonize land, because of its key roles in regulating plant water status and providing protection from biotic and abiotic stresses. Much has been learned about cuticle composition and structure through genetic and biochemical studies of angiosperms, as well as underlying genetic pathways, but little is known about the cuticles of early diverging plant lineages. Here, we demonstrate that the moss Physcomitrella patens, an extant relative of the earliest terrestrial plants, has a cuticle that is analogous in both structure and chemical composition to those of angiosperms. To test whether the underlying cuticle biosynthetic pathways were also shared among distant plant lineages, we generated a genetic knockout of the moss ATP binding cassette subfamily G (ABCG) transporter Pp-ABCG7, a putative ortholog of Arabidopsis thaliana ABCG transporters involved in cuticle precursor trafficking. We show that this mutant is severely deficient in cuticular wax accumulation and has a reduced tolerance of desiccation stress compared with the wild type. This work provides evidence that the cuticle was an adaptive feature present in the first terrestrial plants and that the genes involved in their formation have been functionally conserved for over 450 million years. PMID:24163310

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

    SciTech Connect

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

    2015-01-01

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

  6. Comparison of the expression and function of ATP binding cassette transporters in Caco-2 and T84 cells on stimulation by selected endogenous compounds and xenobiotics.

    PubMed

    Naruhashi, Kazumasa; Kurahashi, Yuko; Fujita, Yukari; Kawakita, Eri; Yamasaki, Yuna; Hattori, Kana; Nishimura, Asako; Shibata, Nobuhito

    2011-01-01

    Caco-2 and T84 cells are intestinal epithelial model cells. Caco-2 cells are more commonly used in drug transport studies, whereas only a few studies have used T84 cells, and the two cell lines have not been compared. We cultured Caco-2 and T84 cells on plastic dishes or polycarbonate Transwell filters and compared the expression and function of ATP binding cassette (ABC) transporters, including multidrug resistance protein (MDR) 1 and multidrug resistance-associated protein (MRP) 2 and MRP3, in response to various compounds. Overall, the pattern of change in transporter mRNA expression in response to compounds was very similar regardless of culture conditions (plastic dish or polycarbonate filter) and cell line (Caco-2 or T84), and changes in MDR1 function was accompanied by expression changes. The cells cultured on Transwell filters were more sensitive to the tested compounds, regardless of the cell line. On comparing the two cell lines, the intrinsic function of MDR1 was stronger in Caco-2 cells, while sensitivity to the tested compounds was more prominent in T84 cells. These results suggest that Caco-2 cells are more suitable for identifying whether MDR1 mediates drug transport, while T84 cells are more useful for assessing the induction capacity of compounds. PMID:21127384

  7. Residues of a proposed gate region in type I ATP-binding cassette import systems are crucial for function as revealed by mutational analysis.

    PubMed

    Weidlich, Daniela; Wiesemann, Nicole; Heuveling, Johanna; Wardelmann, Kristina; Landmesser, Heidi; Sani, Katayoun Behnam; Worth, Catherine L; Preissner, Robert; Schneider, Erwin

    2013-09-01

    The type I ATP-binding cassette (ABC) importer for positively charged amino acids of the thermophilic bacterium Geobacillus stearothermophilus consists of the extracellular solute binding protein, ArtJ, and a homodimer each of the transmembrane subunit, ArtM, and the nucleotide-binding and -hydrolyzing subunit, ArtP. We have investigated the functional consequences of mutations affecting conserved residues from two peptide regions in ArtM, recently proposed to form a 'gate' by which access of a substrate to the translocation path is controlled (Hollenstein et al., 2007 [14]). Transporter variants were reconstituted into proteoliposomes and assayed for ArtJ/arginine-stimulated ATPase activity. Replacement of residues from region 1 (Arg-63, Pro-66) caused no or only moderate reduction in ATPase activity. In contrast, mutating residues from gate region 2 (Lys-159, Leu-163) resulted in a substantial increase in ATPase activity which, however, as demonstrated for variants ArtM(K159I) and ArtM(K159E), is not coupled to transport. Replacing homologous residues in the closely related histidine transporter of Salmonella enterica serovar Typhimurium (HisJ-QMP2) caused different phenotypes. Mutation to isoleucine of HisQ(K163) or HisM(H172), both homologous to ArtM(K159), abolished ATPase activity. The mutations most likely caused a structural change as revealed by limited proteolysis. In contrast, substantial, albeit reduced, enzymatic activity was observed with variants of HisQ(L167→G) or HisM(L176→G), both homologous to ArtM(L163). Our study provides the first experimental evidence in favor of a crucial role of residues from the proposed gate region in type I ABC importer function. PMID:23747295

  8. ATP-Binding Cassette Transporter G26 Is Required for Male Fertility and Pollen Exine Formation in Arabidopsis1[W][OA

    PubMed Central

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

    2010-01-01

    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. PMID:20732973

  9. A functional study on polymorphism of the ATP-binding cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport.

    PubMed Central

    Mitomo, Hideyuki; Kato, Ryo; Ito, Akiko; Kasamatsu, Shiho; Ikegami, Yoji; Kii, Isao; Kudo, Akira; Kobatake, Eiry; Sumino, Yasuhiro; Ishikawa, Toshihisa

    2003-01-01

    Overexpression of the ATP-binding cassette transporter ABCG2 reportedly causes multidrug resistance, whereas altered drug-resistance profiles and substrate specificity are implicated for certain variant forms of ABCG2. At least three variant forms of ABCG2 have been hitherto documented on the basis of their amino acid moieties (i.e., arginine, glycine and threonine) at position 482. In the present study we have generated those ABCG2 variants by site-directed mutagenesis and expressed them in HEK-293 cells. Exogenous expression of the Arg(482), Gly(482), and Thr(482) variant forms of ABCG2 conferred HEK-293 cell resistance toward mitoxantrone 15-, 47- and 54-fold, respectively, as compared with mock-transfected HEK-293 cells. The transport activity of those variants was examined by using plasma-membrane vesicles prepared from ABCG2-overexpressing HEK-293 cells. [Arg(482)]ABCG2 transports [(3)H]methotrexate in an ATP-dependent manner; however, no transport activity was observed with the other variants (Gly(482) and Thr(482)). Transport of methotrexate by [Arg(482)]ABCG2 was significantly inhibited by mitoxantrone, doxorubicin and rhodamine 123, but not by S -octylglutathione. Furthermore, ABCG2 was found to exist in the plasma membrane as a homodimer bound via cysteinyl disulphide bond(s). Treatment with mercaptoethanol decreased its apparent molecular mass from 140 to 70 kDa. Nevertheless, ATP-dependent transport of methotrexate by [Arg(482)]ABCG2 was little affected by such mercaptoethanol treatment. It is concluded that Arg(482) is a critical amino acid moiety in the substrate specificity and transport of ABCG2 for certain drugs, such as methotrexate. PMID:12741957

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

    PubMed Central

    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

  11. Vacuolar Transport of Abscisic Acid Glucosyl Ester Is Mediated by ATP-Binding Cassette and Proton-Antiport Mechanisms in Arabidopsis1[W][OPEN

    PubMed Central

    Burla, Bo; Pfrunder, Stefanie; Nagy, Réka; Francisco, Rita Maria; Lee, Youngsook; Martinoia, Enrico

    2013-01-01

    Abscisic acid (ABA) is a key plant hormone involved in diverse physiological and developmental processes, including abiotic stress responses and the regulation of stomatal aperture and seed germination. Abscisic acid glucosyl ester (ABA-GE) is a hydrolyzable ABA conjugate that accumulates in the vacuole and presumably also in the endoplasmic reticulum. Deconjugation of ABA-GE by the endoplasmic reticulum and vacuolar β-glucosidases allows the rapid formation of free ABA in response to abiotic stress conditions such as dehydration and salt stress. ABA-GE further contributes to the maintenance of ABA homeostasis, as it is the major ABA catabolite exported from the cytosol. In this work, we identified that the import of ABA-GE into vacuoles isolated from Arabidopsis (Arabidopsis thaliana) mesophyll cells is mediated by two distinct membrane transport mechanisms: proton gradient-driven and ATP-binding cassette (ABC) transporters. Both systems have similar Km values of approximately 1 mm. According to our estimations, this low affinity appears nevertheless to be sufficient for the continuous vacuolar sequestration of ABA-GE produced in the cytosol. We further demonstrate that two tested multispecific vacuolar ABCC-type ABC transporters from Arabidopsis exhibit ABA-GE transport activity when expressed in yeast (Saccharomyces cerevisiae), which also supports the involvement of ABC transporters in ABA-GE uptake. Our findings suggest that the vacuolar ABA-GE uptake is not mediated by specific, but rather by several, possibly multispecific, transporters that are involved in the general vacuolar sequestration of conjugated metabolites. PMID:24028845

  12. AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites: functional comparisons with Atmrp1.

    PubMed Central

    Lu, Y P; Li, Z S; Drozdowicz, Y M; Hortensteiner, S; Martinoia, E; Rea, P A

    1998-01-01

    Three ATP binding cassette (ABC) transporter-like activities directed toward large amphipathic organic anions have recently been identified on the vacuolar membrane of plant cells. These are the Mg-ATP-energized, vanadate-inhibitable vacuolar accumulation of glutathione S-conjugates (GS conjugates), chlorophyll catabolites, and bile acids, respectively. Although each of these activities previously had been assigned to distinct pumps in native plant membranes, we describe here the molecular cloning, physical mapping, and heterologous expression of a gene, AtMRP2, from Arabidopsis thaliana that encodes a multispecific ABC transporter competent in the transport of both GS conjugates and chlorophyll catabolites. Unlike its isoform, AtMRP1, which transports the model Brassica napus chlorophyll catabolite transporter substrate Bn-NCC-1 at low efficiency, heterologously expressed AtMRP2 has the facility for simultaneous high-efficiency parallel transport of GS conjugates and Bn-NCC-1. The properties of AtMRP2 therefore establish a basis for the manipulation of two previously identified plant ABC transporter activities and provide an explanation for how the comparable transporter in native plant membranes would be systematically mistaken for two distinct transporters. These findings are discussed with respect to the functional organization of AtMRP2, the inability of AtMRP2 and AtMRP1 to transport the model bile acid transporter substrate taurocholate (despite the pronounced sensitivity of both to direct inhibition by this agent), the differential patterns of expression of their genes in the intact plant, and the high capacity of AtMRP2 for the transport of glutathionated herbicides and anthocyanins. PMID:9490749

  13. Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways1[OPEN

    PubMed Central

    Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Doubský, Jan; Schneeberger, Korbinian; Schulze-Lefert, Paul

    2015-01-01

    Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes. PMID:26023163

  14. Human Immunodeficiency Virus Protease Inhibitors Interact with ATP Binding Cassette Transporter 4/Multidrug Resistance Protein 4: A Basis for Unanticipated Enhanced Cytotoxicity

    PubMed Central

    Fukuda, Yu; Takenaka, Kazumasa; Sparreboom, Alex; Cheepala, Satish B.; Wu, Chung-Pu; Ekins, Sean; Ambudkar, Suresh V.

    2013-01-01

    Human immunodeficiency virus (HIV) pharmacotherapy, by combining different drug classes such as nucleoside analogs and HIV protease inhibitors (PIs), has increased HIV-patient life expectancy. Consequently, among these patients, an increase in non-HIV–associated cancers has produced a patient cohort requiring both HIV and cancer chemotherapy. We hypothesized that multidrug resistance protein 4/ATP binding cassette transporter 4 (MRP4/ABCC4), a widely expressed transporter of nucleoside-based antiviral medications as well as cancer therapeutics might interact with PIs. Among the PIs evaluated (nelfinavir, ritonavir, amprenavir, saquinavir, and indinavir), only nelfinavir both effectively stimulated MRP4 ATPase activity and inhibited substrate-stimulated ATPase activity. Saos2 and human embryonic kidney 293 cells engineered to overexpress MRP4 were then used to assess transport and cytotoxicity. MRP4 expression reduced intracellular accumulation of nelfinavir and consequently conferred survival advantage to nelfinavir cytotoxicity. Nelfinavir blocked Mrp4-mediated export, which is consistent with its ability to increase the sensitivity of MRP4-expressing cells to methotrexate. In contrast, targeted inactivation of Abcc4/Mrp4 in mouse cells specifically enhanced nelfinavir and 9-(2-phosphonylmethoxyethyl) adenine cytotoxicity. These results suggest that nelfinavir is both an inhibitor and substrate of MRP4. Because nelfinavir is a new MRP4/ABCC4 substrate, we developed a MRP4/ABCC4 pharmacophore model, which showed that the nelfinavir binding site is shared with chemotherapeutic substrates such as adefovir and methotrexate. Our studies reveal, for the first time, that nelfinavir, a potent and cytotoxic PI, is both a substrate and inhibitor of MRP4. These findings suggest that HIV-infected cancer patients receiving nelfinavir might experience both enhanced antitumor efficacy and unexpected adverse toxicity given the role of MRP4/ABCC4 in exporting nucleoside

  15. The ATP-binding cassette subunit of the maltose transporter MalK antagonizes MalT, the activator of the Escherichia coli mal regulon.

    PubMed

    Panagiotidis, C H; Boos, W; Shuman, H A

    1998-11-01

    Transcription of the mal regulon of Escherichia coli K-12 is regulated by the positive activator, MalT. In the presence of ATP and maltotriose, MalT binds to decanucleotide MalT boxes that are found upstream of mal promoters and activates transcription at these sites. The earliest studies of the mal regulon, however, suggested a negative role for the MalK protein, the ATP-binding cassette subunit of the maltose transporter, in regulating mal gene expression. More recently, it was found that overexpression of the MalK protein resulted in very low levels of mal gene transcription. In this report we describe the use of tagged versions of MalT to provide evidence that it physically interacts with the MalK protein both in vitro and in vivo. In addition, we show that a novel malK mutation, malK941, results in an increased ability of MalK to down-modulate MalT activity in vivo. The fact that the MalK941 protein binds but does not hydrolyse ATP suggests that the MalK941 mutant protein mimics the inactive, ATP-bound form of the normal MalK protein. In contrast, cells with high levels of MalK ATPase show a reduced ability to down-modulate MalT and express several mal genes constitutively. These results are consistent with a model in which the inactive form of MalK down-modulates MalT and decreases transcription, whereas the active form of MalK does not. This model suggests that bacteria may be able to couple information about extracellular substrate availability to the transcriptional apparatus via the levels of ATP hydrolysis associated with transport. PMID:9822819

  16. The ATP-Binding Cassette Transporter-2 (ABCA2) Overexpression Modulates Sphingosine Levels and Transcription of the Amyloid Precursor Protein (APP) Gene.

    PubMed

    Davis, Warren

    2015-01-01

    The ATP-binding cassette transporter-2 (ABCA2) is a member of a family of multipass transmembrane proteins that use the energy of ATP hydrolysis to transport substrates across membrane bilayers. ABCA2 has also been genetically linked with Alzheimer's disease but the molecular mechanisms are unknown. In this report, we hypothesized that ABCA2 modulation of sphingolipid metabolism activates a signaling pathway that regulates amyloid precursor protein transcription. We found that ABCA2 overexpression in N2a cells was associated with increased mass of the sphingolipid sphingosine, derived from the catabolism of ceramide. ABCA2 overexpression increased in vitro alkaline and acid ceramidase activity. Sphingosine is a physiological inhibitor of protein kinase C (PKC) activity. Pharmacological inhibition of ceramidase activity or activation PKC activity with 12-myristate 13-acetate (PMA) or diacylglycerol (DAG) decreased endogenous APP mRNA levels in ABCA2 overexpressing cells. Treatment with PMA also decreased the expression of a transfected human APP promoter reporter construct, while treatment with a general PKC inhibitor, GF109203x, increased APP promoter activity. In N2a cells, chromatin immunoprecipitation experiments revealed that a repressive complex forms at the AP-1 site in the human APP promoter, consisting of c-jun, c-jun dimerization protein 2 (JDP2) and HDAC3 and this complex was reduced in ABCA2 overexpressing cells. Activation of the human APP promoter in A2 cells was directed by the upstream stimulatory factors USF-1 and USF-2 that bound to an E-box element in vivo. These findings indicate that ABCA2 overexpression modulates sphingosine levels and regulates transcription of the endogenous APP gene. PMID:26510981

  17. The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

    PubMed

    Guo, D; Bowden, M G; Pershad, R; Kaplan, H B

    1996-03-01

    A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521. PMID:8626291

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

    PubMed Central

    Li, Dong; Xiong, Qinghui; Peng, Jin; Hu, Bin; Li, Wanzhen; Zhu, Yizhun; Shen, Xiaoyan

    2016-01-01

    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. PMID:27136542

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

    PubMed

    Li, Dong; Xiong, Qinghui; Peng, Jin; Hu, Bin; Li, Wanzhen; Zhu, Yizhun; Shen, Xiaoyan

    2016-01-01

    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. PMID:27136542

  20. A subset of bone marrow stromal cells regulate ATP-binding cassette gene expression via insulin-like growth factor-I in a leukemia cell line.

    PubMed

    Benabbou, Nadia; Mirshahi, Pezhman; Bordu, Camille; Faussat, Anne-Marie; Tang, Ruoping; Therwath, Amu; Soria, Jeannette; Marie, Jean-Pierre; Mirshahi, Massoud

    2014-10-01

    The importance of the insulin-like growth factor, IGF, as a signaling axis in cancer development, progression and metastasis is highlighted by its effects on cancer cells, notably proliferation and acquired resistance. The role of the microenvironment within which cancer cells evolve and which mediates this effect is far from clear. Here, the involvement of IGF-I in inducing multidrug resistance in a myeloid leukemia cell line, grown in the presence of bone marrow-derived stromal cells called 'Hospicells' (BMH), is demonstrated. We found that i) drug sensitive as well as resistant leukemia cells express IGF-I and its receptor IGF-IR. However, the resistant cells were found to secrete high levels of IGF-I. ii) Presence of exogenous IGF-I promoted cell proliferation, which decreased when an inhibitor of IGF-IR (picropodophyllin, PPP) was added. iii) BMH and IGF-I are both involved in the regulation of genes of the ATP binding cassette (ABC) related to resistance development (MDR1, MRP1, MRP2, MRP3 and BCRP). iv) The levels of ABC gene expression by leukemia cells were found to increase in the presence of increasing numbers of BMH. However, these levels decreased when IGF-IR was inhibited by addition of PPP. v) Co-culture of the drug-sensitive leukemia cells with BMH induced protection against the action of daunorubicin. This chemoresistance was amplified by the presence of IGF-I whereas it decreased when IGF-IR was inhibited. Our results underline the role of microenvironment in concert with the IGF-1 pathway in conferring drug resistance to leukemia cells. PMID:25095896

  1. The Myxococcus xanthus rfbABC operon encodes an ATP-binding cassette transporter homolog required for O-antigen biosynthesis and multicellular development.

    PubMed Central

    Guo, D; Bowden, M G; Pershad, R; Kaplan, H B

    1996-01-01

    A wild-type sasA locus is critical for Myxococcus xanthus multicellular development. Mutations in the sasA locus cause defective fruiting body formation, reduce sporulation, and restore developmental expression of the early A-signal-dependent gene 4521 in the absence of A signal. The wild-type sasA locus has been located on a 14-kb cloned fragment of the M. xanthus chromosome. The nucleotide sequence of a 7-kb region containing the complete sasA locus was determined. Three open reading frames encoded by the genes, designated rfbA, B and C were identified. The deduced amino acid sequences of rfbA and rfbB show identity to the integral membrane domains and ATPase domains, respectively, of the ATP-binding cassette (ABC) transporter family. The highest identities are to a set of predicted ABC transporters required for the biosynthesis of lipopolysaccharide O-antigen in certain gram-negative bacteria. The rfbC gene encodes a predicted protein of 1,276 amino acids. This predicted protein contains a region of 358 amino acids that is 33.8% identical to the Yersinia enterocolitica O3 rfbH gene product, which is also required for O-antigen biosynthesis. Immunoblot analysis revealed that the sasA1 mutant, which was found to encode a nonsense codon in the beginning of rfbA, produced less O-antigen than sasA+ strains. These data indicate that the sasA locus is required for the biosynthesis of O-antigen and, when mutated, results in A-signal-independent expression of 4521. PMID:8626291

  2. A Sensory Complex Consisting of an ATP-binding Cassette Transporter and a Two-component Regulatory System Controls Bacitracin Resistance in Bacillus subtilis*

    PubMed Central

    Dintner, Sebastian; Heermann, Ralf; Fang, Chong; Jung, Kirsten; Gebhard, Susanne

    2014-01-01

    Resistance against antimicrobial peptides in many Firmicutes bacteria is mediated by detoxification systems that are composed of a two-component regulatory system (TCS) and an ATP-binding cassette (ABC) transporter. The histidine kinases of these systems depend entirely on the transporter for sensing of antimicrobial peptides, suggesting a novel mode of signal transduction where the transporter constitutes the actual sensor. The aim of this study was to investigate the molecular mechanisms of this unusual signaling pathway in more detail, using the bacitracin resistance system BceRS-BceAB of Bacillus subtilis as an example. To analyze the proposed communication between TCS and the ABC transporter, we characterized their interactions by bacterial two-hybrid analyses and could show that the permease BceB and the histidine kinase BceS interact directly. In vitro pulldown assays confirmed this interaction, which was found to be independent of bacitracin. Because it was unknown whether BceAB-type transporters could detect their substrate peptides directly or instead recognized the peptide-target complex in the cell envelope, we next analyzed substrate binding by the transport permease, BceB. Direct and specific binding of bacitracin by BceB was demonstrated by surface plasmon resonance spectroscopy. Finally, in vitro signal transduction assays indicated that complex formation with the transporter influenced the autophosphorylation activity of the histidine kinase. Taken together, our findings clearly show the existence of a sensory complex composed of TCS and ABC transporters and provide the first functional insights into the mechanisms of stimulus perception, signal transduction, and antimicrobial resistance employed by Bce-like detoxification systems. PMID:25118291

  3. Remote communication through solute carriers and ATP binding cassette drug transporter pathways: an update on the remote sensing and signaling hypothesis.

    PubMed

    Wu, Wei; Dnyanmote, Ankur V; Nigam, Sanjay K

    2011-05-01

    Recent data from knockouts, human disease, and transport studies suggest that solute carrier (SLC) and ATP binding cassette (ABC) multispecific "drug" transporters maintain effective organ and body fluid concentrations of key nutrients, signaling molecules, and antioxidants. These processes involve transcellular movement of solutes across epithelial barriers and fluid compartments (e.g., blood, cerebrospinal fluid, urine, bile) via "matching" or homologous sets of SLC (e.g., SLC21, SLC22, SLC47) and ABC transporters. As described in the "Remote Sensing and Signaling Hypothesis" (Biochem Biophys Res Commun 323:429-436, 2004; Biochem Biophys Res Commun 351:872-876, 2006; J Biol Chem 282:23841-23853, 2007; Nat Clin Pract Nephrol 3:443-448, 2007; Mol Pharmacol 76:481-490, 2009), highly regulated transporter networks with overlapping substrate preferences are involved in sensing and signaling to maintain homeostasis in response to environmental changes (e.g., substrate imbalance and injury). They function in parallel with (and interact with) the endocrine and autonomic systems. Uric acid (urate), carnitine, prostaglandins, conjugated sex steroids, cGMP, odorants, and enterobiome metabolites are discussed here as examples. Xenobiotics hitchhike on endogenous carrier systems, sometimes leading to toxicity and side effects. By regulation of the expression and/or function of various remote organ multispecific transporters after injury, the overall transport capacity of the remote organ to handle endogenous toxins, metabolites, and signaling molecules may change, aiding in recovery. Moreover, these transporters may play a role in communication between organisms. The specific cellular components involved in sensing and altering transporter abundance or functionality depend upon the metabolite in question and probably involve different types of sensors as well as epigenetic regulation. PMID:21325265

  4. Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism.

    PubMed

    Mann, Evan; Ovchinnikova, Olga G; King, Jerry D; Whitfield, Chris

    2015-10-16

    Lysogenic bacteriophages may encode enzymes that modify the structures of lipopolysaccharide O-antigen glycans, altering the structure of the bacteriophage receptor and resulting in serotype conversion. This can enhance virulence and has implications for antigenic diversity and vaccine development. Side chain glucosylation is a common modification strategy found in a number of bacterial species. To date, glucosylation has only been observed in O-antigens synthesized by Wzy-dependent pathways, one of the two most prevalent O-antigen synthesis systems. Here we exploited a heterologous system to study the glucosylation potential of a model O-antigen produced in an ATP-binding cassette (ABC) transporter-dependent system. Although O-antigen production is cryptic in Escherichia coli K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an α-l-Rha-(1→3)-d-GlcNAc motif found in the original O16 antigen. Raoultella terrigena ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. E. coli harboring the R. terrigena O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native R. terrigena repeat structure, indicative of an altered chemical structure. Structural determination using NMR revealed the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from the E. coli chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways. PMID:26330553

  5. Characterization of the Role of a Highly Conserved Sequence in ATP Binding Cassette Transporter G (ABCG) Family in ABCG1 Stability, Oligomerization, and Trafficking

    PubMed Central

    2013-01-01

    ATP-binding cassette transporter G1 (ABCG1) mediates cholesterol and oxysterol efflux onto lipidated lipoproteins and plays an important role in macrophage reverse cholesterol transport. Here, we identified a highly conserved sequence present in the five ABCG transporter family members. The conserved sequence is located between the nucleotide binding domain and the transmembrane domain and contains five amino acid residues from Asn at position 316 to Phe at position 320 in ABCG1 (NPADF). We found that cells expressing mutant ABCG1, in which Asn316, Pro317, Asp319, and Phe320 in the conserved sequence were replaced with Ala simultaneously, showed impaired cholesterol efflux activity compared with wild type ABCG1-expressing cells. A more detailed mutagenesis study revealed that mutation of Asn316 or Phe 320 to Ala significantly reduced cellular cholesterol and 7-ketocholesterol efflux conferred by ABCG1, whereas replacement of Pro317 or Asp319 with Ala had no detectable effect. To confirm the important role of Asn316 and Phe320, we mutated Asn316 to Asp (N316D) and Gln (N316Q), and Phe320 to Ile (F320I) and Tyr (F320Y). The mutant F320Y showed the same phenotype as wild type ABCG1. However, the efflux of cholesterol and 7-ketocholesterol was reduced in cells expressing ABCG1 mutant N316D, N316Q, or F320I compared with wild type ABCG1. Further, mutations N316Q and F320I impaired ABCG1 trafficking while having no marked effect on the stability and oligomerization of ABCG1. The mutant N316Q and F320I could not be transported to the cell surface efficiently. Instead, the mutant proteins were mainly localized intracellularly. Thus, these findings indicate that the two highly conserved amino acid residues, Asn and Phe, play an important role in ABCG1-dependent export of cellular cholesterol, mainly through the regulation of ABCG1 trafficking. PMID:24320932

  6. A Member of the Second Carbohydrate Uptake Subfamily of ATP-Binding Cassette Transporters Is Responsible for Ribonucleoside Uptake in Streptococcus mutans▿

    PubMed Central

    Webb, Alexander J.; Hosie, Arthur H. F.

    2006-01-01

    Streptococcus mutans has a significant number of transporters of the ATP-binding cassette (ABC) superfamily. Members of this superfamily are involved in the translocation of a diverse range of molecules across membranes. However, the functions of many of these members remain unknown. We have investigated the role of the single S. mutans representative of the second subfamily of carbohydrate uptake transporters (CUT2) of the ABC superfamily. The genetic context of genes encoding this transporter indicates that it may have a role in ribonucleoside scavenging. Inactivation of rnsA (ATPase) or rnsB (solute binding protein) resulted in strains resistant to 5-fluorocytidine and 5-fluorouridine (toxic ribonucleoside analogues). As other ribonucleosides including cytidine, uridine, adenosine, 2-deoxyuridine, and 2-deoxycytidine protected S. mutans from 5-fluorocytidine and 5-fluorouridine toxicity, it is likely that this transporter is involved in the uptake of these molecules. Indeed, the rnsA and rnsB mutants were unable to transport [2-14C]cytidine or [2-14C]uridine and had significantly reduced [8-14C]adenosine uptake rates. Characterization of this transporter in wild-type S. mutans indicates that it is a high-affinity (Km = 1 to 2 μM) transporter of cytidine, uridine, and adenosine. The inhibition of [14C]cytidine uptake by a range of structurally related molecules indicates that the CUT2 transporter is involved in the uptake of most ribonucleosides, including 2-deoxyribonucleosides, but not ribose or nucleobases. The characterization of this permease has directly shown for the first time that an ABC transporter is involved in the uptake of ribonucleosides and extends the range of substrates known to be transported by members of the ABC transporter superfamily. PMID:16997965

  7. Down-regulation of ATP-binding cassette C2 protein expression in HepG2 cells after rifampicin treatment is mediated by microRNA-379.

    PubMed

    Haenisch, Sierk; Laechelt, Sandra; Bruckmueller, Henrike; Werk, Anneke; Noack, Andreas; Bruhn, Oliver; Remmler, Cornelia; Cascorbi, Ingolf

    2011-08-01

    microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3'-untranslated region-interference, have been shown to be involved in the regulation of ATP-binding cassette (ABC) membrane transporters. The aim of this study was to investigate whether ABCC2, an important efflux transporter for various endogenous and exogenous compounds at several compartment barriers, is subject to miRNA-mediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 μM rifampicin [a pregnane X receptor (PXR) ligand] or vehicle using reverse transcription-polymerase chain reaction-based low-density arrays. Specific miRNA, ABCC2 mRNA, and protein expression were monitored in HepG2 cells undergoing rifampicin treatment for 72 h. Loss- and gain-of-function experiments and reporter gene assays were performed for further confirmation. Highly deregulated miRNAs compared with in silico data revealed miRNA (miR) 379 as candidate miRNA targeting ABCC2 mRNA. Under rifampicin treatment, ABCC2 mRNA increased significantly, with a maximal fold change of 1.56 ± 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 ± 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 ± 0.08 after 72 h. In contrast, transfection of miR-379 inhibitor led to an elevation of ABCC2 protein expression after rifampicin incubation for 48 h. We identify a miRNA negatively regulating ABCC2 on the post-transcriptional level and provide evidence that this miRNA impedes overexpression of ABCC2 protein after a PXR-mediated external transcriptional stimulus in HepG2 cells. PMID:21540293

  8. ARRY-334543 reverses multidrug resistance by antagonizing the activity of ATP-binding cassette subfamily G member 2

    PubMed Central

    Wang, De-Shen; Patel, Atish; Sim, Hong-May; Zhang, Yun-Kai; Wang, Yi-Jun; Kathawala, Rishil J.; Zhang, Hui; Talele, Tanaji T.; Ambudkar, Suresh V.; Xu, Rui-Hua; Chen, Zhe-Sheng

    2014-01-01

    Background ARRY-334543 is a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases. We conducted this study to determine whether ARRY-334543 can enhance the efficacy of conventional anticancer drugs through interaction with ABC transporters. Methods Lung cancer cell line NCI-H460 and its ABCG2-overexpressing NCI-H460/MX20, as well as the ABCG2-, ABCB1-, and ABCC10-overexpressing transfected cell lines were used for the reversal study. Results Our results demonstrate that ARRY-334543 (1.0 μM) significantly reversed ABCG2-mediated multidrug resistance (MDR) by directly inhibiting the drug efflux function of ABCG2, resulting in the elevated intracellular accumulation of chemotherapeutic drugs in the ABCG2-overexpressing cell lines. In addition, in isolated membranes, ARRY-334543 stimulated ATPase activity and inhibited photolabeling of ABCG2 with [125I]-iodoarylazidoprazosin in a concentration-dependent manner indicating that this drug directly interacts at the drug-binding pocket of this transporter. ARRY-334543 (1.0 μM) only slightly reversed ABCB1- and partially reversed ABCC10-mediated MDR suggesting that it exhibits high affinity towards ABCG2. Moreover, homology modeling predicted the binding conformation of ARRY-334543 at Arg482 centroid-based grid of ABCG2. However, ARRY-334543 at reversal concentration did not affect the expression level of ABCG2, AKT and ERK1/2 and regulate the re-localization of ABCG2. Conclusion We conclude that ARRY-334543 significantly reverses drug resistance mediated by ABCG2. PMID:24939447

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

    PubMed

    Schäfer-Somi, S; Palme, N

    2016-04-01

    The composition of seminal plasma and the localization of the ATP-binding cassette transporter A1 (ABCA1) in spermatozoa from good and bad freezers were compared to frozen-thawed spermatozoa from the same dog. Ejaculates were obtained from 31 stud dogs, and the sperm-rich fraction (SRF) was kept for analysis. One aliquot was used for the analysis of concentration, progressive motility (P; CASA), viability (V; CASA) and leucocyte count, and the analysis was performed by flow cytometry (FITC-PNA/PI), SCSA and HOST. In seminal plasma, concentration of albumin, cholesterol, calcium, inorganic phosphate, sodium, potassium, zinc and copper was measured. Semen smears were prepared and evaluated for the expression of ABCA1. The remainder of each ejaculate was frozen. After thawing, the quality assessment was repeated and further smears were prepared. According to post-thaw semen quality, dogs were assigned to good freezers (n = 20) or bad freezers (n = 11), the latter were defined as < 50% progressive motility and/or > 40% morphologically abnormal sperm and/or < 50% viability. Bad freezers were older than good freezers (5.3 vs 3.4 years, p < 0.05). In bad freezers, the percentage of sperm with ABCA1 signal in the acrosome was lower (26.3% vs 35.7%, p < 0.01) and the percentage of sperm with complete loss of ABCA1 signal higher (46.7% vs 30%, p < 0.01); the percentage of dead spermatozoa was higher (36.1% vs 25.5%, p < 0.05), and the concentration of cholesterol and sodium in seminal plasma was lower than in good freezers (p < 0.05). We conclude that in thawed bad freezer sperm, an increase in acrosome damages coincided with an increased loss of cholesterol transporters and cell death, and a lower cholesterol concentration in seminal plasma. Follow-up studies revealed whether a relation exists between these findings. PMID:26848092

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

    PubMed

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

    2011-09-01

    Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (± 0.25), 3.6- (± 0.42), 4.1- (± 0.5), and 1.75- (± 0.43) fold, respectively, and Abcg1 by 2.1- (± 0.26), 2.2- (± 0.33), 2.5- (± 0.23), and 2.2- (± 0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions. PMID:21628419

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

    SciTech Connect

    Xu, Yanni; Lai, Fangfang; Xu, Yang; Wu, Yexiang; Liu, Qi; Li, Ni; Wei, Yuzhen; Feng, Tingting; Zheng, Zhihui; Jiang, Wei; Yu, Liyan; Hong, Bin; Si, Shuyi

    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.

  12. Evidence for a molecular diode-based mechanism in a multispecific ATP-binding cassette (ABC) exporter: SER-1368 as a gatekeeping residue in the yeast multidrug transporter Pdr5.

    PubMed

    Mehla, Jitender; Ernst, Robert; Moore, Rachel; Wakschlag, Adina; Marquis, Mary Kate; Ambudkar, Suresh V; Golin, John

    2014-09-19

    ATP-binding cassette multidrug efflux pumps transport a wide range of substrates. Current models suggest that a drug binds relatively tightly to a transport site in the transmembrane domains when the protein is in the closed inward facing conformation. Upon binding of ATP, the transporter can switch to an outward facing (drug off or drug releasing) structure of lower affinity. ATP hydrolysis is critically important for remodeling the drug-binding site to facilitate drug release and to reset the transporter for a new transport cycle. We characterized the novel phenotype of an S1368A mutant that lies in the putative drug-binding pocket of the yeast multidrug transporter Pdr5. This substitution created broad, severe drug hypersensitivity, although drug binding, ATP hydrolysis, and intradomain signaling were indistinguishable from the wild-type control. Several different rhodamine 6G efflux and accumulation assays yielded evidence consistent with the possibility that Ser-1368 prevents reentry of the excluded drug. PMID:25112867

  13. A Unified View of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gating: Combining the Allosterism of a Ligand-gated Channel with the Enzymatic Activity of an ATP-binding Cassette (ABC) Transporter*

    PubMed Central

    Kirk, Kevin L.; Wang, Wei

    2011-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ion channel in that its gating is coupled to an intrinsic enzymatic activity (ATP hydrolysis). This enzymatic activity derives from the evolutionary origin of CFTR as an ATP-binding cassette transporter. CFTR gating is distinct from that of a typical ligand-gated channel because its ligand (ATP) is usually consumed during the gating cycle. However, recent findings indicate that CFTR gating exhibits allosteric properties that are common to conventional ligand-gated channels (e.g. unliganded openings and constitutive mutations). Here, we provide a unified view of CFTR gating that combines the allosterism of a ligand-gated channel with its unique enzymatic activity. PMID:21296873

  14. Effects of a fixed-intensity of endurance training and pistacia atlantica supplementation on ATP-binding cassette G4 expression

    PubMed Central

    2013-01-01

    Background Adenosine triphosphate-cassette binding protein (ABC) type G is considered as a part of reverse cholesterol transport (RCT) process in modification and metabolism of plasma and tissue cholesterol. This study aims to evaluate the effect of endurance training with or without Pistacia atlantica (Baneh) supplementation on the female rat tissues ABC type G expression and its correlation with plasma high-density lipoprotein cholesterol (HDL-C) concentration. Methods Twenty Wistar rats (six to eight weeks old, 125–135 g weight) were arbitrarily allocated into training (n = 10) and control (n = 10) groups and further divided into saline-control (n = 5), saline-training (n = 5), Baneh-control (n = 5), and Baneh-training (n = 5). The training groups were given exercise on a motor-driven treadmill at 25 m/min (0% grade) for 60 min/day, 5 days/week for eight weeks. The rats were fed orally with Baneh extract and saline for six weeks. Seventy-two hours after the last training session, the rats were sacrificed and their tissues were excised for tissues ABCG4 expression which was detected by Real-time PCR method. Results The ABCG4 gene expressions were significantly higher in liver (P = 02), small intestine (P = 06), and visceral fat tissues (P = 04) of the trained rats compared to the tissues of the control rats, but were lower in Baneh treated rats (liver P = 045, small intestine P = 06 and visceral fat P = 004) with lower HDL-C concentrations (P = 008). Conclusions The Baneh administration lowered tissues ABCG4 expression and plasma HDL-C concentrations while endurance training increased the expression in female rat tissues. PMID:24267473

  15. A Member of the PLEIOTROPIC DRUG RESISTANCE Family of ATP Binding Cassette Transporters Is Required for the Formation of a Functional Cuticle in Arabidopsis[W

    PubMed Central

    Bessire, Michael; Borel, Sandra; Fabre, Guillaume; Carraça, Luis; Efremova, Nadia; Yephremov, Alexander; Cao, Yan; Jetter, Reinhard; Jacquat, Anne-Claude; Métraux, Jean-Pierre; Nawrath, Christiane

    2011-01-01

    Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ω-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell. PMID:21628525

  16. The hypocholesterolemic activity of açaí (Euterpe oleracea Mart.) is mediated by the enhanced expression of the ATP-binding cassette, subfamily G transporters 5 and 8 and low-density lipoprotein receptor genes in the rat.

    PubMed

    de Souza, Melina Oliveira; Souza E Silva, Lorena; de Brito Magalhães, Cíntia Lopes; de Figueiredo, Bianca Barros; Costa, Daniela Caldeira; Silva, Marcelo Eustáquio; Pedrosa, Maria Lúcia

    2012-12-01

    Previous studies have demonstrated that the ingestion of açaí pulp can improve serum lipid profile in various animal models; therefore, we hypothesized that açaí pulp (Euterpe oleracea Mart.) may modulate the expression of the genes involved in cholesterol homeostasis in the liver and increase fecal excretion, thus reducing serum cholesterol. To test this hypothesis, we analyzed the expression of 7α-hydroxylase and ATP-binding cassette, subfamily G transporters (ABCG5 and ABCG8), which are genes involved with the secretion of cholesterol in the rat. We also evaluated the expression of sterol regulatory element-binding protein 2, 3-hydroxy-3-methylglutaryl CoA reductase, low-density lipoprotein receptor (LDL-R), and apolipoprotein B100, which are involved in cholesterol biosynthesis. Female Fischer rats were divided into 4 groups: the C group, which was fed a standard AIN-93 M diet; the CA group, which was fed a standard diet supplemented with 2% açaí pulp; the H group, which was fed a hypercholesterolemic diet (25% soy oil and 1% cholesterol); and the HA group, which was fed a hypercholesterolemic diet supplemented with 2% açaí pulp. At the end of the experimental period, the rats were euthanized, and their blood and livers were collected. The HA group exhibited a significant decrease in serum total cholesterol, low-density lipoprotein cholesterol, and atherogenic index and also had increased high-density lipoprotein cholesterol and cholesterol excretion in feces compared with the H group. In addition, the expression of the LDL-R, ABCG5, and ABCG8 genes was significantly increased by the presence of açaí pulp. These results suggest that açaí pulp promotes a hypocholesterolemic effect in a rat model of dietary-induced hypercholesterolemia through an increase in the expression of ATP-binding cassette, subfamily G transporters, and LDL-R genes. PMID:23244543

  17. Genetic variant of V825I in the ATP-binding cassette transporter A1 gene and serum lipid levels in the Guangxi Bai Ku Yao and Han populations

    PubMed Central

    2011-01-01

    Background Several genetic variants in the ATP-binding cassette transporter A1 (ABCA1) gene have associated with modifications of serum high-density lipoprotein cholesterol (HDL-C) levels and the susceptibility for coronary heart disease, but the findings are still controversial in diverse racial/ethnic groups. Bai Ku Yao is an isolated subgroup of the Yao minority in southern China. The present study was undertaken to detect the possible association of V825I (rs2066715) polymorphism in the ABCA1 gene and several environmental factors with serum lipid levels in the Guangxi Bai Ku Yao and Han populations. Methods A total of 677 subjects of Bai Ku Yao and 646 participants of Han Chinese were randomly selected from our previous stratified randomized cluster samples. Polymerase chain reaction and restriction fragment length polymorphism assay combined with gel electrophoresis were performed for the genotyping of V825I variant, and then confirmed by direct sequencing. Results The levels of serum total cholesterol (TC), HDL-C, apolipoprotein (Apo) AI and ApoB were lower in Bai Ku Yao than in Han (P < 0.01 for all). The frequency of G and A alleles was 57.4% and 42.6% in Bai Ku Yao, and 57.7% and 42.3% in Han (P > 0.05); respectively. The frequency of GG, GA and AA genotypes was 33.7%, 47.4% and 18.9% in Bai Ku Yao, and 33.4%, 48.6% and 18.0% in Han (P > 0.05); respectively. There was no difference in the genotypic and allelic frequencies between males and females in the both ethnic groups. The subjects with AA genotype in Bai Ku Yao had higher serum TC levels than the subjects with GG and GA genotypes (P < 0.05). The participants with AA genotype in Han had lower serum HDL-C and ApoAI levels than the participants with GG and GA genotypes (P < 0.05 for each), but these results were found in males but not in females. Multivariate linear regression analysis showed that the levels of TC in Bai Ku Yao and HDL-C and ApoAI in male Han were correlated with genotypes (P < 0

  18. Macrophage-activating lipopeptide-2 downregulates the expression of ATP-binding cassette transporter A1 by activating the TLR2/NF-кB/ZNF202 pathway in THP-1 macrophages.

    PubMed

    Peng, Liangjie; Zhang, Zizhen; Zhang, Min; Yu, Xiaohua; Yao, Feng; Tan, Yulin; Liu, Dan; Gong, Duo; Chong, Huang; Liu, Xiaoyan; Zheng, Xilong; Tian, Guoping; Tang, Chaoke

    2016-04-01

    Macrophage-activating lipopeptide-2 (MALP-2) has been shown to promote the development of atherosclerosis. ATP-binding cassette transporter A1 (ABCA1), a transmembrane protein, plays a critical role in mediating cholesterol export from macrophages to apolipoprotein A-I (apoA-I). However, whether MALP-2 can regulate the expression of ABCA1 is still largely unknown. The aim of this study was to explore the effects of MALP-2 on ABCA1 expression in THP-1 macrophages and the underlying mechanisms. Our results showed that the treatment of cells with MALP-2 decreased ABCA1 level and suppressed cholesterol efflux in both concentration- and time-dependent manners. The contents of intracellular cholesterol were significantly increased in the presence of MALP-2. Moreover, MALP-2-mediated inhibition of ABCA1 expression was abolished by siRNA of either Toll-like receptor 2 (TLR2) or nuclear factor κB (NF-κB). A similar effect was produced by treatment with the NF-κB inhibitor pyrrolidine dithiocarbamate. In addition, MALP-2-induced activation of NF-κB markedly increased zinc finger protein 202 (ZNF202) level, and ZNF202 siRNA impaired the effects of MALP-2 on ABCA1 expression. Taken together, these results suggest that MALP-2 can decrease ABCA1 expression and subsequent cholesterol efflux through activation of the TLR2/NF-κB/ZNF202 signaling pathway in THP-1 macrophages. PMID:26922321

  19. Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism1[OPEN

    PubMed Central

    Cho, Misuk; Henry, Elizabeth M.; Lewis, Daniel R.; Wu, Guosheng; Muday, Gloria K.

    2014-01-01

    Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target. PMID:25324509

  20. Impaired 2',3'-dideoxy-3'-thiacytidine accumulation in T-lymphoblastoid cells as a mechanism of acquired resistance independent of multidrug resistant protein 4 with a possible role for ATP-binding cassette C11.

    PubMed Central

    Turriziani, O; Schuetz, J D; Focher, F; Scagnolari, C; Sampath, J; Adachi, M; Bambacioni, F; Riva, E; Antonelli, G

    2002-01-01

    Cellular factors may contribute to the decreased efficacy of chemotherapy in HIV infection. Indeed, prolonged treatment with nucleoside analogues, such as azidothymidine (AZT), 2',3'-deoxycytidine or 9-(2-phosphonylmethoxyethyl)adenine, induces cellular resistance. We have developed a human T lymphoblastoid cell line (CEM 3TC) that is selectively resistant to the antiproliferative effect of 2',3'-dideoxy-3'-thiacytidine (3TC) because the CEM 3TC cells were equally sensitive to AZT, as well as the antimitotic agent, vinblastine. The anti-retroviral activity of 3TC against HIV-1 was also severely impaired in the CEM 3TC cells. Despite similar deoxycytidine kinase activity and unchanged uptake of nucleosides such as AZT and 2'-deoxycytidine, CEM 3TC had profoundly impaired 3TC accumulation. Further studies indicated that CEM 3TC retained much less 3TC. However, despite a small overexpression of multidrug resistance protein (MRP) 4, additional studies with cells specifically engineered to overexpress MRP4 demonstrated there was no impact on either 3TC accumulation or efflux. Finally, an increased expression of the MRP5 homologue, ATP-binding cassette C11 (ABCC11) was observed in the CEM 3TC cells. We speculate that the decreased 3TC accumulation in the CEM 3TC might be due to the upregulation of ABCC11. PMID:12133003

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

    PubMed

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

    2016-01-01

    Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization. PMID:26595095

  2. ATP-Binding Cassette Transporter G1 and High-Density Lipoprotein Promote Endothelial NO Synthesis Through a Decrease in the Interaction of Caveolin-1 and Endothelial NO Synthase

    PubMed Central

    Terasaka, Naoki; Westerterp, Marit; Koetsveld, Joris; Fernández-Hernando, Carlos; Yvan-Charvet, Laurent; Wang, Nan; Sessa, William C.; Tall, Alan R.

    2016-01-01

    Objective To investigate whether cholesterol efflux to high-density lipoprotein (HDL) via ATP-binding cassette transporter G1 (ABCG1) modulates the interaction of caveolin (Cav) 1 and endothelial NO synthase (eNOS). Methods and Results ABCG1 promotes cholesterol and 7-oxysterol efflux from endothelial cells (ECs) to HDL. It was previously reported that ABCG1 protects against dietary cholesterol-induced endothelial dysfunction by promoting the efflux of 7-oxysterols to HDL. Increased cholesterol loading in ECs is known to cause an inhibitory interaction between Cav-1 and eNOS and impaired NO release. In human aortic ECs, free cholesterol loading promoted the interaction of Cav-1 with eNOS, reducing eNOS activity. These effects of cholesterol loading were reversed by HDL in an ABCG1-dependent manner. HDL also reversed the inhibition of eNOS by cholesterol loading in murine lung ECs, but this effect of HDL was abolished in Cav-1–deficient murine lung ECs. Increased interaction of Cav-1 with eNOS was also detected in aortic homogenates of high-cholesterol diet–fed Abcg1−/− mice, paralleling a decrease in eNOS activity and impaired endothelial function. Conclusion The promotion of cholesterol efflux via ABCG1 results in a reduced inhibitory interaction of eNOS with Cav-1. PMID:20798376

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

    PubMed

    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. PMID:23230133

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

    PubMed

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

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

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

    PubMed Central

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

    2015-01-01

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

  6. Protein Kinase C Is Involved in the Induction of ATP-Binding Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor Agonist in Human Macrophages.

    PubMed

    Huwait, Etimad A; Singh, Nishi N; Michael, Daryn R; Davies, Thomas S; Moss, Joe W E; Ramji, Dipak P

    2015-09-01

    The transcription of the ATP-binding cassette transporter A1 (ABCA1) gene, which plays a key anti-atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) in the LXRs-mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have, therefore, investigated a potential role for PKC in the action of LXR/RXR agonist 22-(R)-hydroxycholesterol (22-(R)-HC)/9-cis-retinoic acid (9cRA) in THP-1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein-1 (AP-1) DNA binding activity in macrophages treated with 22-(R)-HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype-specific inhibitors. These studies, therefore, reveal a potentially important role for PKC in the action of 22-(R)-HC and 9cRA in human macrophages. PMID:25752819

  7. Novel mechanism of transcriptional repression of the human ATP binding cassette transporter A1 gene in hepatic cells by the winged helix/forkhead box transcription factor A2.

    PubMed

    Thymiakou, Efstathia; Kardassis, Dimitris

    2014-06-01

    ATP binding cassette transporter A1 (ABCA1) plays a key role in the biogenesis of HDL by promoting the efflux of cellular cholesterol and phospholipids to lipid free apoA-I. Mutations in the ABCA1 gene cause Tangier disease which is characterized by near or complete absence of circulating plasma HDL. In the present study we show that the winged helix/forkhead box containing transcription factor A2 (FOXA2) shown previously to play a role in glucose and bile acid homeostasis in the liver and in energy utilization in adipose tissue is a negative modulator of ABCA1 gene expression in hepatic cells. We show that the ABCA1 promoter contains three FOXA2 binding elements in the proximal region. Two of the sites are localized in a region of the ABCA1 promoter enriched in binding elements for transcriptional repressor proteins whereas the third site is the core of the TATA element of the ABCA1 promoter. Inhibition of FOXA2 binding to the ABCA1 promoter by site-directed mutagenesis or FOXA2 gene expression by siRNA was associated with increased ABCA1 promoter activity and protein levels. Overexpression of FOXA2 inhibited both the constitutive ABCA1 gene expression as well as ABCA1 gene induction by oxysterols and retinoids via nuclear receptors LXRα/RXRα. In summary, the present study identifies transcription factor FOXA2 as a negative modulator of ABCA1 gene expression in hepatic cells and reveals a novel mechanism of transcriptional repression by FOXA2 which involves the TATA element of the ABCA1 gene. PMID:24807696

  8. Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins

    PubMed Central

    Tseng, Pei-Chi; Lee, Tzong-Shyuan; Lee, Wen-Jane; Chang, Pey-Jium; Chiang, An-Na

    2016-01-01

    Metabolic syndrome (MetS) is a complicated health problem that encompasses a variety of metabolic disorders. In this study, we analyzed the relationship between the major biochemical parameters associated with MetS and circulating levels of microRNA (miR)-33, miR-103, and miR-155. We found that miRNA-33 levels were positively correlated with levels of fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, LDL-cholesterol, and triacylglycerol, but negatively correlated with HDL-cholesterol levels. In the cellular study, miR-33 levels were increased in macrophages treated with high glucose and cholesterol-lowering drugs atorvastatin and pitavastatin. miR-33 has been reported to play an essential role in cholesterol homeostasis through ATP-binding cassette transporter A1 (ABCA1) regulation and reverse cholesterol transport. However, the molecular mechanism underlying the linkage between miR-33 and statin treatment remains unclear. In the present study, we investigated whether atorvastatin and pitavastatin exert their functions through the modulation of miR-33 and ABCA1-mediated cholesterol efflux from macrophages. The results showed that treatment of the statins up-regulated miR-33 expression, but down-regulated ABCA1 mRNA levels in RAW264.7 cells and bone marrow-derived macrophages. Statin-mediated ABCA1 regulation occurs at the post-transcriptional level through targeting of the 3′-UTR of the ABCA1 transcript by miR-33. Additionally, we found significant down-regulation of ABCA1 protein expression in macrophages treated with statins. Finally, we showed that high glucose and statin treatment significantly suppressed cholesterol efflux from macrophages. These findings have highlighted the complexity of statins, which may exert detrimental effects on metabolic abnormalities through regulation of miR-33 target genes. PMID:27139226

  9. 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.

    PubMed

    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. PMID:27057678

  10. Modulation of microRNA Expression in Subjects with Metabolic Syndrome and Decrease of Cholesterol Efflux from Macrophages via microRNA-33-Mediated Attenuation of ATP-Binding Cassette Transporter A1 Expression by Statins.

    PubMed

    Chen, Wei-Ming; Sheu, Wayne H-H; Tseng, Pei-Chi; Lee, Tzong-Shyuan; Lee, Wen-Jane; Chang, Pey-Jium; Chiang, An-Na

    2016-01-01

    Metabolic syndrome (MetS) is a complicated health problem that encompasses a variety of metabolic disorders. In this study, we analyzed the relationship between the major biochemical parameters associated with MetS and circulating levels of microRNA (miR)-33, miR-103, and miR-155. We found that miRNA-33 levels were positively correlated with levels of fasting blood glucose, glycosylated hemoglobin A1c, total cholesterol, LDL-cholesterol, and triacylglycerol, but negatively correlated with HDL-cholesterol levels. In the cellular study, miR-33 levels were increased in macrophages treated with high glucose and cholesterol-lowering drugs atorvastatin and pitavastatin. miR-33 has been reported to play an essential role in cholesterol homeostasis through ATP-binding cassette transporter A1 (ABCA1) regulation and reverse cholesterol transport. However, the molecular mechanism underlying the linkage between miR-33 and statin treatment remains unclear. In the present study, we investigated whether atorvastatin and pitavastatin exert their functions through the modulation of miR-33 and ABCA1-mediated cholesterol efflux from macrophages. The results showed that treatment of the statins up-regulated miR-33 expression, but down-regulated ABCA1 mRNA levels in RAW264.7 cells and bone marrow-derived macrophages. Statin-mediated ABCA1 regulation occurs at the post-transcriptional level through targeting of the 3'-UTR of the ABCA1 transcript by miR-33. Additionally, we found significant down-regulation of ABCA1 protein expression in macrophages treated with statins. Finally, we showed that high glucose and statin treatment significantly suppressed cholesterol efflux from macrophages. These findings have highlighted the complexity of statins, which may exert detrimental effects on metabolic abnormalities through regulation of miR-33 target genes. PMID:27139226

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

    PubMed Central

    2010-01-01

    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 expression likely plays a

  12. ATP-Binding Cassette Systems of Brucella

    PubMed Central

    Jenner, Dominic C.; Dassa, Elie; Whatmore, Adrian M.; Atkins, Helen S.

    2009-01-01

    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. PMID:20169092

  13. Aldehyde dehydrogenase and ATP binding cassette transporter G2 (ABCG2) functional assays isolate different populations of prostate stem cells where ABCG2 function selects for cells with increased stem cell activity

    PubMed Central

    2013-01-01

    Introduction High expression of aldehyde dehydrogenase1A1 (ALDH1A1) is observed in many organs and tumors and may identify benign and cancer stem cell populations. Methods In the current study, the stem cell characteristics were determined in cells isolated from human prostate cell lines and clinical prostate specimens based upon the ALDEFLUOR™ assay. Cells isolated based on the ALDEFLUOR™ assay were compared to cells isolated based on ATP binding cassette transporter G2 (ABCG2) activity using the side population assay. To test for stem cell characteristics of self-renewal and multipotency, cells with high and low ALDH1A1 activity, based on the ALDEFLUOR™ assay (ALDHHi and ALDHLow), were isolated from prostate clinical specimens and were recombined with rat urogenital sinus mesenchyme to induce prostate gland formation. Results The percentage of ALDHHi cells in prostate cell lines (RWPE-1, RWPE-2, CWR-R1, and DU-145) was 0.5 to 6%, similarly in non-tumor and tumor clinical specimens the percentage of ALDHHi cells was 0.6 to 4%. Recombinants using ALDHHi cells serially generated prostate tissue up to three generations with as few as 250 starting cells. Immunohistochemical analysis of the recombinants using ALDHHi cells contained prostatic glands frequently expressing androgen receptor (AR), p63, chromogranin A, ALDH1A1, ABCG2, and prostate specific antigen (PSA), compared to their ALDHLow counterparts. Inhibition of ALDH resulted in the reduction of sphere formation capabilities in the CWR-R1, but not in the RWPE-2 and DU-145, prostate cell lines. ABCG2 inhibition resulted in a more robust decrease of sphere formation in androgen sensitive cell lines, CWR-R1 and RWPE-2, but not androgen insensitive DU-145. ALDH1A1 expression was enriched in ALDHHi cells and non-side population cells. ABCG2 expression was only enriched in side population cells. Conclusions The percentage of ALDHHi cells in prostate cell lines and prostate tissue was consistently higher compared

  14. Co-expression of pregnane X receptor and ATP-binding cassette sub-family B member 1 in peripheral blood: A prospective indicator for drug resistance prediction in non-small cell lung cancer

    PubMed Central

    KONG, QINGNUAN; HAN, ZENGLEI; ZUO, XIAOLI; WEI, HONGJUN; HUANG, WEIQING

    2016-01-01

    The aim of the present study was to investigate the protein expression profiling of pregnane X receptor (PXR) and ATP-binding cassette sub-family B member 1 (ABCB1; also known as MDR1 or P-gp), present in the peripheral blood mononuclear cells (PBMCs) and cancerous tissues of cases of non-small cell lung cancer (NSCLC). Furthermore, the study aimed to assess the feasibility of predicting drug resistance through the medium of PBMCs. Of the subjects included in the study, 37 were histopathologically diagnosed with NSCLC and 17 were control patients without cancer. ThinPrep liquid-based smears with cytosine were applied in the examination of the PBMCs and proved quite effective in preserving the morphology and surface antigens of the lymphocytes. Measurements of expression levels in the PBMCs and cancerous tissues were obtained by immunohistochemical means. The results showed that, with the exception of the selective PXR expression in the normal lung tissues, the two types of proteins existed extensively throughout the PBMCs, normal tissues and tumors. Among the cancer patients, prior to chemotherapy, a significant rise in ABCB1 expression could be observed in the PBMCs, together with a similar rise in ABCB1 and PXR expression in the tumor specimens. Marked upregulation of the two proteins was detected in the PBMCs following 1 cycle of first-line chemotherapy. ABCB1 expression, correlated with PXR, persisted mostly in the PBMCs and tissue samples. When bound to and activated by ligands, PXR translocates from the cytoplasm to the nucleus of the cells. PXR subsequently binds to its DNA response elements as a heterodimer with the retinoid X receptor. A PXR translocation of moderate or low differentiation was identified in 3 cases of adenocarcinoma, which were co-expressing the two genes in the PBMCs prior to chemotherapy. During follow-up visits, tumor recurrence was observed within 3 months in 5 cases, which were characterized by PXR translocation. These findings

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

    PubMed

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

    2014-08-01

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

  16. Mutant cycles at CFTR's non-canonical ATP-binding site support little interface separation during gating.

    PubMed

    Szollosi, Andras; Muallem, Daniella R; Csanády, László; Vergani, Paola

    2011-06-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily. ABC proteins share a common molecular mechanism that couples ATP binding and hydrolysis at two nucleotide-binding domains (NBDs) to diverse functions. This involves formation of NBD dimers, with ATP bound at two composite interfacial sites. In CFTR, intramolecular NBD dimerization is coupled to channel opening. Channel closing is triggered by hydrolysis of the ATP molecule bound at composite site 2. Site 1, which is non-canonical, binds nucleotide tightly but is not hydrolytic. Recently, based on kinetic arguments, it was suggested that this site remains closed for several gating cycles. To investigate movements at site 1 by an independent technique, we studied changes in thermodynamic coupling between pairs of residues on opposite sides of this site. The chosen targets are likely to interact based on both phylogenetic analysis and closeness on structural models. First, we mutated T460 in NBD1 and L1353 in NBD2 (the corresponding site-2 residues become energetically coupled as channels open). Mutation T460S accelerated closure in hydrolytic conditions and in the nonhydrolytic K1250R background; mutation L1353M did not affect these rates. Analysis of the double mutant showed additive effects of mutations, suggesting that energetic coupling between the two residues remains unchanged during the gating cycle. We next investigated pairs 460-1348 and 460-1375. Although both mutations H1348A and H1375A produced dramatic changes in hydrolytic and nonhydrolytic channel closing rates, in the corresponding double mutants these changes proved mostly additive with those caused by mutation T460S, suggesting little change in energetic coupling between either positions 460-1348 or positions 460-1375 during gating. These results provide independent support for a gating model in which ATP-bound composite site 1 remains

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

    PubMed

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

    2013-01-10

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP binding cassette (ABC) protein superfamily. Currently, it remains unclear how ATP binding causes the opening of the channel gate at the molecular level. To clarify this mechanism, we first constructed an atomic model of the inward-facing CFTR using the X-ray structures of other ABC proteins. Molecular dynamics (MD) simulations were then performed to explore the structure and dynamics of the inward-facing CFTR in a membrane environment. In the MgATP-bound state, two nucleotide-binding domains (NBDs) formed a head-to-tail type of dimer, in which the ATP molecules were sandwiched between the Walker A and signature motifs. Alternatively, one of the final MD structures in the apo state was similar to that of a "closed-apo" conformation found in the X-ray analysis of ATP-free MsbA. Principal component analysis for the MD trajectory indicated that NBD dimerization causes significant structural and dynamical changes in the transmembrane domains (TMDs), which is likely indicative of the formation of a chloride ion access path. This study suggests that the free energy gain from ATP binding acts as a driving force not only for NBD dimerization but also for NBD-TMD concerted motions. PMID:23214920

  18. The two ATP binding sites of cystic fibrosis transmembrane conductance regulator (CFTR) play distinct roles in gating kinetics and energetics.

    PubMed

    Zhou, Zhen; Wang, Xiaohui; Liu, Hao-Yang; Zou, Xiaoqin; Li, Min; Hwang, Tzyh-Chang

    2006-10-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase-dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing. PMID:16966475

  19. The Two ATP Binding Sites of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Play Distinct Roles in Gating Kinetics and Energetics

    PubMed Central

    Zhou, Zhen; Wang, Xiaohui; Liu, Hao-Yang; Zou, Xiaoqin; Li, Min; Hwang, Tzyh-Chang

    2006-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC (ATP binding cassette) transporter family, is a chloride channel whose activity is controlled by protein kinase–dependent phosphorylation. Opening and closing (gating) of the phosphorylated CFTR is coupled to ATP binding and hydrolysis at CFTR's two nucleotide binding domains (NBD1 and NBD2). Recent studies present evidence that the open channel conformation reflects a head-to-tail dimerization of CFTR's two NBDs as seen in the NBDs of other ABC transporters (Vergani et al., 2005). Whether these two ATP binding sites play an equivalent role in the dynamics of NBD dimerization, and thus in gating CFTR channels, remains unsettled. Based on the crystal structures of NBDs, sequence alignment, and homology modeling, we have identified two critical aromatic amino acids (W401 in NBD1 and Y1219 in NBD2) that coordinate the adenine ring of the bound ATP. Conversion of the W401 residue to glycine (W401G) has little effect on the sensitivity of the opening rate to [ATP], but the same mutation at the Y1219 residue dramatically lowers the apparent affinity for ATP by >50-fold, suggesting distinct roles of these two ATP binding sites in channel opening. The W401G mutation, however, shortens the open time constant. Energetic analysis of our data suggests that the free energy of ATP binding at NBD1, but not at NBD2, contributes significantly to the energetics of the open state. This kinetic and energetic asymmetry of CFTR's two NBDs suggests an asymmetric motion of the NBDs during channel gating. Opening of the channel is initiated by ATP binding at the NBD2 site, whereas separation of the NBD dimer at the NBD1 site constitutes the rate-limiting step in channel closing. PMID:16966475

  20. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel.

    PubMed

    Tsai, Ming-Feng; Li, Min; Hwang, Tzyh-Chang

    2010-05-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR's opening-closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N(3)-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N(3)-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1-NBD2 interface. The open state of CFTR has been shown to represent a two-ATP-bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a "partial NBD dimer" state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and "partial" separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR's NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed. PMID:20421370

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

    PubMed

    Bompadre, Silvia G; Hwang, Tzyh-Chang

    2007-08-25

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that belongs to the ATP-binding cassette (ABC) transporter superfamily. Defective function of CFTR is responsible for cystic fibrosis (CF), the most common lethal autosomal recessive disorder in Caucasian populations. The disease is manifested in defective chloride transport across the epithelial cells in various tissues. To date, more than 1400 different mutations have been identified as CF-associated. CFTR is regulated by phosphorylation in its regulatory (R) domain, and gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBD1 and NBD2). Recent studies reveal that the NBDs of CFTR may dimerize as observed in other ABC proteins. Upon dimerization of CFTR's two NBDs, in a head-to-tail configuration, the two ATP-binding pockets (ABP1 and ABP2) are formed by the canonical Walker A and B motifs from one NBD and the signature sequence from the partner NBD. Mutations of the amino acids that interact with ATP reveal that the two ABPs play distinct roles in controlling ATP-dependent gating of CFTR. It was proposed that binding of ATP to the ABP2, which is formed by the Walker A and B in NBD2 and the signature sequence in NBD1, is critical for catalyzing channel opening. While binding of ATP to the ABP1 alone may not increase the opening rate, it does contribute to the stabilization of the open channel conformation. Several disease-associated mutations of the CFTR channel are characterized by gating defects. Understanding how CFTR's two NBDs work together to gate the channel could provide considerable mechanistic information for future pharmacological studies, which could pave the way for tailored drug design for therapeutical interventions in CF. PMID:17700963

  2. Role of ATP binding and hydrolysis in assembly of MacAB-TolC macrolide transporter.

    PubMed

    Lu, Shuo; Zgurskaya, Helen I

    2012-12-01

    MacB is a founding member of the Macrolide Exporter family of transporters belonging to the ATP-Binding Cassette superfamily. These proteins are broadly represented in genomes of both Gram-positive and Gram-negative bacteria and are implicated in virulence and protection against antibiotics and peptide toxins. MacB transporter functions together with MacA, a periplasmic membrane fusion protein, which stimulates MacB ATPase. In Gram-negative bacteria, MacA is believed to couple ATP hydrolysis to transport of substrates across the outer membrane through a TolC-like channel. In this study, we report a real-time analysis of concurrent ATP hydrolysis and assembly of MacAB-TolC complex. MacB binds nucleotides with a low millimolar affinity and fast on- and off-rates. In contrast, MacA-MacB complex is formed with a nanomolar affinity, which further increases in the presence of ATP. Our results strongly suggest that association between MacA and MacB is stimulated by ATP binding to MacB but remains unchanged during ATP hydrolysis cycle. We also found that the large periplasmic loop of MacB plays the major role in coupling reactions separated in two different membranes. This loop is required for MacA-dependent stimulation of MacB ATPase and at the same time, contributes to recruitment of TolC into a trans-envelope complex. PMID:23057817

  3. Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

    PubMed Central

    Tsai, Ming-Feng; Li, Min

    2010-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N3-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N3-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed. PMID:20421370

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

    PubMed Central

    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

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

    PubMed

    Yu, Fang; De Luca, Vincenzo

    2013-09-24

    The Madagascar periwinkle (Catharanthus roseus) is highly specialized for the biosynthesis of many different monoterpenoid indole alkaloids (MIAs), many of which have powerful biological activities. Such MIAs include the commercially important chemotherapy drugs vinblastine, vincristine, and other synthetic derivatives that are derived from the coupling of catharanthine and vindoline. However, previous studies have shown that biosynthesis of these MIAs involves extensive movement of metabolites between specialized internal leaf cells and the leaf epidermis that require the involvement of unknown secretory processes for mobilizing catharanthine to the leaf surface and vindoline to internal leaf cells. Spatial separation of vindoline and catharanthine provides a clear explanation for the low levels of dimers that accumulate in intact plants. The present work describes the molecular cloning and functional identification of a unique catharanthine transporter (CrTPT2) that is expressed predominantly in the epidermis of young leaves. CrTPT2 gene expression is activated by treatment with catharanthine, and its in planta silencing redistributes catharanthine to increase the levels of catharanthine-vindoline drug dimers in the leaves. Phylogenetic analysis shows that CrTPT2 is closely related to a key transporter involved in cuticle assembly in plants and that may be unique to MIA-producing plant species, where it mediates secretion of alkaloids to the plant surface. PMID:24019465

  6. Marine medaka ATP-binding cassette (ABC) superfamily and new insight into teleost Abch nomenclature

    PubMed Central

    Jeong, Chang-Bum; Kim, Bo-Mi; Kang, Hye-Min; Choi, Ik-Young; Rhee, Jae-Sung; Lee, Jae-Seong

    2015-01-01

    The ABC gene family is recognized as one of the largest gene families in all kingdoms of life. Although many genes involved in the ABC superfamily have been annotated from several fish species, information on large sets of the ABC superfamily and their evolutionary characterization are still unclear. In the marine medaka Oryzias melastigma, 50 ABC transporters were identified with bioinformatics-aided in silico analyses, and their full-length cDNA sequences were characterized. Phylogenetic analysis revealed that they could be classified into the eight subfamilies (A–H) that include all members of all ABC subfamilies. Interestingly, several teleosts’ Abcg members were closely clustered with Abch members in a distinctive clade. The abch gene was also observed in the coelacanth and the spotted gar, suggesting that this gene was retained from a bilaterian ancestor and that a gene loss event recently occurred in the tetrapod lineage. In teleosts, the nomenclature of previously annotated abcg genes should be considered carefully, as they form a distinctive clade with the marine medaka abch subfamily and other teleost abch genes, but not with the members of the Abcg subfamily. PMID:26472499

  7. Role of ATP-binding cassette (ABC) transporters in interactions between natural products and drugs.

    PubMed

    Aszalos, Adorjan

    2008-12-01

    Medicinal use of natural products such as extracts of plants has existed for many years in China and in other countries and they are now available worldwide. Citrus fruit juices are consumed on a daily basis around the world. Modern medicine provides well-tested compounds or drugs for most sicknesses. However, the simultaneous consumption of plant extracts, food supplements, and fruit juices with drugs can create metabolic aberrations in humans. Interactions between drugs used simultaneously are regulated by government agencies. Not regulated, but warned against in drug inserts are potential interactions between drugs and food and food-additives containing certain compounds with potential side effects. Summarized here are the results of investigations that point out possible interactions at the level of transporter molecules by drugs and compounds of natural origin. These transporter molecules play important roles in absorption in the intestines, at the blood brain barrier, in the liver, the kidney and in some other parts of the human body. Drugs and metabolites pass through these pumps and may compete with compounds from food supplements. The most studied natural compounds that are potential modulators of these transport molecules are flavonoids, found in fruit juices, vegetables, flowers and tea. Mycotoxins found in cereal grains are also shown to modulate transporter proteins. We detail here how such constituents of natural origin were shown to modulate three types of the major transporter molecules, P-glycoprotein (ABCB1), multidrug resistance proteins (ABCCs) and breast cancer resistance protein (ABCG2). Interference of these natural compounds with drugs at the transporter level is also discussed. PMID:19075617

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

    PubMed

    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. PMID:25260902

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  10. ATP binding turns plant cryptochrome into an efficient natural photoswitch.

    PubMed

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

    2014-01-01

    Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH(·) radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·(-), from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396(-). Its negative charge could trigger conformational changes necessary for signal transduction. PMID:24898692

  11. Optimization of the degenerated interfacial ATP binding site improves the function of disease-related mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels.

    PubMed

    Tsai, Ming-Feng; Jih, Kang-Yang; Shimizu, Hiroyasu; Li, Min; Hwang, Tzyh-Chang

    2010-11-26

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed. PMID:20861014

  12. Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels*♦

    PubMed Central

    Tsai, Ming-Feng; Jih, Kang-Yang; Shimizu, Hiroyasu; Li, Min; Hwang, Tzyh-Chang

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed. PMID:20861014

  13. ATP binding to the pseudokinase domain of JAK2 is critical for pathogenic activation.

    PubMed

    Hammarén, Henrik M; Ungureanu, Daniela; Grisouard, Jean; Skoda, Radek C; Hubbard, Stevan R; Silvennoinen, Olli

    2015-04-14

    Pseudokinases lack conserved motifs typically required for kinase activity. Nearly half of pseudokinases bind ATP, but only few retain phosphotransfer activity, leaving the functional role of nucleotide binding in most cases unknown. Janus kinases (JAKs) are nonreceptor tyrosine kinases with a tandem pseudokinase-kinase domain configuration, where the pseudokinase domain (JAK homology 2, JH2) has important regulatory functions and harbors mutations underlying hematological and immunological diseases. JH2 of JAK1, JAK2, and TYK2 all bind ATP, but the significance of this is unclear. We characterize the role of nucleotide binding in normal and pathogenic JAK signaling using comprehensive structure-based mutagenesis. Disruption of JH2 ATP binding in wild-type JAK2 has only minor effects, and in the presence of type I cytokine receptors, the mutations do not affect JAK2 activation. However, JH2 mutants devoid of ATP binding ameliorate the hyperactivation of JAK2 V617F. Disrupting ATP binding in JH2 also inhibits the hyperactivity of other pathogenic JAK2 mutants, as well as of JAK1 V658F, and prevents induction of erythrocytosis in a JAK2 V617F myeloproliferative neoplasm mouse model. Molecular dynamic simulations and thermal-shift analysis indicate that ATP binding stabilizes JH2, with a pronounced effect on the C helix region, which plays a critical role in pathogenic activation of JAK2. Taken together, our results suggest that ATP binding to JH2 serves a structural role in JAKs, which is required for aberrant activity of pathogenic JAK mutants. The inhibitory effect of abrogating JH2 ATP binding in pathogenic JAK mutants may warrant novel therapeutic approaches. PMID:25825724

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

    SciTech Connect

    Hattori, Motoyuki; Gouaux, Eric

    2012-10-24

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

  15. Bis(monoacylglycero)phosphate accumulation in macrophages induces intracellular cholesterol redistribution, attenuates liver-X receptor/ATP-Binding cassette transporter A1/ATP-binding cassette transporter G1 pathway, and impairs cholesterol efflux

    PubMed Central

    Luquain-Costaz, Céline; Lefai, Etienne; Arnal-Levron, Maud; Markina, Daria; Sakaï, Shota; Euthine, Vanessa; Makino, Asami; Guichardant, Michel; Yamashita, Shizuya; Kobayashi, Toshihide; Lagarde, Michel; Moulin, Philippe; Delton-Vandenbroucke, Isabelle

    2013-01-01

    Objective Endosomal signature phospholipid bis(monoacylglycero)phosphate (BMP) has been involved in the regulation of cellular cholesterol homeostasis. Accumulation of BMP is a hallmark of lipid storage disorders and was recently reported as a noticeable feature of oxidized LDL-laden macrophages. This study was designed to delineate the consequences of macrophage BMP accumulation on intracellular cholesterol distribution, metabolism and efflux and to unravel the underlying molecular mechanisms. Methods and results We have developed an experimental design to specifically increase BMP content in RAW macrophages. Following BMP accumulation, cell cholesterol distribution was markedly altered despite no change in LDL uptake and hydrolysis, cholesterol esterification, or total cell cholesterol content. The expression of cholesterol regulated genes SREBP2 and HMGCoAR was decreased by 40%, indicative of an increase of endoplasmic reticulum associated-cholesterol. Cholesterol delivery to plasma membrane was reduced as evidenced by the 20% decrease of efflux by cyclodextrin. Functionally, BMP accumulation reduced cholesterol efflux to both apoA1 and HDL by 40%, correlated with a 40% decrease in mRNA contents of ABCA1 and ABCG1 transporters and LXR α and β. Foam cell formation induced by oxidized LDL exposure was exacerbated in BMP enriched cells. Conclusion The present work shows for the first time a strong functional link between BMP and cholesterol regulating genes involved in both intracellular metabolism and efflux. We propose that accumulation of cellular BMP might contribute to the deregulation of cholesterol homeostasis in atheromatous macrophages. PMID:23788762

  16. Learning our ABCs: Rad50 directs MRN repair functions via adenylate kinase activity from the conserved ATP binding cassette.

    PubMed

    Williams, R Scott; Tainer, John A

    2007-03-23

    In groundbreaking work, Bhaskara et al. (2007) demonstrate in a recent issue of Molecular Cell that the Mre11/Rad50/Nbs1 (MRN) complex harbors distinct, yet chemically related, ATPase and adenylate kinase catalytic activities that together orchestrate multiple requisite MRN functional and conformational states in dsDNA break repair sensing and signaling with general implications for ABC ATPases. PMID:17386254

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  18. Genotype/Phenotype analysis of a photoreceptor-specific ATP-binding cassette transporter gene, ABCR, in Stargardt disease.

    PubMed Central

    Lewis, R A; Shroyer, N F; Singh, N; Allikmets, R; Hutchinson, A; Li, Y; Lupski, J R; Leppert, M; Dean, M

    1999-01-01

    Mutation scanning and direct DNA sequencing of all 50 exons of ABCR were completed for 150 families segregating recessive Stargardt disease (STGD1). ABCR variations were identified in 173 (57%) disease chromosomes, the majority of which represent missense amino acid substitutions. These ABCR variants were not found in 220 unaffected control individuals (440 chromosomes) but do cosegregate with the disease in these families with STGD1, and many occur in conserved functional domains. Missense amino acid substitutions located in the amino terminal one-third of the protein appear to be associated with earlier onset of the disease and may represent misfolding alleles. The two most common mutant alleles, G1961E and A1038V, each identified in 16 of 173 disease chromosomes, composed 18.5% of mutations identified. G1961E has been associated previously, at a statistically significant level in the heterozygous state, with age-related macular degeneration (AMD). Clinical evaluation of these 150 families with STGD1 revealed a high frequency of AMD in first- and second-degree relatives. These findings support the hypothesis that compound heterozygous ABCR mutations are responsible for STGD1 and that some heterozygous ABCR mutations may enhance susceptibility to AMD. PMID:9973280

  19. Do ATP-binding cassette transporters cause pharmacoresistance in epilepsy? Problems and approaches in determining which antiepileptic drugs are affected.

    PubMed

    Löscher, Wolfgang; Luna-Tortós, Carlos; Römermann, Kerstin; Fedrowitz, Maren

    2011-01-01

    Resistance to multiple antiepileptic drugs (AEDs) is a common problem in epilepsy, affecting at least 30% of patients. One prominent hypothesis to explain this resistance suggests an inadequate penetration or excess efflux of AEDs across the blood - brain barrier (BBB) as a result of overexpressed efflux transporters such as P-glycoprotein (Pgp), the encoded product of the multidrug resistance- 1 (MDR1, ABCB1) gene. Pgp and MDR1 are markedly increased in epileptogenic brain tissue of patients with AED-resistant partial epilepsy and following seizures in rodent models of partial epilepsy. In rodent models, AED-resistant rats exhibit higher Pgp levels than responsive animals; increased Pgp expression is associated with lower brain levels of AEDs; and, most importantly, co-administration of Pgp inhibitors reverses AED resistance. Thus, it is reasonable to conclude that Pgp plays a significant role in mediating resistance to AEDs in rodent models of epilepsy - however, whether this phenomenon extends to at least some human refractory epilepsy remains unclear, particularly because it is still a matter of debate which AEDs, if any, are transported by human Pgp. The difficulty in determining which AEDs are substrates of human Pgp is mainly a consequence of the fact that AEDs are highly permeable compounds, which are not easily identified as Pgp substrates in in vitro models of the BBB, such as monolayer (Transwell(®)) efflux assays. By using a modified assay (concentration equilibrium transport assay; CETA), which minimizes the influence of high transcellular permeability, two groups have recently demonstrated that several major AEDs are transported by human Pgp. Importantly, it was demonstrated in these studies that Pgp-mediated transport highly depends on the AED concentration and may not be identified if concentrations below or above the therapeutic range are used. In addition to the efflux transporters, seizure-induced alterations in BBB integrity and activity of drug metabolizing enzymes (CYPs) affect the brain uptake of AEDs. For translating these findings to the clinical arena, in vivo imaging studies using positron emission tomography (PET) with (11)C-labelled AEDs in epileptic patients are under way. PMID:21827408

  20. Apolipoprotein M expression increases the size of nascent pre beta HDL formed by ATP binding cassette transporter A1.

    PubMed

    Mulya, Anny; Seo, Jeongmin; Brown, Amanda L; Gebre, Abraham K; Boudyguina, Elena; Shelness, Gregory S; Parks, John S

    2010-03-01

    Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1. PMID:19767535

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

    PubMed Central

    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

  2. Probing the ATP-binding site of P1 ParA: partition and repression have different requirements for ATP binding and hydrolysis

    PubMed Central

    Fung, Emma; Bouet, Jean-Yves; Funnell, Barbara E.

    2001-01-01

    The ParA family of proteins is involved in partition of a variety of plasmid and bacterial chromosomes. P1 ParA plays two roles in partition: it acts as a repressor of the par operon and has an undefined yet indispensable role in P1 plasmid localization. We constructed seven mutations in three putative ATP-binding motifs of ParA. Three classes of phenotypes resulted, each represented by mutations in more than one motif. Three mutations created ‘super-repressors’, in which repressor activity was much stronger than in wild-type ParA, while the remainder damaged repressor activity. All mutations eliminated partition activities, but two showed a plasmid stability defect that was worse than that of a null mutation. Four mutant ParAs, two super-repressors and two weak repressors, were analyzed biochemically, and all exhibited damaged ATPase activity. The super-repressors bound site-specifically to the par operator sequence, and this activity was strongly stimulated by ATP and ADP. These results support the proposal that ATP binding is essential but hydrolysis is inhibitory for ParA’s repressor activity and suggest that ATP hydrolysis is essential for plasmid localization. PMID:11532954

  3. Cassette Books.

    ERIC Educational Resources Information Center

    Library of Congress, Washington, DC. National Library Service for the Blind and Physically Handicapped.

    This catalog lists cassette books produced by the National Library Service for the Blind and Physically Handicapped during 1989. Books are listed alphabetically within subject categories under nonfiction and fiction headings. Nonfiction categories include: animals and wildlife, the arts, bestsellers, biography, blindness and physical handicaps,…

  4. The ATP-binding site of Ca(2+)-ATPase revealed by electron image analysis.

    PubMed Central

    Yonekura, K; Stokes, D L; Sasabe, H; Toyoshima, C

    1997-01-01

    The location of the ATP-binding site of a P-type ion pump, Ca(2+)-ATPase from rabbit sarcoplasmic reticulum, was examined by cryoelectron microscopy. A nonhydrolyzable analog of ATP, beta, gamma-bidentate chromium (III) complex of ATP (CrATP), was used to stabilize the enzyme in the Ca(2+)-occluded state. Tubular crystals were then induced by vanadate in the presence of EGTA, keeping CrATP bound to the enzyme. The three-dimensional structures of the crystals were determined at 14 A resolution by cryoelectron microscopy and helical image analysis. Statistical comparison of the structures with and without CrATP showed clear and significant differences at the groove proposed previously as the ATP-binding pocket. Images FIGURE 3 FIGURE 6 FIGURE 7 PMID:9138598

  5. Inactivation of Multiple Bacterial Histidine Kinases by Targeting the ATP-Binding Domain

    PubMed Central

    2015-01-01

    Antibacterial agents that exploit new targets will be required to combat the perpetual rise of bacterial resistance to current antibiotics. We are exploring the inhibition of histidine kinases, constituents of two-component systems. Two-component systems are the primary signaling pathways that bacteria utilize to respond to their environment. They are ubiquitous in bacteria and trigger various pathogenic mechanisms. To attenuate these signaling pathways, we sought to broadly target the histidine kinase family by focusing on their highly conserved ATP-binding domain. Development of a fluorescence polarization displacement assay facilitated high-throughput screening of ∼53 000 diverse small molecules for binding to the ATP-binding pocket. Of these compounds, nine inhibited the catalytic activity of two or more histidine kinases. These scaffolds could provide valuable starting points for the design of broadly effective HK inhibitors, global reduction of bacterial signaling, and ultimately, a class of antibiotics that function by a new mechanism of action. PMID:25531939

  6. The rad50 signature motif: essential to ATP binding and biological function.

    PubMed

    Moncalian, Gabriel; Lengsfeld, Bettina; Bhaskara, Venugopal; Hopfner, Karl-Peter; Karcher, Annette; Alden, Erinn; Tainer, John A; Paull, Tanya T

    2004-01-23

    The repair of double-strand breaks in DNA is an essential process in all organisms, and requires the coordinated activities of evolutionarily conserved protein assemblies. One of the most critical of these is the Mre11/Rad50 (M/R) complex, which is present in all three biological kingdoms, but is not well-understood at the biochemical level. Previous structural analysis of a Rad50 homolog from archaebacteria illuminated the catalytic core of the enzyme, an ATP-binding domain related to the ABC transporter family of ATPases. Here, we present the crystallographic structure of the Rad50 mutant S793R. This missense signature motif mutation changes the key serine residue in the signature motif that is conserved among Rad50 homologs and ABC ATPases. The S793R mutation is analogous to the mutation S549R in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in cystic fibrosis. We show here that the serine to arginine change in the Rad50 protein prevents ATP binding and disrupts the communication among the other ATP-binding loops. This structural change, in turn, alters the communication between Rad50 monomers and thus prevents Rad50 dimerization. The equivalent mutation was made in the human Rad50 gene, and the resulting mutant protein did form a complex with Mre11 and Nbs1, but was specifically deficient in all ATP-dependent enzymatic activities. This signature motif structure-function homology extends to yeast, because the same mutation introduced into the Saccharomyces cerevisiae RAD50 gene generated an allele that failed to complement a rad50 deletion strain in DNA repair assays in vivo. These structural and biochemical results extend our understanding of the Rad50 catalytic domain and validate the use of the signature motif mutant to test the role of Rad50 ATP binding in diverse organisms. PMID:14698290

  7. Regulation of CFTR Cl- channel gating by ATP binding and hydrolysis.

    PubMed

    Ikuma, M; Welsh, M J

    2000-07-18

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg(2+) prevents hydrolysis. We found that even in the absence of Mg(2+), ATP could support channel activity, albeit at a reduced level compared with the presence of Mg(2+). Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg(2+) further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg(2+), divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  8. Regulation of CFTR Cl− channel gating by ATP binding and hydrolysis

    PubMed Central

    Ikuma, Mutsuhiro; Welsh, Michael J.

    2000-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is regulated by the interaction of ATP with its two cytoplasmic nucleotide-binding domains (NBD). Although ATP hydrolysis by the NBDs is required for normal gating, the influence of ATP binding versus hydrolysis on specific steps in the gating cycle remains uncertain. Earlier work showed that the absence of Mg2+ prevents hydrolysis. We found that even in the absence of Mg2+, ATP could support channel activity, albeit at a reduced level compared with the presence of Mg2+. Application of ATP with a divalent cation, including the poorly hydrolyzed CaATP complex, increased the rate of opening. Moreover, in CFTR variants with mutations that disrupt hydrolysis, ATP alone opened the channel and Mg2+ further enhanced ATP-dependent opening. These data suggest that ATP alone can open the channel and that divalent cations increase ATP binding. Consistent with this conclusion, when we mutated an aspartate thought to bind Mg2+, divalent cations failed to increase activity compared with ATP alone. Two observations suggested that divalent cations also stabilize the open state. In wild-type CFTR, CaATP generated a long duration open state, whereas ATP alone did not. With a CFTR variant in which hydrolysis was disrupted, MgATP, but not ATP alone, produced long openings. These results suggest a gating cycle for CFTR in which ATP binding opens the channel and either hydrolysis or dissociation leads to channel closure. In addition, the data suggest that ATP binding and hydrolysis by either NBD can gate the channel. PMID:10880569

  9. Unique Functional and Structural Properties of the LRRK2 Protein ATP-binding Pocket*

    PubMed Central

    Liu, Zhiyong; Galemmo, Robert A.; Fraser, Kyle B.; Moehle, Mark S.; Sen, Saurabh; Volpicelli-Daley, Laura A.; DeLucas, Lawrence J.; Ross, Larry J.; Valiyaveettil, Jacob; Moukha-Chafiq, Omar; Pathak, Ashish K.; Ananthan, Subramaniam; Kezar, Hollis; White, E. Lucile; Gupta, Vandana; Maddry, Joseph A.; Suto, Mark J.; West, Andrew B.

    2014-01-01

    Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult. PMID:25228699

  10. On the ATP binding site of the ε subunit from bacterial F-type ATP synthases.

    PubMed

    Krah, Alexander; Takada, Shoji

    2016-04-01

    F-type ATP synthases are reversible machinery that not only synthesize adenosine triphosphate (ATP) using an electrochemical gradient across the membrane, but also can hydrolyze ATP to pump ions under certain conditions. To prevent wasteful ATP hydrolysis, subunit ε in bacterial ATP synthases changes its conformation from the non-inhibitory down- to the inhibitory up-state at a low cellular ATP concentration. Recently, a crystal structure of the ε subunit in complex with ATP was solved in a non-biologically relevant dimeric form. Here, to derive the functional ATP binding site motif, we carried out molecular dynamics simulations and free energy calculations. Our results suggest that the ATP binding site markedly differs from the experimental resolved one; we observe a reorientation of several residues, which bind to ATP in the crystal structure. In addition we find that an Mg(2+) ion is coordinated by ATP, replacing interactions of the second chain in the crystal structure. Thus we demonstrate more generally the influence of crystallization effects on ligand binding sites and their respective binding modes. Furthermore, we propose a role for two highly conserved residues to control the ATP binding/unbinding event, which have not been considered before. Additionally our results provide the basis for the rational development of new biosensors based on subunit ε, as shown previously for novel sensors measuring the ATP concentration in cells. PMID:26780667

  11. Recombinant preparation and functional studies of EspI ATP binding domain from Mycobacterium tuberculosis.

    PubMed

    Chen, Hanyu; Wang, Huilin; Sun, Tao; Tian, Shuangliang; Lin, Donghai; Guo, Chenyun

    2016-07-01

    The ESX-1 secretion system of Mycobacterium tuberculosis is required for the virulence of tubercle bacillus. EspI, the ESX-1 secretion-associated protein in Mycobacterium tuberculosis (MtEspI), is involved in repressing the activity of ESX-1-mediated secretion when the cellular ATP level is low. The ATP binding domain of MtEspI plays a crucial role in this regulatory process. However, further structural and functional studies of MtEspI are hindered due to the bottleneck of obtaining stable and pure recombinant protein. In this study, we systematically analyzed the structure and function of MtEspI using bioinformatics tools and tried various expression constructs to recombinantly express full-length and truncated MtEspI ATP binding domain. Finally, we prepared pure and stable MtEspI ATP binding domain, MtEspI415-493, in Escherichia coli by fusion expression and purification with dual tag, Glutathione S-transferase (GST) tag and (His)6 tag. (31)P NMR titration assay indicated that MtEspI415-493 possessed a moderate affinity (∼μM) for ATP and the residue K425 was located at the binding site. The protocol described here may provide a train of thought for recombinant preparation of other ESX-1 secretion-associated proteins. PMID:27017992

  12. Cowpox virus protein CPXV012 eludes CTLs by blocking ATP binding to TAP.

    PubMed

    Luteijn, Rutger D; Hoelen, Hanneke; Kruse, Elisabeth; van Leeuwen, Wouter F; Grootens, Jennine; Horst, Daniëlle; Koorengevel, Martijn; Drijfhout, Jan W; Kremmer, Elisabeth; Früh, Klaus; Neefjes, Jacques J; Killian, Antoinette; Lebbink, Robert Jan; Ressing, Maaike E; Wiertz, Emmanuel J H J

    2014-08-15

    CD8(+) CTLs detect virus-infected cells through recognition of virus-derived peptides presented at the cell surface by MHC class I molecules. The cowpox virus protein CPXV012 deprives the endoplasmic reticulum (ER) lumen of peptides for loading onto newly synthesized MHC class I molecules by inhibiting the transporter associated with Ag processing (TAP). This evasion strategy allows the virus to avoid detection by the immune system. In this article, we show that CPXV012, a 9-kDa type II transmembrane protein, prevents peptide transport by inhibiting ATP binding to TAP. We identified a segment within the ER-luminal domain of CPXV012 that imposes the block in peptide transport by TAP. Biophysical studies show that this domain has a strong affinity for phospholipids that are also abundant in the ER membrane. We discuss these findings in an evolutionary context and show that a frameshift deletion in the CPXV012 gene in an ancestral cowpox virus created the current form of CPXV012 that is capable of inhibiting TAP. In conclusion, our findings indicate that the ER-luminal domain of CPXV012 inserts into the ER membrane, where it interacts with TAP. CPXV012 presumably induces a conformational arrest that precludes ATP binding to TAP and, thus, activity of TAP, thereby preventing the presentation of viral peptides to CTLs. PMID:25024387

  13. Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins

    PubMed Central

    Atherton, Joseph; Farabella, Irene; Yu, I-Mei; Rosenfeld, Steven S; Houdusse, Anne; Topf, Maya; Moores, Carolyn A

    2014-01-01

    Kinesins are a superfamily of microtubule-based ATP-powered motors, important for multiple, essential cellular functions. How microtubule binding stimulates their ATPase and controls force generation is not understood. To address this fundamental question, we visualized microtubule-bound kinesin-1 and kinesin-3 motor domains at multiple steps in their ATPase cycles—including their nucleotide-free states—at ∼7 Å resolution using cryo-electron microscopy. In both motors, microtubule binding promotes ordered conformations of conserved loops that stimulate ADP release, enhance microtubule affinity and prime the catalytic site for ATP binding. ATP binding causes only small shifts of these nucleotide-coordinating loops but induces large conformational changes elsewhere that allow force generation and neck linker docking towards the microtubule plus end. Family-specific differences across the kinesin–microtubule interface account for the distinctive properties of each motor. Our data thus provide evidence for a conserved ATP-driven mechanism for kinesins and reveal the critical mechanistic contribution of the microtubule interface. DOI: http://dx.doi.org/10.7554/eLife.03680.001 PMID:25209998

  14. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR

    PubMed Central

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min

    2016-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  15. A stable ATP binding to the nucleotide binding domain is important for reliable gating cycle in an ABC transporter CFTR.

    PubMed

    Shimizu, Hiroyasu; Yu, Ying-Chun; Kono, Koichi; Kubota, Takahiro; Yasui, Masato; Li, Min; Hwang, Tzyh-Chang; Sohma, Yoshiro

    2010-09-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, a member of ABC transporter superfamily, gates following ATP-dependent conformational changes of the nucleotide binding domains (NBD). Reflecting the hundreds of milliseconds duration of the channel open state corresponding to the dimerization of two NBDs, macroscopic WT-CFTR currents usually showed a fast, single exponential relaxation upon removal of cytoplasmic ATP. Mutations of tyrosine1219, a residue critical for ATP binding in second NBD (NBD2), induced a significant slow phase in the current relaxation, suggesting that weakening ATP binding affinity at NBD2 increases the probability of the stable open state. The slow phase was effectively diminished by a higher affinity ATP analogue. These data suggest that a stable binding of ATP to NBD2 is required for normal CFTR gating cycle, andthat the instability of ATP binding frequently halts the gating cycle in the open state presumably through a failure of ATP hydrolysis at NBD2. PMID:20628841

  16. ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase.

    PubMed Central

    Zalk, Ran; Shoshan-Barmatz, Varda

    2003-01-01

    VCP (valosin-containing protein) or p97 is a member of the AAA family (ATPases associated with a variety of cellular activities family), a diverse group of proteins sharing a key conserved AAA module containing duplicate putative ATP-binding sites. Although the functions of the AAA family are related to their putative ATP-binding sites, the binding of ATP to these sites has not yet been demonstrated. In the present study, the ATP-binding site(s) of brain VCP was characterized using the photoreactive ATP analogue, BzATP [3'- O -(4-benzoylbenzoyl)ATP]. Photo-activation of Bz-[alpha-(32)P]ATP resulted in its covalent binding to a 97-kDa purified soluble or membrane-associated protein, identified by amino acid sequencing as VCP. Bz-[alpha-(32)P]ATP covalently bound to the purified homo-hexameric VCP with an apparent high affinity (74-111 nM). A molar stoichiometry of 2.23+/-0.14 BzATP bound per homo-hexameric VCP (n =6) was determined using different methods for analysis of radiolabelling and protein determination. Nucleotides inhibited the binding of Bz-[alpha-(32)P]ATP to VCP with the following efficiency: BzATP>ATP>ADP>>adenosine 5'-[beta,gamma-imido]triphosphate>or=adenosine 5'-[beta,gamma-methylene]triphosphate, whereas AMP, GTP and CTP were ineffective. VCP was observed to possess very low ATPase activity, with nucleotide specificity similar to that for BzATP binding. Conformational changes induced by an alternating site mechanism for ATP binding are suggested as a molecular mechanism for coupling ATP binding to the diverse activities of the AAA family. PMID:12747802

  17. Study of the ATP-binding site of helicase IV from Escherichia coli.

    PubMed

    Dubaele, Sandy; Lourdel, Claude; Chène, Patrick

    2006-03-17

    Helicases contain conserved motifs involved in ATP/magnesium/nucleic acid binding and in the mechanisms coupling nucleotide hydrolysis to duplex unwinding. None of these motifs are located at the adenine-binding pocket of the protein. We show here that the superfamily I helicase, helicase IV from Escherichia coli, utilizes a conserved glutamine and conserved aromatic residue to interact with ATP. Other superfamily I helicases such as, UvrD/Rep/PcrA also possess these residues but in addition they interact with adenine via a conserved arginine, which is replaced by a serine in helicase IV. Mutation of this serine residue in helicase IV into histidine or methionine leads to proteins with unaffected ATPase and DNA-binding activities but with low helicase activity. This suggests that residues located at the adenine-binding pocket, in addition to be involved in ATP-binding, are important for efficient coupling between ATP hydrolysis and DNA unwinding. PMID:16442499

  18. Inhibitors of Ketohexokinase: Discovery of Pyrimidinopyrimidines with Specific Substitution that Complements the ATP-Binding Site

    PubMed Central

    2011-01-01

    Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket. PMID:24900346

  19. Critical roles of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase.

    PubMed

    Clausen, Johannes D; Holdensen, Anne Nyholm; Andersen, Jens Peter

    2014-10-17

    ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca(2+)-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca(2+) transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser(186) and Asp(203) interact with Glu(439) (N-domain) and Arg(678) (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp(203) and Arg(678) rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser(186) and Glu(439). By taking advantage of the ability of wild type and mutant Ca(2+)-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca(2+)-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp(203)-Arg(678) and Ser(186)-Glu(439) interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser(186)-Glu(439) bond are mutually exclusive in the E2P ground state. PMID:25193668

  20. Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase*

    PubMed Central

    Clausen, Johannes D.; Holdensen, Anne Nyholm; Andersen, Jens Peter

    2014-01-01

    ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca2+-ATPase. Upon binding to the Ca2E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca2+ transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser186 and Asp203 interact with Glu439 (N-domain) and Arg678 (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp203 and Arg678 rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser186 and Glu439. By taking advantage of the ability of wild type and mutant Ca2+-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca2+-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp203-Arg678 and Ser186-Glu439 interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser186-Glu439 bond are mutually exclusive in the E2P ground state. PMID:25193668

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

    PubMed

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

    2014-09-17

    Cryptochromes are flavoproteins encountered in most vegetal and animal species. They play a role of blue-light receptors in plants and in invertebrates. The putative resting state of the FAD cofactor in these proteins is its fully oxidized form, FADox. Upon blue-light excitation, the isoalloxazine ring (ISO) may undergo an ultrafast reduction by a nearby tryptophan residue W400. This primary reduction triggers a cascade of electron and proton transfers, ultimately leading to the formation of the FADH° radical. A recent experimental study has shown that the yield of FADH° formation in Arabidopsis cryptochrome can be strongly modulated by ATP binding and by pH, affecting the protonation state of D396 (proton donor to FAD°(-)). Here we provide a detailed molecular analysis of these effects by means of combined classical molecular dynamics simulations and time-dependent density functional theory calculations. When ATP is present and D396 protonated, FAD remains in close contact with W400, thereby enhancing electron transfer (ET) from W400 to ISO*. In contrast, deprotonation of D396 and absence of ATP introduce flexibility to the photoactive site prior to FAD excitation, with the consequence of increased ISO-W400 distance and diminished tunneling rate by almost two orders of magnitude. We show that under these conditions, ET from the adenine moiety of FAD becomes a competitive relaxation pathway. Overall, our data suggest that the observed effects of ATP and pH on the FAD photoreduction find their roots in the earliest stage of the photoreduction process; i.e., ATP binding and the protonation state of D396 determine the preferred pathway of ISO* relaxation. PMID:25157750

  2. A Chemical Proteomics Approach to Profiling the ATP-binding Proteome of Mycobacterium tuberculosis *

    PubMed Central

    Wolfe, Lisa M.; Veeraraghavan, Usha; Idicula-Thomas, Susan; Schürer, Stephan; Wennerberg, Krister; Reynolds, Robert; Besra, Gurdyal S.; Dobos, Karen M.

    2013-01-01

    Tuberculosis, caused by Mycobacterium tuberculosis, remains one of the leading causes of death worldwide despite extensive research, directly observed therapy using multidrug regimens, and the widespread use of a vaccine. The majority of patients harbor the bacterium in a state of metabolic dormancy. New drugs with novel modes of action are needed to target essential metabolic pathways in M. tuberculosis; ATP-competitive enzyme inhibitors are one such class. Previous screening efforts for ATP-competitive enzyme inhibitors identified several classes of lead compounds that demonstrated potent anti-mycobacterial efficacy as well as tolerable levels of toxicity in cell culture. In this report, a probe-based chemoproteomic approach was used to selectively profile the M. tuberculosis ATP-binding proteome in normally growing and hypoxic M. tuberculosis. From these studies, 122 ATP-binding proteins were identified in either metabolic state, and roughly 60% of these are reported to be essential for survival in vitro. These data are available through ProteomeXchange with identifier PXD000141. Protein families vital to the survival of the tubercle bacillus during hypoxia emerged from our studies. Specifically, along with members of the DosR regulon, several proteins involved in energy metabolism (Icl/Rv0468 and Mdh/Rv1240) and lipid biosynthesis (UmaA/Rv0469, DesA1/Rv0824c, and DesA2/Rv1094) were found to be differentially abundant in hypoxic versus normal growing cultures. These pathways represent a subset of proteins that may be relevant therapeutic targets for development of novel ATP-competitive antibiotics. PMID:23462205

  3. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain.

    PubMed

    Berger, Allan L; Ikuma, Mutsuhiro; Welsh, Michael J

    2005-01-11

    ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [alpha-(32)P]8-N(3)-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating. PMID:15623556

  4. Normal gating of CFTR requires ATP binding to both nucleotide-binding domains and hydrolysis at the second nucleotide-binding domain

    PubMed Central

    Berger, Allan L.; Ikuma, Mutsuhiro; Welsh, Michael J.

    2005-01-01

    ATP interacts with the two nucleotide-binding domains (NBDs) of CFTR to control gating. However, it is unclear whether gating involves ATP binding alone, or also involves hydrolysis at each NBD. We introduced phenylalanine residues into nonconserved positions of each NBD Walker A motif to sterically prevent ATP binding. These mutations blocked [α-32P]8-N3-ATP labeling of the mutated NBD and reduced channel opening rate without changing burst duration. Introducing cysteine residues at these positions and modifying with N-ethylmaleimide produced the same gating behavior. These results indicate that normal gating requires ATP binding to both NBDs, but ATP interaction with one NBD is sufficient to support some activity. We also studied mutations of the conserved Walker A lysine residues (K464A and K1250A) that prevent hydrolysis. By combining substitutions that block ATP binding with Walker A lysine mutations, we could differentiate the role of ATP binding vs. hydrolysis at each NBD. The K1250A mutation prolonged burst duration; however, blocking ATP binding prevented the long bursts. These data indicate that ATP binding to NBD2 allowed channel opening and that closing was delayed in the absence of hydrolysis. The corresponding NBD1 mutations showed relatively little effect of preventing ATP hydrolysis but a large inhibition of blocking ATP binding. These data suggest that ATP binding to NBD1 is required for normal activity but that hydrolysis has little effect. Our results suggest that both NBDs contribute to channel gating, NBD1 binds ATP but supports little hydrolysis, and ATP binding and hydrolysis at NBD2 are key for normal gating. PMID:15623556

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

    PubMed

    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. PMID:26143398

  6. A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

    PubMed Central

    Dierks, T; Volkmer, J; Schlenstedt, G; Jung, C; Sandholzer, U; Zachmann, K; Schlotterhose, P; Neifer, K; Schmidt, B; Zimmermann, R

    1996-01-01

    Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates. Images PMID:9003769

  7. Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes

    SciTech Connect

    C Simmons; C Magee; D Smith; L Lauman; J Chaput; J Allen

    2011-12-31

    The creation of synthetic enzymes with predefined functions represents a major challenge in future synthetic biology applications. Here, we describe six structures of de novo proteins that have been determined using protein crystallography to address how simple enzymes perform catalysis. Three structures are of a protein, DX, selected for its stability and ability to tightly bind ATP. Despite the addition of ATP to the crystallization conditions, the presence of a bound but distorted ATP was found only under excess ATP conditions, with ADP being present under equimolar conditions or when crystallized for a prolonged period of time. A bound ADP cofactor was evident when Asp was substituted for Val at residue 65, but ATP in a linear configuration is present when Phe was substituted for Tyr at residue 43. These new structures complement previously determined structures of DX and the protein with the Phe 43 to Tyr substitution [Simmons, C. R., et al. (2009) ACS Chem. Biol. 4, 649-658] and together demonstrate the multiple ADP/ATP binding modes from which a model emerges in which the DX protein binds ATP in a configuration that represents a transitional state for the catalysis of ATP to ADP through a slow, metal-free reaction capable of multiple turnovers. This unusual observation suggests that design-free methods can be used to generate novel protein scaffolds that are tailor-made for catalysis.

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

    PubMed Central

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

    2014-01-01

    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, while 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. PMID:25129028

  9. Trapping the ATP binding state leads to a detailed understanding of the F1-ATPase mechanism

    PubMed Central

    Nam, Kwangho; Pu, Jingzhi; Karplus, Martin

    2014-01-01

    The rotary motor enzyme FoF1-ATP synthase uses the proton-motive force across a membrane to synthesize ATP from ADP and Pi (H2PO4−) under cellular conditions that favor the hydrolysis reaction by a factor of 2 × 105. This remarkable ability to drive a reaction away from equilibrium by harnessing an external force differentiates it from an ordinary enzyme, which increases the rate of reaction without shifting the equilibrium. Hydrolysis takes place in the neighborhood of one conformation of the catalytic moiety F1-ATPase, whose structure is known from crystallography. By use of molecular dynamics simulations we trap a second structure, which is rotated by 40° from the catalytic dwell conformation and represents the state associated with ATP binding, in accord with single-molecule experiments. Using the two structures, we show why Pi is not released immediately after ATP hydrolysis, but only after a subsequent 120° rotation, in agreement with experiment. A concerted conformational change of the α3β3 crown is shown to induce the 40° rotation of the γ-subunit only when the βE subunit is empty, whereas with Pi bound, βE serves as a latch to prevent the rotation of γ. The present results provide a rationalization of how F1-ATPase achieves the coupling between the small changes in the active site of βDP and the 40° rotation of γ. PMID:25453082

  10. Evaluation of Cassette Braille.

    ERIC Educational Resources Information Center

    VSE Corp., Alexandria, VA.

    A study conducted to determine user acceptability of cassette braille systems addressed three concerns: (1) user reaction to the basic concept of cassette braille, (2) the determination of the strong and weak features of the cassette braille machines currently available, and (3) the determination of those features of braille machines which would…

  11. Video Cartridges and Cassettes.

    ERIC Educational Resources Information Center

    Kletter, Richard C.; Hudson, Heather

    The economic and social significance of video cassettes (viewer-controlled playback system) is explored in this report. The potential effect of video cassettes on industrial training, education, libraries, and television is analyzed in conjunction with the anticipated hardware developments. The entire video cassette industry is reviewed firm by…

  12. Validation of BKV Large T-antigen ATP-Binding Site as a Target for Drug Discovery

    PubMed Central

    Zheng, Gang; Bueno, Marta; Camachos, Carlos J; Randhawa, Parmjeet

    2009-01-01

    Summary BK virus large T antigen (LTA) is a hexameric protein with a helicase activity that is powered by ATP hydrolysis. A mutant virus with Lys420Ala, Arg421Ala, and Asp504Ala mutations at the ATP binding sites showed marked reduction in viral fitness. This observation indicates that high throughput screening for ATPase inhibitors will be valid strategy to discover anti-BKV drugs. PMID:19084558

  13. Parametrisation of the free energy of ATP binding to wild-type and mutant Kir6.2 potassium channels.

    PubMed

    Moran, Oscar; Grottesi, Alessandro; Chadburn, Andrew J; Tammaro, Paolo

    2013-01-01

    ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.x and regulatory SURx subunits, play important roles in many cellular functions; because of their sensitivity to inhibition by intracellular ATP, K(ATP) channels provide a link between cell metabolism and membrane electrical activity. We constructed structural homology models of Kir6.2 and a series of Kir6.2 channels carrying mutations within the putative ATP-binding site. Computational docking was carried out to determine the conformation of ATP in its binding site. The Linear Interaction Energy (LIE) method was used to estimate the free-energy of ATP binding to wild-type and mutant Kir6.2 channels. Comparisons of the theoretical binding free energies for ATP with those determined from mutational experiments enabled the identification of the most probable conformation of ATP bound to the Kir6.2 channel. A set of LIE parameters was defined that may enable prediction of the effects of additional Kir6.2 mutations within the ATP binding site on the affinity for ATP. PMID:23219002

  14. Cardiac myosin isoforms exhibit differential rates of MgADP release and MgATP binding detected by myocardial viscoelasticity.

    PubMed

    Wang, Yuan; Tanner, Bertrand C W; Lombardo, Andrew T; Tremble, Sarah M; Maughan, David W; Vanburen, Peter; Lewinter, Martin M; Robbins, Jeffrey; Palmer, Bradley M

    2013-01-01

    We measured myosin crossbridge detachment rate and the rates of MgADP release and MgATP binding in mouse and rat myocardial strips bearing one of the two cardiac myosin heavy chain (MyHC) isoforms. Mice and rats were fed an iodine-deficient, propylthiouracil diet resulting in ~100% expression of β-MyHC in the ventricles. Ventricles of control animals expressed ~100% α-MyHC. Chemically-skinned myocardial strips prepared from papillary muscle were subjected to sinusoidal length perturbation analysis at maximum calcium activation pCa 4.8 and 17°C. Frequency characteristics of myocardial viscoelasticity were used to calculate crossbridge detachment rate over 0.01 to 5mM [MgATP]. The rate of MgADP release, equivalent to the asymptotic value of crossbridge detachment rate at high MgATP, was highest in mouse α-MyHC (111.4±6.2s(-1)) followed by rat α-MyHC (65.0±7.3s(-1)), mouse β-MyHC (24.3±1.8s(-1)) and rat β-MyHC (15.5±0.8s(-1)). The rate of MgATP binding was highest in mouse α-MyHC (325±32 mM(-1) s(-1)) then mouse β-MyHC (152±23 mM(-1) s(-1)), rat α-MyHC (108±10 mM(-1) s(-1)) and rat β-MyHC (55±6 mM(-1) s(-1)). Because the events of MgADP release and MgATP binding occur in a post power-stroke state of the myosin crossbridge, we infer that MgATP release and MgATP binding must be regulated by isoform- and species-specific structural differences located outside the nucleotide binding pocket, which is identical in sequence for these four myosins. We postulate that differences in the stiffness profile of the entire myosin molecule, including the thick filament and the myosin-actin interface, are primarily responsible for determining the strain on the nucleotide binding pocket and the subsequent differences in the rates of nucleotide release and binding observed among the four myosins examined here. PMID:23123290

  15. Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

    PubMed

    Cho, Ha Yeon; Lee, Young-Hoon; Bae, Young-Seuk; Kim, Eungbin; Kang, Beom Sik

    2013-05-01

    The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity. PMID:23486471

  16. Characterization of DalS, an ATP-binding cassette transporter for D-alanine, and its role in pathogenesis in Salmonella enterica.

    PubMed

    Osborne, Suzanne E; Tuinema, Brian R; Mok, Mac C Y; Lau, Pui Sai; Bui, Nhat Khai; Tomljenovic-Berube, Ana M; Vollmer, Waldemar; Zhang, Kun; Junop, Murray; Coombes, Brian K

    2012-05-01

    Expansion into new host niches requires bacterial pathogens to adapt to changes in nutrient availability and to evade an arsenal of host defenses. Horizontal acquisition of Salmonella Pathogenicity Island (SPI)-2 permitted the expansion of Salmonella enterica serovar Typhimurium into the intracellular environment of host cells by allowing it to deliver bacterial effector proteins across the phagosome membrane. This is facilitated by the SsrA-SsrB two-component regulatory system and a type III secretion system encoded within SPI-2. SPI-2 acquisition was followed by evolution of existing regulatory DNA, creating an expanded SsrB regulon involved in intracellular fitness and host infection. Here, we identified an SsrB-regulated operon comprising an ABC transporter in Salmonella. Biochemical and structural studies determined that the periplasmic solute-binding component, STM1633/DalS, transports D-alanine and that DalS is required for intracellular survival of the bacteria and for fitness in an animal host. This work exemplifies the role of nutrient exchange at the host-pathogen interface as a critical determinant of disease outcome. PMID:22418438

  17. Tandutinib (MLN518/CT53518) targeted to stem-like cells by inhibiting the function of ATP-binding cassette subfamily G member 2.

    PubMed

    Zhao, Xiao-qin; Dai, Chun-ling; Ohnuma, Shinobu; Liang, Yong-ju; Deng, Wen; Chen, Jun-Jiang; Zeng, Mu-Sheng; Ambudkar, Suresh V; Chen, Zhe-Sheng; Fu, Li-Wu

    2013-06-14

    Tandutinib is a novel inhibitor of tyrosine kinases FLT3, PDGFR and KIT. Our study was to explore the capability of tandutinib to reverse ABC transporter-mediated multidrug resistance. Tandutinib reversed ABCG2-mediated drug resistance in ABCG2-482-R2, ABCG2-482-G2, ABCG2-482-T7 and S1-M1-80 cells and increased the accumulation of doxorubicin, rhodamine 123 and [H(3)] mitoxantrone in ABCG2-overexpressing cells. Importantly, tandutinib selectively sensitized side population cells to mitoxantrone. Taken together, our results advocate the potency of tandutinib as an ABCG2 modulator and stem-like cells targeted agent to increase efficiency of anticancer drugs. PMID:23619284

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-01-01

    Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED)-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype for AED-resistant epilepsy); juvenile myoclonic epilepsy (JME) (prototype for AED-responsive epilepsy); and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T) rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004). This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004) and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05) cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE-HS in south Indian ancestry from Kerala. PMID:24586633

  20. Inhibitory Potential of Antifungal Drugs on ATP-Binding Cassette Transporters P-Glycoprotein, MRP1 to MRP5, BCRP, and BSEP.

    PubMed

    Lempers, Vincent J C; van den Heuvel, Jeroen J M W; Russel, Frans G M; Aarnoutse, Rob E; Burger, David M; Brüggemann, Roger J; Koenderink, Jan B

    2016-06-01

    Inhibition of ABC transporters is a common mechanism underlying drug-drug interactions (DDIs). We determined the inhibitory potential of antifungal drugs currently used for invasive fungal infections on ABC transporters P-glycoprotein (P-gp), MRP1 to MRP5, BCRP, and BSEP in vitro Membrane vesicles isolated from transporter-overexpressing HEK 293 cells were used to investigate the inhibitory potential of antifungal drugs (250 μM) on transport of model substrates. Concentration-inhibition curves were determined if transport inhibition was >60%. Fifty percent inhibitory concentrations (IC50s) for P-gp and BCRP were both 2 μM for itraconazole, 5 and 12 μM for hydroxyitraconazole, 3 and 6 μM for posaconazole, and 3 and 11 μM for isavuconazole, respectively. BSEP was strongly inhibited by itraconazole and hydroxyitraconazole (3 and 17 μM, respectively). Fluconazole and voriconazole did not inhibit any transport for >60%. Micafungin uniquely inhibited all transporters, with strong inhibition of MRP4 (4 μM). Anidulafungin and caspofungin showed strong inhibition of BCRP (7 and 6 μM, respectively). Amphotericin B only weakly inhibited BCRP-mediated transport (127 μM). Despite their wide range of DDIs, azole antifungals exhibit selective inhibition on efflux transporters. Although echinocandins display low potential for clinically relevant DDIs, they demonstrate potent in vitro inhibitory activity. This suggests that inhibition of ABC transporters plays a crucial role in the inexplicable (non-cytochrome P450-mediated) DDIs with antifungal drugs. PMID:27001813

  1. Encapsulated Brucella ovis Lacking a Putative ATP-Binding Cassette Transporter (ΔabcBA) Protects against Wild Type Brucella ovis in Rams

    PubMed Central

    Silva, Ana Patrícia C.; Macêdo, Auricélio A.; Costa, Luciana F.; Rocha, Cláudia E.; Garcia, Luize N. N.; Farias, Jade R. D.; Gomes, Priscilla P. R.; Teixeira, Gustavo C.; Fonseca, Kessler W. J.; Maia, Andréa R. F.; Neves, Gabriela G.; Romão, Everton L.; Silva, Teane M. A.; Mol, Juliana P. S.; Oliveira, Renata M.; Araújo, Márcio S. S.; Nascimento, Ernane F.; Martins-Filho, Olindo A.; Brandão, Humberto M.; Paixão, Tatiane A.; Santos, Renato L.

    2015-01-01

    This study aimed to evaluate protection induced by the vaccine candidate B. ovis ΔabcBA against experimental challenge with wild type B. ovis in rams. Rams were subcutaneously immunized with B. ovis ΔabcBA encapsulated with sterile alginate or with the non encapsulated vaccine strain. Serum, urine, and semen samples were collected during two months after immunization. The rams were then challenged with wild type B. ovis (ATCC25840), and the results were compared to non immunized and experimentally challenged rams. Immunization, particularly with encapsulated B. ovis ΔabcBA, prevented infection, secretion of wild type B. ovis in the semen and urine, shedding of neutrophils in the semen, and the development of clinical changes, gross and microscopic lesions induced by the wild type B. ovis reference strain. Collectively, our data indicates that the B. ovis ΔabcBA strain is an exceptionally good vaccine strain for preventing brucellosis caused by B. ovis infection in rams. PMID:26317399

  2. 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

    PubMed Central

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

    2014-01-01

    Epilepsy constitutes a heterogeneous group of disorders that is characterized by recurrent unprovoked seizures due to widely different etiologies. Multidrug resistance remains a major issue in clinical epileptology, where one third of patients with epilepsy continue to have seizures. Role of efflux transporters in multidrug resistant epilepsy has been attributed to drug-resistant epilepsy although, with discrepant observation in genetic studies. These discrepancies could be attributed to variety of factors such as variable definition of the anti-epileptic drug (AED)-resistance, variable epilepsy phenotypes and ethnicities among the studies. In the present study we inquired the role of multidrug transporters ABCB1 and ABCG2 variants in determining AED-resistance and susceptibility to epilepsy in three well-characterized cohorts comprising of mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype for AED-resistant epilepsy); juvenile myoclonic epilepsy (JME) (prototype for AED-responsive epilepsy); and healthy non-epileptic controls, in 738 subjects of Malayalam speaking south Indian ancestry. ABCB1 and ABCG2 variants were not found to be associated with drug resistance when AED-resistant and AED-responsive cohorts were compared. However, a significant association was observed between ABCB1 (C3435T) rs1045642 and risk of having epilepsy (MTLE-HS and JME pooled cohort; genotypic p-value = 0.0002; allelic p-value = 0.004). This association was seen persistent with MTLE-HS (genotypic p-value = 0.0008; allelic p-value = 0.004) and also with JME (genotypic p-value = 0.01; allelic p-value = 0.05) cohort individually. In-silico functional prediction indicated that ABCB1 rs1045642 has a deleterious impact on protein coding function and in splicing regulation. We conclude that the ABCB1 and ABCG2 variants do not confer to AED-resistance in the study population. However, ABCB1 rs1045642 increases vulnerability to epilepsy with greater tendency for MTLE-HS in south Indian ancestry from Kerala. PMID:24586633

  3. Polymorphisms of ATP binding cassette G5 and G8 transporters: their effect on cholesterol metabolism after moderate weight loss in overweight and obese hyperlipidemic women

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To determine the effect of polymorphisms ABCG5 and ABCG8 transporters on changes in lipid levels, cholesterol absorption rate (ABS), fractional synthesis rate (FSR), and turnover (TO) after moderate weight loss (WtL) in women. Cholesterol metabolism was measured pre and post WtL in 35 hyperlipidemic...

  4. Construction of Listeria monocytogenes mutants with in-frame deletions in putative ATP-binding cassette (ABC) transporters and analysis of their growth under stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate since it can survive under multiple stress conditions such as low pH and low temperature. Understanding its survival under stress conditions is important to control this pathogen in food. ABC transporters have been shown...

  5. The Tomato R Gene Products I-2 and Mi-1 Are Functional ATP Binding Proteins with ATPase Activity

    PubMed Central

    Tameling, Wladimir I. L.; Elzinga, Sandra D. J.; Darmin, Patricia S.; Vossen, Jack H.; Takken, Frank L. W.; Haring, Michel A.; Cornelissen, Ben J. C.

    2002-01-01

    Most plant disease resistance (R) genes known today encode proteins with a central nucleotide binding site (NBS) and a C-terminal Leu-rich repeat (LRR) domain. The NBS contains three ATP/GTP binding motifs known as the kinase-1a or P-loop, kinase-2, and kinase-3a motifs. In this article, we show that the NBS of R proteins forms a functional nucleotide binding pocket. The N-terminal halves of two tomato R proteins, I-2 conferring resistance to Fusarium oxysporum and Mi-1 conferring resistance to root-knot nematodes and potato aphids, were produced as glutathione S-transferase fusions in Escherichia coli. In a filter binding assay, purified I-2 was found to bind ATP rather than other nucleoside triphosphates. ATP binding appeared to be fully dependent on the presence of a divalent cation. A mutant I-2 protein containing a mutation in the P-loop showed a strongly reduced ATP binding capacity. Thin layer chromatography revealed that both I-2 and Mi-1 exerted ATPase activity. Based on the strong conservation of NBS domains in R proteins of the NBS-LRR class, we propose that they all are capable of binding and hydrolyzing ATP. PMID:12417711

  6. ATP-Binding Pocket-Targeted Suppression of Src and Syk by Luteolin Contributes to Its Anti-Inflammatory Action

    PubMed Central

    Lee, Jeong-Oog; Jeong, Deok; Kim, Mi-Yeon; Cho, Jae Youl

    2015-01-01

    Luteolin is a flavonoid identified as a major anti-inflammatory component of Artemisia asiatica. Numerous reports have demonstrated the ability of luteolin to suppress inflammation in a variety of inflammatory conditions. However, its exact anti-inflammatory mechanism has not been fully elucidated. In the present study, the anti-inflammatory mode of action in activated macrophages of luteolin from Artemisia asiatica was examined by employing immunoblotting analysis, a luciferase reporter gene assay, enzyme assays, and an overexpression strategy. Luteolin dose-dependently inhibited the secretion of nitric oxide (NO) and prostaglandin E2 (PGE2) and diminished the levels of mRNA transcripts of inducible NO synthase (iNOS), tumor necrosis factor- (TNF-) α, and cyclooxygenase-2 (COX-2) in lipopolysaccharide- (LPS-) and pam3CSK-treated macrophage-like RAW264.7 cells without displaying cytotoxicity. Luteolin displayed potent NO-inhibitory activity and also suppressed the nuclear translocation of NF-κB (p65 and p50) via blockade of Src and Syk, but not other mitogen-activated kinases. Overexpression of wild type Src and point mutants thereof, and molecular modelling studies, suggest that the ATP-binding pocket may be the luteolin-binding site in Src. These results strongly suggest that luteolin may exert its anti-inflammatory action by suppressing the NF-κB signaling cascade via blockade of ATP binding in Src and Syk. PMID:26236111

  7. Naturally occurring germline and tumor-associated mutations within the ATP-binding motifs of PTEN lead to oxidative damage of DNA associated with decreased nuclear p53

    PubMed Central

    He, Xin; Ni, Ying; Wang, Yu; Romigh, Todd; Eng, Charis

    2011-01-01

    Somatic and germline mutations in PTEN (phosphatase and tensin homolog deleted on chromosome 10) are found in sporadic cancers and Cowden syndrome patients, respectively. Recent identification of naturally occurring cancer and germline mutations within the ATP-binding motifs of PTEN (heretofore referred to as PTEN ATP-binding mutations) has revealed that these mutations disrupted the subcellular localization and tumor-suppressor activity of PTEN. However, very little is known about the underlying mechanisms of PTEN ATP-binding mutations in tumorigenesis. Here we show that these mutations impair PTEN's function both qualitatively and quantitatively. On the one hand, PTEN ATP-binding mutants lose their phosphatase activity and the effect of downregulation of cyclin D1. On the other, the mislocalized mutant PTEN results in a significantly decreased nuclear p53 protein level and transcriptional activity, enhanced production of reactive oxygen species, induction of Cu/Zn superoxide dismutase as well as dramatically increased DNA double-strand breaks (DSBs). When compared with wild-type PTEN, the ATP-binding mutant PTEN has reduced half-life in vitro and decreased protein expression levels in vivo. Our data, thus, reveal a novel mechanism of tumorigenesis in patients with germline or somatic mutations affecting PTEN ATP-binding motifs, i.e. qualitative and quantitative impairment of PTEN due to the loss of its phosphatase activity, and nuclear mislocalization, resulting in rapid PTEN protein degradation, suppression of p53-mediated transcriptional activity, loss of protection against oxidative stress as well as accumulation of spontaneous DNA DSBs. PMID:20926450

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

    PubMed Central

    Ding, Hao; Guo, Manhong; Vidhyasagar, Venkatasubramanian; Talwar, Tanu; Wu, Yuliang

    2015-01-01

    Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase. PMID:26474416

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

    PubMed Central

    Bilder, Patrick; Sun, Meihao; Lim, Jihyeon; Bielefeldt-Ohmann, Helle; Basaraba, Randall; So, Melvin; Zhu, Guofeng; Tufariello, JoAnn M.; Izzo, Angelo A.; Orme, Ian M.; Almo, Steve C.; Leyh, Thomas S.; Chan, John

    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 tuberculosis universal 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-Å-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. PMID:19478878

  10. Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP-binding Protein Superfamily†,‡

    PubMed Central

    Morar, Mariya; Anand, Ruchi; Hoskins, Aaron A.; Stubbe, JoAnne; Ealick, Steven E.

    2008-01-01

    Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. Small PurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the apo form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analog, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. Availability of a ternary complex enabled mapping of the active site thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. A surprising discovery, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculations about the regulatory role of the auxiliary site in PurLSQ complex formation as well as the evolutionary relationship of PurL's from different organisms. PMID:17154526

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

    SciTech Connect

    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.

  12. Automatic cassette to cassette radiant impulse processor

    NASA Astrophysics Data System (ADS)

    Sheets, Ronald E.

    1985-01-01

    Single wafer rapid annealing using high temperature isothermal processing has become increasingly popular in recent years. In addition to annealing, this process is also being investigated for suicide formation, passivation, glass reflow and alloying. Regardless of the application, there is a strong necessity to automate in order to maintain process control, repeatability, cleanliness and throughput. These requirements have been carefully addressed during the design and development of the Model 180 Radiant Impulse Processor which is a totally automatic cassette to cassette wafer processing system. Process control and repeatability are maintained by a closed loop optical pyrometer system which maintains the wafer at the programmed temperature-time conditions. Programmed recipes containing up to 10 steps may be easily entered on the computer keyboard or loaded in from a recipe library stored on a standard 5 {1}/{4″} floppy disk. Cold wall heating chamber construction, controlled environment (N 2, A, forming gas) and quartz wafer carriers prevent contamination of the wafer during high temperature processing. Throughputs of 150-240 wafers per hour are achieved by quickly heating the wafer to temperature (450-1400°C) in 3-6 s with a high intensity, uniform (± 1%) radiant flux of 100 {W}/{cm 2}, parallel wafer handling system and a wafer cool down stage.

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

    SciTech Connect

    Rhee, Dong Keun; Cho, Bon A; Kim, Hyong Bai . E-mail: hbkim5212@hotmail.com

    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.

  14. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    SciTech Connect

    Nakai, A.; Hirayama, C.; Ohtsuka, K.; Hirayoshi, K.; Nagata, K. )

    1990-06-01

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with (35S)methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of (32P)orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

  15. Automated cassette-to-cassette substrate handling system

    DOEpatents

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

    2014-03-18

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

  16. Blockade of ATP binding site of P2 purinoceptors in rat parotid acinar cells by isothiocyanate compounds.

    PubMed

    Soltoff, S P; McMillian, M K; Talamo, B R; Cantley, L C

    1993-05-01

    Extracellular ATP activates a P2Z-type purinergic receptor (purinoceptor) in rat parotid acinar cells that increases the intracellular free Ca2+ concentration via the entry of extracellular Ca2+ through an ATP-sensitive cation channel (Soltoff et al., Am J Physiol 262: C934-C940, 1992). To learn more about the ATP binding site of the purinoceptor, we examined the effects of several stilbene isothiocyanate analogs of DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid), which block the binding of [32P]ATP to intact parotid cells (McMillian et al., Biochem J 255:291-300, 1988) and blocked the activation of the P2Z purinoceptor. The ATP-stimulated 45Ca2+ uptake was blocked by DIDS, H2DIDS (dihydro-DIDS; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid), and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid), but not by DNDS (4,4'-dinitrostilbene-2,2'-disulfonic acid), a stilbene disulfonate compound lacking isothiocyanate (SCN-) groups, or by KSCN. The potency of the stilbene disulfonates was related to the number of isothiocyanate groups on each compound. Under the experimental conditions, the IC50 value of DIDS (approximately 35 microM), which has two SCN-groups, was much lower than that of SITS (approximately 125 microM), which has only one SCN-group. The inhibitory effects of DIDS appeared to be much more potent than those of SITS due to the kinetics of their binding to the purinoceptors. Eosin-5-isothiocyanate (EITC) and fluorescein-5-isothiocyanate (FITC), non-stilbene isothiocyanate compounds with single SCN-groups, also blocked the response to ATP and were less potent than DIDS. Trinitrophenyl-ATP (TNP-ATP), an ATP derivative that is not an effective agonist of the parotid P2Z receptor, blocked the covalent binding of DIDS to the plasma membrane, suggesting that ATP and DIDS bind to the same site. Reactive Blue 2 (Cibacron Blue 3GA), an anthraquinone-sulfonic acid derivative that is a noncovalent purinergic antagonist, also blocked

  17. Phosphorylation of the TOR ATP binding domain by AGC kinase constitutes a novel mode of TOR inhibition.

    PubMed

    Hálová, Lenka; Du, Wei; Kirkham, Sara; Smith, Duncan L; Petersen, Janni

    2013-11-25

    TOR (target of rapamycin) signaling coordinates cell growth, metabolism, and cell division through tight control of signaling via two complexes, TORC1 and TORC2. Here, we show that fission yeast TOR kinases and mTOR are phosphorylated on an evolutionarily conserved residue of their ATP-binding domain. The Gad8 kinase (AKT homologue) phosphorylates fission yeast Tor1 at this threonine (T1972) to reduce activity. A T1972A mutation that blocked phosphorylation increased Tor1 activity and stress resistance. Nitrogen starvation of fission yeast inhibited TOR signaling to arrest cell cycle progression in G1 phase and promoted sexual differentiation. Starvation and a Gad8/T1972-dependent decrease in Tor1 (TORC2) activity was essential for efficient cell cycle arrest and differentiation. Experiments in human cell lines recapitulated these yeast observations, as mTOR was phosphorylated on T2173 in an AKT-dependent manner. In addition, a T2173A mutation increased mTOR activity. Thus, TOR kinase activity can be reduced through AGC kinase-controlled phosphorylation to generate physiologically significant changes in TOR signaling. PMID:24247430

  18. Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene.

    PubMed Central

    Kerem, B S; Zielenski, J; Markiewicz, D; Bozon, D; Gazit, E; Yahav, J; Kennedy, D; Riordan, J R; Collins, F S; Rommens, J M

    1990-01-01

    Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probably affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation (delta F508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-delta F508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward. Images PMID:2236053

  19. Identification of mutations in regions corresponding to the two putative nucleotide (ATP)-binding folds of the cystic fibrosis gene

    SciTech Connect

    Kerem, B.; Zielenski, J.; Markiewicz, D.; Bozon, D.; Kennedy, D.; Rommens, J.M. ); Gazit, E. ); Yahav, J. ); Riordan, J.R. ); Collins, F.S. ); Tsui, Lapchee Univ. of Toronto, Ontario )

    1990-11-01

    Additional mutations in the cystic fibrosis (CF) gene were identified in the regions corresponding to the two putative nucleotide (ATP)-binding folds (NBFs) of the predicted polypeptide. The patient cohort included 46 Canadian CF families with well-characterized DNA marker haplotypes spanning the disease locus and several other families from Israel. Eleven mutations were found in the first NBF, 2 were found in the second NBF, but none was found in the R-domain. Seven of the mutations were of the missense type affecting some of the highly conserved amino acid residues in the first NBF; 3 were nonsense mutations; 2 would probably affect mRNA splicing; 2 corresponded to small deletions, including another 3-base-pair deletion different from the major mutation ({delta}F508), which could account for 70% of the CF chromosomes in the population. Nine of these mutations accounted for 12 of the 31 non-{delta}F508 CF chromosomes in the Canadian families. The highly heterogeneous nature of the remaining CF mutations provides important insights into the structure and function of the protein, but it also suggests that DNA-based genetic screening for CF carrier status will not be straightforward.

  20. Hedyotis diffusa Willd overcomes 5-fluorouracil resistance in human colorectal cancer HCT-8/5-FU cells by downregulating the expression of P-glycoprotein and ATP-binding casette subfamily G member 2

    PubMed Central

    LI, QIONGYU; WANG, XIANGFENG; SHEN, ALING; ZHANG, YUCHEN; CHEN, YOUQIN; SFERRA, THOMAS J.; LIN, JIUMAO; PENG, JUN

    2015-01-01

    Previous studies have demonstrated that Hedyotis diffusa Willd (HDW), a traditional Chinese herbal medicine, exhibits potent anticancer activity in models of colorectal cancer (CRC). Aggressive forms of CRC exhibit resistance to widely used chemotherapeutic drugs, including the antimetabolite, 5-fluorouracil (5-FU); however, less is known with regard to the activity of HDW against 5-FU-resistant cancer. In the present study, the mechanism of action and the potency of ethanol extracts of HDW (EEHDW) were investigated on a multidrug-resistant CRC HCT-8/5-FU cell line. Using an MTT cell proliferation assay, EEHDW treatment was shown to significantly reduce the cell viability of HCT-8/5-FU cells in a dose- and time-dependent manner. Furthermore, EEHDW significantly increased the retention of the ATP-binding cassette (ABC) transporter substrate, rhodamine-123, as compared with the untreated controls. To further investigate the molecular mechanisms targeted by EEHDW in the resistant cells, the expression levels of the ABC drug transporter protein, P-glycoprotein (P-gp), and ABC subfamily G member 2 (ABCG2), were analyzed using reverse-transcription polymerase chain reaction and western blot analysis. The mRNA and protein expression levels of P-gp and ABCG2 were reduced in the HCT-8/5-FU cells following EEHDW treatment, indicating that EEHDW inhibits ABCG2-mediated drug resistance by downregulating the expression of ABCG2 and P-gp. Therefore, the potential application of EEHDW as a chemotherapeutic adjuvant represents a promising alternative approach to the treatment of drug-resistant CRC. PMID:26640560

  1. Biophysical changes of ATP binding pocket may explain loss of kinase activity in mutant DAPK3 in cancer: A molecular dynamic simulation analysis.

    PubMed

    Agarwal, Tarun; Annamalai, Nithyanan; Maiti, Tapas Kumar; Arsad, Hasni

    2016-04-10

    DAPK3 belongs to family of DAPK (death-associated protein kinases) and is involved in the regulation of progression of the cell cycle, cell proliferation, apoptosis and autophagy. It is considered as a tumor suppressor kinase, suggesting the loss of its function in case of certain specific mutations. The T112M, D161N and P216S mutations in DAPK3 have been observed in cancer patients. These DAPK3 mutants have been associated with very low kinase activity, which results in the cellular progression towards cancer. However, a clear understanding of the structural and biophysical variations that occur in DAPK3 with these mutations, resulting in the decreased kinase activity has yet not been deciphered. We performed a molecular dynamic simulation study to investigate such structural variations. Our results revealed that mutations caused a significant structural variation in DAPK3, majorly concentrated in the flexible loops that form part of the ATP binding pocket. Interestingly, D161N and P216S mutations collapsed the ATP binding pocket through flexible loops invasion, hindering ATP binding which resulted in very low kinase activity. On the contrary, T112M mutant DAPK3 reduces ATP binding potential through outward distortion of flexible loops. In addition, the mutant lacked characteristic features of the active protein kinase including proper interaction between HR/FD and DFG motifs, well structured hydrophobic spine and Lys42-Glu64 salt bridge interaction. These observations could possibly explain the underlying mechanism associated with the loss of kinase activity with T112M, D161N and P216S mutation in DAPK3. PMID:26748242

  2. Role of the glycine triad in the ATP-binding site of cAMP-dependent protein kinase.

    PubMed

    Hemmer, W; McGlone, M; Tsigelny, I; Taylor, S S

    1997-07-01

    A glycine-rich loop in the ATP-binding site is one of the most highly conserved sequence motifs in protein kinases. Each conserved glycine (Gly-50, Gly-52, and Gly-55) in the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) was replaced with Ser and/or Ala. Active mutant proteins were expressed in Escherichia coli, purified to apparent homogeneity, separated into phosphoisoforms, and characterized. Replacing Gly-55 had minimal effects on steady-state kinetic parameters, whereas replacement of either Gly-50 or Gly-52 had major effects on both Km and kcat values consistent with the prediction of the importance of the tip of the glycine-rich loop for catalysis. Substitution of Gly-50 caused a 5-8-fold reduction in Km (ATP), an 8-12-fold increase in Km (peptide), and a 3-5-fold drop in kcat. The Km (ATP) and Km (peptide) values of C(G52S) were increased 8- and 18-fold, respectively, and the kcat was decreased 6-fold. In contrast to catalytic efficiency, the ATPase rates of C(G50S) and C(G52S) were increased by more than an order of magnitude. The thermostability of each mutant was slightly increased. Unphosphorylated C(G52S) was characterized as well as several isoforms phosphorylated at a single site, Ser-338. All of these phosphorylation-defective mutants displayed a substantial decrease in both enzymatic activity and thermal stability that correlated with the missing phosphate at Thr-197. These results are correlated with the crystal structure, models of the respective mutant proteins, and conservation of the Glys within the protein kinase family. PMID:9202006

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

    PubMed

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

    2016-05-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5-inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

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

    PubMed

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

    2016-03-01

    The ABCC transporter subfamily includes pumps, the long and short multidrug resistance proteins (MRPs), and an ATP-gated anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR). We show that despite their thermodynamic differences, these ABCC transporter subtypes use broadly similar mechanisms to couple their extracellular gates to the ATP occupancies of their cytosolic nucleotide binding domains. A conserved extracellular phenylalanine at this gate was a prime location for producing gain of function (GOF) mutants of a long MRP in yeast (Ycf1p cadmium transporter), a short yeast MRP (Yor1p oligomycin exporter), and human CFTR channels. Extracellular gate mutations rescued ATP binding mutants of the yeast MRPs and CFTR by increasing ATP sensitivity. Control ATPase-defective MRP mutants could not be rescued by this mechanism. A CFTR double mutant with an extracellular gate mutation plus a cytosolic GOF mutation was highly active (single-channel open probability >0.3) in the absence of ATP and protein kinase A, each normally required for CFTR activity. We conclude that all 3 ABCC transporter subtypes use similar mechanisms to couple their extracellular gates to ATP occupancy, and highly active CFTR channels that bypass defects in ATP binding or phosphorylation can be produced.-Wei, S., Roessler, B. C., Icyuz, M., Chauvet, S., Tao, B., Hartman IV, J. L., Kirk, K. L. Long-range coupling between the extracellular gates and the intracellular ATP binding domains of multidrug resistance protein pumps and cystic fibrosis transmembrane conductance regulator channels. PMID:26606940

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

    PubMed Central

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

    2016-01-01

    MAP kinases act as an integration point for multiple biochemical signals and are involved in a wide variety of cellular processes such as proliferation, differentiation, regulation of transcription and development. As a member of the MAP kinase family, ERK5 (MAPK7) is involved in the downstream signalling pathways of various cell-surface receptors, including receptor tyrosine kinases and G protein-coupled receptors. In the current study, five structures of the ERK5 kinase domain co-crystallized with ERK5 inhibitors are reported. Interestingly, three of the compounds bind at a novel allosteric binding site in ERK5, while the other two bind at the typical ATP-binding site. Binding of inhibitors at the allosteric site is accompanied by displacement of the P-loop into the ATP-binding site and is shown to be ATP-competitive in an enzymatic assay of ERK5 kinase activity. Kinase selectivity data show that the most potent allosteric inhibitor exhibits superior kinase selectivity compared with the two inhibitors that bind at the canonical ATP-binding site. An analysis of these structures and comparison with both a previously published ERK5–inhibitor complex structure (PDB entry 4b99) and the structures of three other kinases (CDK2, ITK and MEK) in complex with allosteric inhibitors are presented. PMID:27139631

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  7. 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.

    PubMed

    Maharana, Jitendra; Sahoo, Bikash Ranjan; Bej, Aritra; Jena, Itishree; Parida, Arunima; Sahoo, Jyoti Ranjan; Dehury, Budheswar; Patra, Mahesh Chandra; Martha, Sushma Rani; Balabantray, Sucharita; Pradhan, Sukanta Kumar; Behera, Bijay Kumar

    2015-01-01

    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. PMID:25811192

  8. 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

    PubMed Central

    Maharana, Jitendra; Sahoo, Bikash Ranjan; Bej, Aritra; Sahoo, Jyoti Ranjan; Dehury, Budheswar; Patra, Mahesh Chandra; Martha, Sushma Rani; Balabantray, Sucharita; Pradhan, Sukanta Kumar; Behera, Bijay Kumar

    2015-01-01

    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. PMID:25811192

  9. The histone H4 tail regulates the conformation of the ATP-binding pocket in the SNF2h chromatin remodeling enzyme

    PubMed Central

    Racki, Lisa R.; Naber, Nariman; Pate, Ed; Leonard, John; Cooke, Roger; Narlikar, Geeta J.

    2014-01-01

    The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. Yet the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP- binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps. PMID:24607692

  10. The histone H4 tail regulates the conformation of the ATP-binding pocket in the SNF2h chromatin remodeling enzyme.

    PubMed

    Racki, Lisa R; Naber, Nariman; Pate, Ed; Leonard, John D; Cooke, Roger; Narlikar, Geeta J

    2014-05-15

    The chromatin remodeling complex ACF helps establish the appropriate nucleosome spacing for generating repressed chromatin states. ACF activity is stimulated by two defining features of the nucleosomal substrate: a basic patch on the histone H4 N-terminal tail and the specific length of flanking DNA. However, the mechanisms by which these two substrate cues function in the ACF remodeling reaction is not well understood. Using electron paramagnetic resonance spectroscopy with spin-labeled ATP analogs to probe the structure of the ATP active site under physiological solution conditions, we identify a closed state of the ATP-binding pocket that correlates with ATPase activity. We find that the H4 tail promotes pocket closure. We further show that ATPase stimulation by the H4 tail does not require a specific structure connecting the H4 tail and the globular domain. In the case of many DNA helicases, closure of the ATP-binding pocket is regulated by specific DNA substrates. Pocket closure by the H4 tail may analogously provide a mechanism to directly couple substrate recognition to activity. Surprisingly, the flanking DNA, which also stimulates ATP hydrolysis, does not promote pocket closure, suggesting that the H4 tail and flanking DNA may be recognized in different reaction steps. PMID:24607692

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

    PubMed Central

    Wang, Zhongshan; Xiang, Quanju; Wang, Guangjun; Wang, Haiyan; Zhang, Yizheng

    2011-01-01

    The cloning, expression and purification of the glutathione (sulfur) import system ATP-binding protein (gsiA) was carried out. The coding sequence of Escherichia coli gsiA, which encodes the ATP-binding protein of a glutathione importer, was amplified by PCR, and then inserted into a prokaryotic expression vector pWaldo-GFPe harboring green fluorescent protein (GFP) reporter gene. The resulting recombinant plasmid pWaldo-GFP-GsiA was transformed into various E. coli strains, and expression conditions were optimized. The effect of five E. coli expression strains on the production of the recombinant gsiA protein was evaluated. E. coli BL21 (DE3) was found to be the most productive strain for GsiA-GFP fusion-protein expression, most of which was insoluble fraction. However, results from in-gel and Western blot analysis suggested that expression of recombinant GsiA in Rosetta (DE3) provides an efficient source in soluble form. By using GFP as reporter, the most suitable host strain was conveniently obtained, whereby optimizing conditions for overexpression and purification of the proteins for further functional and structural studies, became, not only less laborious, but also time-saving. PMID:22215971

  12. Conservation of an ATP-binding domain among recA proteins from Proteus vulgaris, erwinia carotovora, Shigella flexneri, and Escherichia coli K-12 and B/r

    SciTech Connect

    Knight, K.L.; Hess, R.M.; McEntee, K.

    1988-06-01

    The purified RecA proteins encoded by the cloned genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri, and Escherichia coli B/r were compared with the RecA protein from E. coli K-12. Each of the proteins hydrolyzed ATP in the presence of single-stranded DNA, and each was covalently modified with the photoaffinity ATP analog 8-azidoadenosine 5'-triphosphate (8N/sub 3/ATP). Two-dimensional tryptic maps of the four heterologous RecA proteins demonstrated considerable structural conservation among these bacterial genera. Moreover, when the (..cap alpha..-/sup 32/P)8N/sub 3/ATP-modified proteins were digested with trypsin and analyzed by high-performance liquid chromatography, a single peak of radioactivity was detected in each of the digests and these peptides eluted identically with the tryptic peptide T/sub 31/ of the E. coli K-12 RecA protein, which was the unique site of 8N/sub 3/ATP photolabeling. Each of the heterologous recA genes hybridized to oligonucleotide probes derived from the ATP-binding domain sequence of the E. coli K-12 gene. These last results demonstrate that the ATP-binding domain of the RecA protein has been strongly conserved for greater than 10/sup 7/ years.

  13. RAC: Repository of Antibiotic resistance Cassettes

    PubMed Central

    Tsafnat, Guy; Copty, Joseph; Partridge, Sally R.

    2011-01-01

    Antibiotic resistance in bacteria is often due to acquisition of resistance genes associated with different mobile genetic elements. In Gram-negative bacteria, many resistance genes are found as part of small mobile genetic elements called gene cassettes, generally found integrated into larger elements called integrons. Integrons carrying antibiotic resistance gene cassettes are often associated with mobile elements and here are designated ‘mobile resistance integrons’ (MRIs). More than one cassette can be inserted in the same integron to create arrays that contribute to the spread of multi-resistance. In many sequences in databases such as GenBank, only the genes within cassettes, rather than whole cassettes, are annotated and the same gene/cassette may be given different names in different entries, hampering analysis. We have developed the Repository of Antibiotic resistance Cassettes (RAC) website to provide an archive of gene cassettes that includes alternative gene names from multiple nomenclature systems and allows the community to contribute new cassettes. RAC also offers an additional function that allows users to submit sequences containing cassettes or arrays for annotation using the automatic annotation system Attacca. Attacca recognizes features (gene cassettes, integron regions) and identifies cassette arrays as patterns of features and can also distinguish minor cassette variants that may encode different resistance phenotypes (aacA4 cassettes and bla cassettes-encoding β-lactamases). Gaps in annotations are manually reviewed and those found to correspond to novel cassettes are assigned unique names. While there are other websites dedicated to integrons or antibiotic resistance genes, none includes a complete list of antibiotic resistance gene cassettes in MRI or offers consistent annotation and appropriate naming of all of these cassettes in submitted sequences. RAC thus provides a unique resource for researchers, which should reduce confusion

  14. Magnetic cassette for radiographic film material

    SciTech Connect

    Dallas, D.

    1985-03-26

    A radiographic film cassette having a plurality of magnet components integral with the cassette holder for adhering the cassette to ferrous material in X-raying for defects in welds or fissures in shipyards, pipe lines, or the like. What is provided is a substantially flexible cassette envelope comprising first and second layers of radiographic intensifying screens with a sheet of radiographic film positioned therebetween. The cassette would be a cassette envelope constructed of waterproof fabric or other suitable material providing a light-free environment, and having the ability to flex around the curvature of the surface of a pipe or the like to be x-rayed. There is further provided a plurality of magnet components, preferably situated in each corner of the cassette envelope and flexibly attached thereto for overall adherence of the envelope to the surface of the pipe or the like to be x-rayed during the process.

  15. Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD.

    PubMed

    Eom, Gyeong Tae; Oh, Joon Young; Park, Ji Hyun; Lim, Hye Jin; Lee, So Jeong; Kim, Eun Young; Choi, Ji-Eun; Jegal, Jonggeon; Song, Bong Keun; Yu, Ju-Hyun; Song, Jae Kwang

    2016-09-01

    An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein. PMID:27033673

  16. Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.

    PubMed Central

    McNicholas, C M; Guggino, W B; Schwiebert, E M; Hebert, S C; Giebisch, G; Egan, M E

    1996-01-01

    We demonstrate here that coexpression of ROMK2, an inwardly rectifying ATP-sensitive renal K+ channel (IKATP) with cystic fibrosis transmembrane regulator (CFTR) significantly enhances the sensitivity of ROMK2 to the sulfonylurea compound glibenclamide. When expressed alone, ROMK2 is relatively insensitive to glibenclamide. The interaction between ROMK2, CFTR, and glibenclamide is modulated by altering the phosphorylation state of either ROMK2, CFTR, or an associated protein, as exogenous MgATP and the catalytic subunit of protein kinase A significantly attenuate the inhibitory effect of glibenclamide on ROMK2. Thus CFTR, which has been demonstrated to interact with both Na+ and Cl- channels in airway epithelium, modulates the function of renal ROMK2 K+ channels. PMID:8755607

  17. The ATP binding site of the chromatin remodeling homolog Lsh is required for nucleosome density and de novo DNA methylation at repeat sequences

    PubMed Central

    Ren, Jianke; Briones, Victorino; Barbour, Samantha; Yu, Weishi; Han, Yixing; Terashima, Minoru; Muegge, Kathrin

    2015-01-01

    Lsh, a chromatin remodeling protein of the SNF2 family, is critical for normal heterochromatin structure. In particular, DNA methylation at repeat elements, a hallmark of heterochromatin, is greatly reduced in Lsh−/− (KO) cells. Here, we examined the presumed nucleosome remodeling activity of Lsh on chromatin in the context of DNA methylation. We found that dynamic CG methylation was dependent on Lsh in embryonic stem cells. Moreover, we demonstrate that ATP function is critical for de novo methylation at repeat sequences. The ATP binding site of Lsh is in part required to promote stable association of the DNA methyltransferase 3b with the repeat locus. By performing nucleosome occupancy assays, we found distinct nucleosome occupancy in KO ES cells compared to WT ES cells after differentiation. Nucleosome density was restored to wild-type level by re-expressing wild-type Lsh but not the ATP mutant in KO ES cells. Our results suggest that ATP-dependent nucleosome remodeling is the primary molecular function of Lsh, which may promote de novo methylation in differentiating ES cells. PMID:25578963

  18. Structures of the CDK12/CycK complex with AMP-PNP reveal a flexible C-terminal kinase extension important for ATP binding

    PubMed Central

    Dixon-Clarke, Sarah E.; Elkins, Jonathan M.; Cheng, S.-W. Grace; Morin, Gregg B.; Bullock, Alex N.

    2015-01-01

    Cyclin-dependent kinase 12 (CDK12) promotes transcriptional elongation by phosphorylation of the RNA polymerase II C-terminal domain (CTD). Structure-function studies show that this activity is dependent on a C-terminal kinase extension, as well as the binding of cyclin K (CycK). To better define these interactions we determined the crystal structure of the human CDK12/CycK complex with and without the kinase extension in the presence of AMP-PNP. The structures revealed novel features for a CDK, including a large β4-β5 loop insertion that contributes to the N-lobe interaction with the cyclin. We also observed two different conformations of the C-terminal kinase extension that effectively open and close the ATP pocket. Most notably, bound AMP-PNP was only observed when trapped in the closed state. Truncation of this C-terminal structure also diminished AMP-PNP binding, as well as the catalytic activity of the CDK12/CycK complex. Further kinetic measurements showed that the full length CDK12/CycK complex was significantly more active than the two crystallised constructs suggesting a critical role for additional domains. Overall, these results demonstrate the intrinsic flexibility of the C-terminal extension in CDK12 and highlight its importance for both ATP binding and kinase activity. PMID:26597175

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

    PubMed Central

    Karlin, Justin; Fischhaber, Paula L.

    2013-01-01

    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. PMID:25346869

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

    PubMed

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

    2016-03-01

    G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein's ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure-function relationships of the YchF subfamily of G proteins in all kingdoms of life. PMID:26912459

  1. Influence of a mutation in the ATP-binding region of Ca2+/calmodulin-dependent protein kinase II on its interaction with peptide substrates.

    PubMed

    Praseeda, Mullasseril; Pradeep, Kurup K; Krupa, Ananth; Krishna, S Sri; Leena, Suseela; Kumar, R Rajeev; Cheriyan, John; Mayadevi, Madhavan; Srinivasan, Narayanaswamy; Omkumar, Ramakrishnapillai V

    2004-03-01

    CaMKII (Ca2+/calmodulin-dependent protein kinase II) is expressed in high concentrations in the brain and is found enriched in the postsynaptic densities. The enzyme is activated by the binding of calmodulin to the autoregulatory domain in the presence of high levels of intracellular Ca2+, which causes removal of auto-inhibition from the N-terminal catalytic domain. Knowledge of the 3D (three-dimensional) structure of this enzyme at atomic resolution is restricted to the association domain, a region at the extreme C-terminus. The catalytic domain of CaMKII shares high sequence similarity with CaMKI. The 3D structure of the catalytic core of CaMKI comprises ATP- and substrate-binding regions in a cleft between two distinct lobes, similar to the structures of all protein kinases solved to date. Mutation of Glu-60, a residue in the ATP-binding region of CaMKII, to glycine exerts different effects on phosphorylation of two peptide substrates, syntide and NR2B ( N -methyl-D-aspartate receptor subunit 2B) 17-mer. Although the mutation caused increases in the Km values for phosphorylation for both the peptide substrates, the effect on the kcat values for each was different. The kcat value decreased in the case of syntide, whereas it increased in the case of the NR2B peptide as a result of the mutation. This resulted in a significant decrease in the apparent kcat/Km value for syntide, but the change was minimal for the NR2B peptide. These results indicate that different catalytic mechanisms are employed by the kinase for the two peptides. Molecular modelling suggests structural changes are likely to occur at the peptide-binding pocket in the active state of the enzyme as a consequence of the Glu-60-->Gly mutation. PMID:14558884

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

    PubMed Central

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

    2016-01-01

    G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP. The YchF subfamily, interestingly, possesses the unique ability to bind both ATP and GTP, and is possibly an ancestral form of G proteins based on phylogenetic studies and is present in all kingdoms of life. However, the biological significance of such a relaxed ligand specificity has long eluded researchers. Here, we have elucidated the different conformational changes caused by the binding of a YchF homolog in rice (OsYchF1) to ATP versus GTP by X-ray crystallography. Furthermore, by comparing the 3D relationships of the ligand position and the various amino acid residues at the binding sites in the crystal structures of the apo-bound and ligand-bound versions, a mechanism for the protein’s ability to bind both ligands is revealed. Mutation of the noncanonical G4 motif of the OsYchF1 to the canonical sequence for GTP specificity precludes the binding/hydrolysis of ATP and prevents OsYchF1 from functioning as a negative regulator of plant-defense responses, while retaining its ability to bind/hydrolyze GTP and its function as a negative regulator of abiotic stress responses, demonstrating the specific role of ATP-binding/hydrolysis in disease resistance. This discovery will have a significant impact on our understanding of the structure–function relationships of the YchF subfamily of G proteins in all kingdoms of life. PMID:26912459

  3. Synthesis and Evaluation of a Novel Deguelin Derivative, L80, which Disrupts ATP Binding to the C-terminal Domain of Heat Shock Protein 90.

    PubMed

    Lee, Su-Chan; Min, Hye-Young; Choi, Hoon; Kim, Ho Shin; Kim, Kyong-Cheol; Park, So-Jung; Seong, Myeong A; Seo, Ji Hae; Park, Hyun-Ju; Suh, Young-Ger; Kim, Kyu-Won; Hong, Hyun-Seok; Kim, Hee; Lee, Min-Young; Lee, Jeewoo; Lee, Ho-Young

    2015-08-01

    The clinical benefit of current anticancer regimens for lung cancer therapy is still limited due to moderate efficacy, drug resistance, and recurrence. Therefore, the development of effective anticancer drugs for first-line therapy and for optimal second-line treatment is necessary. Because the 90-kDa molecular chaperone heat shock protein (Hsp90) contributes to the maturation of numerous mutated or overexpressed oncogenic proteins, targeting Hsp90 may offer an effective anticancer therapy. Here, we investigated antitumor activities and toxicity of a novel deguelin-derived C-terminal Hsp90 inhibitor, designated L80. L80 displayed significant inhibitory effects on the viability, colony formation, angiogenesis-stimulating activity, migration, and invasion of a panel of non-small cell lung cancer cell lines and their sublines with acquired resistance to paclitaxel with minimal toxicity to normal lung epithelial cells, hippocampal cells, vascular endothelial cells, and ocular cells. Biochemical analyses and molecular docking simulation revealed that L80 disrupted Hsp90 function by binding to the C-terminal ATP-binding pocket of Hsp90, leading to the disruption of the interaction between hypoxia-inducible factor (HIF)-1α and Hsp90, downregulation of HIF-1α and its target genes, including vascular endothelial growth factor (VEGF) and insulin-like growth factor 2 (IGF2), and decreased the expression of various Hsp90 client proteins. Consistent with these in vitro findings, L80 exhibited significant antitumor and antiangiogenic activities in H1299 xenograft tumors. These results suggest that L80 represents a novel C-terminal Hsp90 inhibitor with effective anticancer activities with minimal toxicities. PMID:25976766

  4. The three-dimensional structure of MAP kinase p38[beta]: different features of the ATP-binding site in p38[beta] compared with p38[alpha

    SciTech Connect

    Patel, Sangita B.; Cameron, Patricia M.; O'Keefe, Stephen J.; Frantz-Wattley, Betsy; Thompson, Jed; O'Neill, Edward A.; Tennis, Trevor; Liu, Luping; Becker, Joseph W.; Scapin, Giovanna; Merck

    2010-10-18

    The p38 mitogen-activated protein kinases are activated in response to environmental stress and cytokines and play a significant role in transcriptional regulation and inflammatory responses. Of the four p38 isoforms known to date, two (p38{alpha} and p38{beta}) have been identified as targets for cytokine-suppressive anti-inflammatory drugs. Recently, it was reported that specific inhibition of the p38{alpha} isoform is necessary and sufficient for anti-inflammatory efficacy in vivo, while further inhibition of p38{beta} may not provide any additional benefit. In order to aid the development of p38{alpha}-selective compounds, the three-dimensional structure of p38{beta} was determined. To do so, the C162S and C119S,C162S mutants of human MAP kinase p38{beta} were cloned, expressed in Escherichia coli and purified. Initial screening hits in crystallization trials in the presence of an inhibitor led upon optimization to crystals that diffracted to 2.05 {angstrom} resolution and allowed structure determination (PDB codes 3gc8 and 3gc9 for the single and double mutant, respectively). The structure of the p38{alpha} C162S mutant in complex with the same inhibitor is also reported (PDB code 3gc7). A comparison between the structures of the two kinases showed that they are highly similar overall but that there are differences in the relative orientation of the N- and C-terminal domains that causes a reduction in the size of the ATP-binding pocket in p38{beta}. This difference in size between the two pockets could be exploited in order to achieve selectivity.

  5. Cassette for handling banknotes or the like

    DOEpatents

    Lundblad, Leif

    1981-08-11

    A cassette for banknotes and like valuable articles is provided with a displaceable lid (6) and locking means (10) for latching the lid of the cassette when the cassette is located outside a housing (25) in which it is intended to be placed. An operating means (8) is arranged to co-act with the locking means and with a latching element (15). The latching element is arranged to be released in dependence upon a pre-set program. A signal circuit is arranged to send a code signal to a detector circuit (23) when electrical contact elements on the cassette and the housing co-act with one another, which detector circuit, when the signal coincides with the signal program in the detector circuit, causes a signal to be sent for moving the latching means to a non-latching position.

  6. A disposable microfluidic cassette for DNA amplification and detection.

    PubMed

    Wang, Jing; Chen, Zongyuan; Corstjens, Paul L A M; Mauk, Michael G; Bau, Haim H

    2006-01-01

    A pneumatically driven, disposable, microfluidic cassette comprised of a polymerase chain reaction (PCR) thermal cycler, an incubation chamber to label PCR amplicons with up-converting phosphor (UPT) reporter particles, conduits, temperature-activated, normally closed hydrogel valves, and a lateral flow strip, was constructed and tested. The hydrogel valves, which were opened and closed with the aid of electrically controlled thermoelectric units, provided a simple means to seal the PCR reactor and suppress bubble formation. The hydrogel-based flow control was electronically addressable, leakage-free, and biocompatible. To test the device, a solution laden with genomic DNA isolated from B. cereus was introduced into the microfluidic cassette and a specific 305 bp fragment was amplified. The PCR amplicons were labelled with the phosphor (UPT) reporter particles, applied to the lateral flow strip, bound to pre-immobilized ligands, and detected with an IR laser that scanned the lateral flow strip and excited the phosphor (UPT) particles that, in turn, emitted light in the visible spectrum. The UPT particles do not bleach, they provide a permanent record, and they readily facilitate the filtering of background noise. The cassette described herein will be used for rapid testing at the point of care. PMID:16372068

  7. Books (on Cassette) Are Better Than Ever.

    ERIC Educational Resources Information Center

    Davis, Bryan

    1984-01-01

    Describes introduction of books on tape at Oskaloosa (Iowa) Public Library, highlighting determination of audience and use, display of recorded books, packaging of tapes, cataloging, and quality of tapes. A list of 19 production companies and six distributors noting address, telephone number, type of cassettes, and price range is included. (EJS)

  8. 21 CFR 892.1850 - Radiographic film cassette.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Radiographic film cassette. 892.1850 Section 892...) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette. (a) Identification. A radiographic film cassette is a device intended for use during diagnostic x-ray procedures...

  9. 21 CFR 892.1860 - Radiographic film/cassette changer.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Radiographic film/cassette changer. 892.1860... (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A radiographic film/cassette changer is a device intended to be used during...

  10. CryoEM and Molecular Dynamics of the Circadian KaiB–KaiC Complex Indicates KaiB Monomers Interact with KaiC and Block ATP Binding Clefts

    PubMed Central

    Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.; Mori, Tetsuya; Qin, Ximing; Johnson, Carl H.; Egli, Martin; Stewart, Phoebe L.

    2013-01-01

    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 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. PMID:23796516

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

    SciTech Connect

    Villarreal, Seth A.; Pattanayek, Rekha; Williams, Dewight R.; Mori, Tetsuya; Qin, Ximing; Johnson, Carl H.; Egli, Martin; Stewart, Phoebe L.

    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.

  12. A Cassette Based System for Hydrogen Storage and Delivery

    SciTech Connect

    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.

  13. Characterization of a multiple endogenously expressed Adenosine triphosphate-Binding Cassette transporters using nuclear and cellular membrane affinity chromatography columns

    PubMed Central

    Khadeer, M.A.; Shimmo, R.; Wainer, I.W.; Moaddel, R.

    2014-01-01

    Glioblastoma multiforme is an aggressive form of human astrocytoma, with poor prognosis due to multi-drug resistance to a number of anticancer drugs. The observed multi-drug resistance is primarily due to the efflux activity of ATP Binding Cassette (ABC) efflux transporters such as Pgp, MRP1 and BCRP. The expression of these transporters has been demonstrated in nuclear and cellular membranes of the LN-229 human glioblastoma cell line. Nuclear membrane and cellular membrane fragments from LN229 cells were immobilized on the IAM stationary phase to create nuclear and cellular membrane affinity chromatography columns, (NMAC(LN229)) and (CMAC(LN229)), respectively. Pgp, MRP1and BCRP transporters co-immobilized on both columns was characterized and compared by establishing the binding affinities for estrone-3-sulfate (3.8 vs 3.7μM), verapamil (0.6 vs 0.7μM) and prazosin (0.099 vs 0.033μM) on each column and no significant differences were observed. Since the marker ligands had overlapping selectivities, the selective characterization of each transporter was carried out by saturation of the binding sites of the non-targeted transporters. The addition of verapamil (Pgp and MRP1 substrate) to the mobile phase allowed the comparative screening of 8 compounds at the nuclear and cellular BCRP using etoposide as the marker ligand. AZT increased the retention of etoposide (+15%), a positive allosteric interaction, on the CMAC(LN229) column and decreased it (−5%) on the NMAC(LN229), while the opposite effect was produced by rhodamine. The results indicate that there are differences between the cellular and nuclear membrane expressed BCRP and that NMAC and CMAC columns can be used to probe these differences. PMID:24642394

  14. 21 CFR 892.1850 - Radiographic film cassette.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Radiographic film cassette. 892.1850 Section 892.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette....

  15. 21 CFR 892.1850 - Radiographic film cassette.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Radiographic film cassette. 892.1850 Section 892.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette....

  16. 21 CFR 892.1850 - Radiographic film cassette.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Radiographic film cassette. 892.1850 Section 892.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette....

  17. 21 CFR 892.1850 - Radiographic film cassette.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Radiographic film cassette. 892.1850 Section 892.1850 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1850 Radiographic film cassette....

  18. Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

    PubMed Central

    Fonseca, Érica L.; Vicente, Ana Carolina Paulo

    2015-01-01

    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-bla GES-1 /aacA4 gene cassette array, which harbours a fused gene cassette represented by bla GES-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 bla GES-1 /aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, bla GES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette bla GES-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 bla GES-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 bla GES-1/ aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-bla GES-1/ aacA4 free circular forms, but not to bla GES-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

  19. Apparatus and method for loading and unloading multiple digital tape cassettes utilizing a removable magazine

    DOEpatents

    Lindenmeyer, Carl W.

    1993-01-01

    An apparatus and method to automate the handling of multiple digital tape cassettes for processing by commercially available cassette tape readers and recorders. A removable magazine rack stores a plurality of tape cassettes, and cooperates with a shuttle device that automatically inserts and removes cassettes from the magazine to the reader and vice-versa. Photocells are used to identify and index to the desired tape cassette. The apparatus allows digital information stored on multiple cassettes to be processed without significant operator intervention.

  20. Apparatus and method for loading and unloading multiple digital tape cassettes utilizing a removable magazine

    DOEpatents

    Lindenmeyer, C.W.

    1993-01-26

    An apparatus and method to automate the handling of multiple digital tape cassettes for processing by commercially available cassette tape readers and recorders. A removable magazine rack stores a plurality of tape cassettes, and cooperates with a shuttle device that automatically inserts and removes cassettes from the magazine to the reader and vice-versa. Photocells are used to identify and index to the desired tape cassette. The apparatus allows digital information stored on multiple cassettes to be processed without significant operator intervention.

  1. The Lethal(1)tw-6(cs) Mutation of Drosophila Melanogaster Is a Dominant Antimorphic Allele of Nod and Is Associated with a Single Base Change in the Putative Atp-Binding Domain

    PubMed Central

    Rasooly, R. S.; New, C. M.; Zhang, P.; Hawley, R. S.; Baker, B. S.

    1991-01-01

    The l(1)TW-6(cs) mutation is a cold-sensitive recessive lethal mutation in Drosophila melanogaster, that affects both meiotic and mitotic chromosome segregation. We report the isolation of three revertants of this mutation. All three revert both the meiotic and mitotic effects as well as the cold sensitivity, demonstrating that all three phenotypes are due to a single lesion. We further show that these revertants fail to complement an amorphic allele of the nod (no distributive disjunction) locus, which encodes a kinesin-like protein. These experiments demonstrate that l(1)TW-6(cs) is an antimorphic allele of nod, and we rename it nod(DTW). Sequencing of the nod locus on a nod(DTW)-bearing chromosome reveals a single base change in the putative ATP-binding region of the motor domain of nod. Recessive, loss-of-function mutations at the nod locus specifically disrupt the segregation of nonexchange chromosomes in female meiosis. We demonstrate that, at 23.5°, the meiotic defects in nod(DTW)/+ females are similar to those observed in nod/nod females; that is, the segregation of nonexchange chromosomes is abnormal. However, in nod(DTW)/nod(DTW) females, or in nod(DTW)/+ females at 18°, we observe a more severe meiotic defect that apparently affects the segregation of both exchange and nonexchange chromosomes. In addition, nod(DTW) homozygotes and hemizygous males have previously been shown to exhibit mitotic defects including somatic chromosome breakage and loss. We propose that the defective protein encoded by the nod(DTW) allele interferes with proper chromosome movement during both meiosis and mitosis, perhaps by binding irreversibly to microtubules. PMID:1743485

  2. Cell cycle-dependent nuclear accumulation of the p94fer tyrosine kinase is regulated by its NH2 terminus and is affected by kinase domain integrity and ATP binding.

    PubMed

    Ben-Dor, I; Bern, O; Tennenbaum, T; Nir, U

    1999-02-01

    p94fer and p51ferT are two tyrosine kinases that are encoded by differentially spliced transcripts of the FER locus in the mouse. The two tyrosine kinases share identical SH2 and kinase domains but differ in their NH2-terminal amino acid sequence. Unlike p94fer, the presence of which has been demonstrated in most mammalian cell lines analyzed, the expression of p51ferT is restricted to meiotic cells. Here, we show that the two related tyrosine kinases also differ in their subcellular localization profiles. Although p51ferT accumulates constitutively in the cell nucleus, p94fer is cytoplasmic in quiescent cells and enters the nucleus concomitantly with the onset of S phase. The nuclear translocation of the FER proteins is driven by a nuclear localization signal (NLS), which is located within the kinase domain of these enzymes. The functioning of that NLS depends on the integrity of the kinase domain but was not affected by inactivation of the kinase activity. The NH2 terminus of p94fer dictated the cell cycle-dependent functioning of the NLS of FER kinase. This process was governed by coiled-coil forming sequences that are present in the NH2 terminus of the kinase. The regulatory effect of the p94fer NH2-terminal sequences was not affected by kinase activity but was perturbed by mutations in the kinase domain ATP binding site. Ectopic expression of the constitutively nuclear p51ferT in CHO cells interfered with S-phase progression in these cells. This was not seen in p94fer-overexpressing cells. The FER tyrosine kinases seem, thus, to be regulated by novel mechanisms that direct their different subcellular distribution profiles and may, consequently, control their cellular functioning. PMID:10074905

  3. Chemical modification with dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate reveals the distance between K480 and K501 in the ATP-binding domain of the Na,K-ATPase.

    PubMed

    Gatto, C; Lutsenko, S; Kaplan, J H

    1997-04-01

    Dihydro-4,4'-diisothiocyanostilbene-2,2'-disulfonate (H2DIDS) inactivates the renal Na,K-ATPase in an ATP- and K-preventable fashion; inactivation results in the covalent incorporation of a single [3H2]DIDS molecule into the Na pump alpha-subunit. K+ protection is observed at low concentrations (< 2 mM) and reversed at higher concentrations. The biphasic effect is also seen with Rb+, to a lesser extent by Cs+, and not at all by Na+ or choline. After extensive tryptic digestion of 3H2DIDS-inactivated enzyme, a single radiolabeled peptide is seen in 16.5% Tricine gels. N-terminal amino acid sequencing revealed two sequences 470IVEIPFNSTNxYQLS and 495HLLVMxGAPER, the unidentified residues were K480 and K501, respectively. These data provide suggestive evidence of cross-linking by H2DIDS between the two lysines. CNBr digestion of 3H2DIDS-labeled alpha-subunit produced a single radioactive band of the predicted 15-kDa mass for cross-linking between K480 an K501 produced by cleavage at known methione residues. The 15-kDa band combined two N-terminal sequences 464RDRYAKIVEI and 501xGAPERILDR which include K480 and K501. Thus K480 and K501 are within approximately 14 A of each other in the Na-bound form of the enzyme and information about the occupancy of the cation binding domain is transmitted to the ATP binding loop of the Na,K-ATPase. PMID:9126281

  4. Kinetics, in silico docking, molecular dynamics, and MM-GBSA binding studies on prototype indirubins, KT5720, and staurosporine as phosphorylase kinase ATP-binding site inhibitors: the role of water molecules examined.

    PubMed

    Hayes, Joseph M; Skamnaki, Vicky T; Archontis, Georgios; Lamprakis, Christos; Sarrou, Josephine; Bischler, Nicolas; Skaltsounis, Alexios-Leandros; Zographos, Spyros E; Oikonomakos, Nikos G

    2011-03-01

    With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC₅₀ > 50 μM), indirubin-3'-oxime (IC₅₀ = 144 nM), KT5720 (K(i) = 18.4 nM) and staurosporine (K(i) = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP-binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MMGBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc-staurosporine complex has the greatest number of receptor-ligand hydrogen bonds, while for the indirubin-3'-oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor-enzyme contacts. The MM-GBSA results revealed the source of staurosporine's low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1-water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schrödinger induced-fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor-ligand contacts. PMID:21287607

  5. Cellular pathways controlling integron cassette site folding.

    PubMed

    Loot, Céline; Bikard, David; Rachlin, Anna; Mazel, Didier

    2010-08-01

    By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single-stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double-stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non-recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination. PMID:20628355

  6. How to Use Both Audio Channels on Video Cassette Recorders.

    ERIC Educational Resources Information Center

    Senour, Robert A.; Campbell, Lloyd

    1980-01-01

    Videotape cassettes have two audio channels which makes possible a wide variety of new instructional techniques. Instructors can put running commentary/critiques on tapes of student counselor training sessions. (Author/TG)

  7. Mini-TV: The Case for Cassettes -- The Far North.

    ERIC Educational Resources Information Center

    Porcaro, Michael

    1977-01-01

    Describes a television system proposed for rural Alaska employing a core of locally selected programs transmitted via satellite plus a cassette machine for program substitutions as a way to maximize local participation. (JMF)

  8. Mitoxantrone targets the ATP-binding site of FAK, binds the FAK kinase domain and decreases FAK, Pyk-2, c-Src, and IGF-1R in vitro kinase activities.

    PubMed

    Golubovskaya, Vita M; Ho, Baotran; Zheng, Min; Magis, Andrew; Ostrov, David; Cance, William G

    2013-05-01

    Focal Adhesion Kinase (FAK) is a non-receptor kinase that is overexpressed in many types of tumors and plays a key role in cell adhesion, spreading, motility, proliferation, invasion, angiogenesis, and survival. Recently, FAK has been proposed as a target for cancer therapy, and we performed computer modeling and screening of the National Cancer Institute (NCI) small molecule compounds database to target the ATP-binding site of FAK, K454. More than 140,000 small molecule compounds were docked into the crystal structure of the kinase domain of FAK in 100 different orientations using DOCK5.1 that identified small molecule compounds, targeting the K454 site, called A-compounds. To find the therapeutic efficacy of these compounds, we examined the effect of twenty small molecule compounds on cell viability by MTT assays in different cancer cell lines. One compound, A18 (1,4-bis(diethylamino)-5,8- dihydroxy anthraquinon) was a mitoxantrone derivative and significantly decreased viability in most of the cells comparable to the to the level of FAK kinase inhibitors TAE-226 (Novartis, Inc) and PF-573,228 (Pfizer). The A18 compound specifically blocked autophosphorylation of FAK like TAE-226 and PF-228. ForteBio Octet Binding assay demonstrated that mitoxantrone (1,4-dihydroxy- 5,8-bis[2-(2-hydroxyethylamino) ethylamino] anthracene-9,10-dione directly binds the FAK-kinase domain. In addition, mitoxantrone significantly decreased the viability of breast cancer cells in a dose-dependent manner and inhibited the kinase activity of FAK and Y56/577 FAK phosphorylation at 10-20 μM. Mitoxantrone did not affect phosphorylation of EGFR, but decreased Pyk-2, c-Src, and IGF-1R kinase activities. The data demonstrate that mitoxantrone decreases cancer viability, binds FAK-Kinase domain, inhibits its kinase activity, and also inhibits in vitro kinase activities of Pyk-2 and IGF-1R. Thus, this novel function of the mitoxantrone drug can be critical for future development of anti

  9. Point Mutations in Human β Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly

    PubMed Central

    Becker, K. David; Gottshall, Kim R.; Hickey, Reed; Perriard, Jean-Claude; Chien, Kenneth R.

    1997-01-01

    Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a

  10. Kanamycin Resistance Cassette for Genetic Manipulation of Treponema denticola

    PubMed Central

    Li, Yuebin; Ruby, John

    2015-01-01

    Treponema denticola has been recognized as an important oral pathogen of the “red complex” bacterial consortium that is associated with the pathogenesis of endodontal and periodontal diseases. However, little is known about the virulence of T. denticola due to its recalcitrant genetic system. The difficulty in genetically manipulating oral spirochetes is partially due to the lack of antibiotic resistance cassettes that are useful for gene complementation following allelic replacement mutagenesis. In this study, a kanamycin resistance cassette was identified and developed for the genetic manipulation of T. denticola ATCC 35405. Compared to the widely used ermF-ermAM cassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistant T. denticola colonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames of T. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional in trans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had the flgE gene coding for PF hook protein inactivated. The integration of the full-length flgE gene into the genome of the flgE mutant rescued all of the defects associated with the flgE mutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation of T. denticola that will facilitate the characterization of virulence factors attributed to this important oral pathogen. PMID:25888173

  11. Encapsulated Energy Transfer Cassettes with Extremely Well Resolved Fluorescent Outputs

    PubMed Central

    Ueno, Yuichiro; Jose, Jiney; Loudet, Aurore; Pérez-Bolívar, César; Anzenbacher, Pavel; Burgess, Kevin

    2010-01-01

    This paper concerns the development of water-compatible fluorescent imaging-probes with tunable photonic properties that can be excited at a single wavelength. Bichromophoric cassettes 1a – 1c consisting of a BODIPY donor and a cyanine acceptor were prepared using a simple synthetic route, and their photophysical properties were investigated. Upon excitation of the BODIPY moiety at 488 nm the excitation energy is transferred through an acetylene bridge to the cyanine dye acceptor, which emits light at approximately 600, 700, and 800 nm, ie with remarkable dispersions. This effect is facilitated by efficient energy transfer that gives a ‘quasi-Stokes’ shift of between 86 – 290 nm opening a huge spectral window for imaging. The emissive properties of the cassettes depend on the energy transfer (ET) mechanism: the faster the transfer, the more efficient it is. Measurements of rates of energy transfer indicate that a through-bond energy transfer takes place in the cassettes 1a and 1b that is two orders of magnitude faster than the classical through-space, Förster, energy transfer (in the case of cassette 1c, however, both mechanisms are possible, and the rate measurements do not allow us to discern between them). Thus the cassettes 1a – 1c are well suited for multiplexing experiments in biotechnological methods that involve a single laser-excitation source. However, for widespread application of these probes their solubility in aqueous media must be improved. Consequently, the probes were encapsulated in calcium phosphate/silicate nanoparticles (diameter ca 22 nm) that are freely dispersible in water. This encapsulation process resulted in only minor changes in the photophysical properties of the cassettes. The system based on cassette 1a was chosen to probe how effectively these nanoparticles could be used to deliver the dyes into cells. Encapsulated cassette 1a permeated Clone 9 rat liver cells where it localized in the mitochondria and fluoresced through

  12. A Portable Analyzer for Pouch-Actuated, Immunoassay Cassettes

    PubMed Central

    Qiu, Xianbo; Liu, Changchun; Mauk, Michael G.; Hart, Robert W.; Chen, Dafeng; Qiu, Jing; Kientz, Terry; Fiene, Jonathan; Bau, Haim H.

    2011-01-01

    A portable, small footprint, light, general purpose analyzer (processor) to control the flow in immunoassay cassettes and to facilitate the detection of test results is described. The durable analyzer accepts disposable cassettes that contain pouches and reaction chambers for various unit operations such as hydration of dry reagents, stirring, and incubation. The analyzer includes individually controlled, linear actuators to compress the pouches in the cassette, which facilitates the pumping and mixing of sample and reagents, and to close diaphragm-based valves for flow control. The same types of actuators are used to compress pouches and actuate valves. The analyzer also houses a compact OEM scanner/reader to excite fluorescence and detect emission from labels. The analyzer is hydraulically isolated from the cassette, reducing the possibility of cross-contamination. The analyzer facilitates programmable, automated execution of a sequence of operations such as pumping and valving in a timely fashion, reducing the level of expertise required from the operator and the possibility for errors. The analyzer’s design is modular and expandable to accommodate cassettes of various complexities and additional functionalities. In this paper, the utility of the analyzer has been demonstrated with the execution of a simple, consecutive, lateral flow assay of a model biological system and the test results were detected with up converting phosphor labels that are excited at infrared frequencies and emit in the visible spectrum. PMID:22125359

  13. The effect of ABCG5/G8 polymorphisms on plasma HDL cholesterol levels depends on the ABCA1 gene variation in the Boston Puerto Rican Health Study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Directory of Spoken-Voice Audio-Cassettes, 1972.

    ERIC Educational Resources Information Center

    McKee, Gerald, Ed.

    Most listings in this catalog, which draws on many sources of production and is not a guide to one company's output, are for programs of college or adult level interest, with the exception of the "Careers" listings, geared toward high school students. The catalog also has lists of producers of children's cassettes and those designed for school…

  15. The Real World French Cassette Program. Script Book.

    ERIC Educational Resources Information Center

    Sternburg, Sheldon G.; Sammarco, Anthony M., Jr.

    This dual cassette package, accompanied by a script book, is designed to give students listening practice in French, particularly for regional differences of pronunciation and for variety in idiomatic constructions. The program may be integrated with texts used in intermediate and advanced levels of instruction. The announcements, jingles, and…

  16. The Real World Spanish Cassette Program. Script Book.

    ERIC Educational Resources Information Center

    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…

  17. 21 CFR 892.1860 - Radiographic film/cassette changer.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Radiographic film/cassette changer. 892.1860 Section 892.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic...

  18. 21 CFR 892.1860 - Radiographic film/cassette changer.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Radiographic film/cassette changer. 892.1860 Section 892.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic...

  19. 21 CFR 892.1860 - Radiographic film/cassette changer.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Radiographic film/cassette changer. 892.1860 Section 892.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic...

  20. 21 CFR 892.1860 - Radiographic film/cassette changer.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Radiographic film/cassette changer. 892.1860 Section 892.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic...

  1. Unprecedented laser action from energy transfer in multichromophoric BODIPY cassettes.

    PubMed

    Xiao, Yi; Zhang, Dakui; Qian, Xuhong; Costela, Angel; Garcia-Moreno, Inmaculada; Martin, Virginia; Perez-Ojeda, M Eugenia; Bañuelos, Jorge; Gartzia, Leire; Arbeloa, Iñigo López

    2011-11-01

    A cassette molecule, featuring direct integration of two donor BODIPY units to one acceptor BODIPY unit, was conveniently developed as the first highly "through-bond energy transfer" (TBET) laser dye. This multicolor absorbing dye exhibited highly efficient and photostable laser action under drastic pumping conditions. PMID:21935560

  2. DIY series of genetic cassettes useful in construction of versatile vectors specific for Alphaproteobacteria.

    PubMed

    Dziewit, Lukasz; Adamczuk, Marcin; Szuplewska, Magdalena; Bartosik, Dariusz

    2011-08-01

    We have developed a DIY (Do It Yourself) series of genetic cassettes, which facilitate construction of novel versatile vectors for Alphaproteobacteria. All the cassettes are based on defined genetic modules derived from three natural plasmids of Paracoccus aminophilus JCM 7686. We have constructed over 50 DIY cassettes, which differ in structure and specific features. All of them are functional in eight strains representing three orders of Alphaproteobacteria: Rhodobacterales, Rhizobiales and Caulobacterales. Besides various replication and stabilization systems, many of the cassettes also contain selective markers appropriate for Alphaproteobacteria (40 cassettes) and genetic modules responsible for mobilization for conjugal transfer (24 cassettes). All the DIY cassettes are bordered by different types of polylinkers, which facilitate vector construction. Using these DIY cassettes, we have created a set of compatible Escherichia coli-Alphaproteobacteria mobilizable shuttle vectors (high or low copy number in E. coli), which will greatly assist the genetic manipulation of Alphaproteobacteria. PMID:21569803

  3. Reaching Out: The Role of Audio Cassette Communication in Rural Development. Occasional Paper 19.

    ERIC Educational Resources Information Center

    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…

  4. 21 CFR 892.1880 - Wall-mounted radiographic cassette holder.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Wall-mounted radiographic cassette holder. 892... SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a) Identification. A wall-mounted radiographic cassette holder is a device...

  5. 21 CFR 892.1870 - Radiographic film/cassette changer programmer.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Radiographic film/cassette changer programmer. 892... SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic film/cassette changer programmer. (a) Identification. A radiographic film/cassette changer programmer is...

  6. Patterns of Availability and Use of Audiotape Cassettes in Special Libraries. Ph.D. Thesis

    NASA Technical Reports Server (NTRS)

    Hughes, J. M., II

    1975-01-01

    The availability and use of audiotape cassettes is studied in terms of user requirements. The following factors were examined: how special libraries utilize audiotape cassettes; who the users of the medium are; how the libraries acquire and maintain their collection; and opinions of librarians as to the value of the audiotape cassette as a medium for dissemination of information.

  7. THE REAL ISSUE WITH WALL DEPOSITS IN CLOSED FILTER CASSETTES - WHAT'S THE SAMPLE?

    SciTech Connect

    Brisson, M.

    2009-09-12

    The measurement of aerosol dusts has long been utilized to assess the exposure of workers to metals. Tools used to sample and measure aerosol dusts have gone through many transitions over the past century. In particular, there have been several different techniques used to sample for beryllium, not all of which might be expected to produce the same result. Today, beryllium samples are generally collected using filters housed in holders of several different designs, some of which are expected to produce a sample that mimics the human capacity for dust inhalation. The presence of dust on the interior walls of cassettes used to hold filters during metals sampling has been discussed in the literature for a number of metals, including beryllium, with widely varying data. It appears that even in the best designs, particulates can enter the sampling cassette and deposit on the interior walls rather than on the sampling medium. The causes are not well understood but are believed to include particle bounce, electrostatic forces, particle size, particle density, and airflow turbulence. Historically, the filter catch has been considered to be the sample, but the presence of wall deposits, and the potential that the filter catch is not representative of the exposure to the worker, puts that historical position into question. This leads to a fundamental question: What is the sample? This article reviews the background behind the issue, poses the above-mentioned question, and discusses options and a possible path forward for addressing that question.

  8. Cassette less SOFC stack and method of assembly

    DOEpatents

    Meinhardt, Kerry D

    2014-11-18

    A cassette less SOFC assembly and a method for creating such an assembly. The SOFC stack is characterized by an electrically isolated stack current path which allows welded interconnection between frame portions of the stack. In one embodiment electrically isolating a current path comprises the step of sealing a interconnect plate to a interconnect plate frame with an insulating seal. This enables the current path portion to be isolated from the structural frame an enables the cell frame to be welded together.

  9. Pouring and running a protein gel by reusing commercial cassettes.

    PubMed

    Hwang, Alexander C; Grey, Paris H; Cuddy, Katrina; Oppenheimer, David G

    2012-01-01

    The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (~$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms. PMID:22349047

  10. Chromosome inversions, adaptive cassettes and the evolution of species' ranges.

    PubMed

    Kirkpatrick, Mark; Barrett, Brian

    2015-05-01

    A chromosome inversion can spread when it captures locally adapted alleles or when it is introduced into a species by hybridization with adapted alleles that were previously absent. We present a model that shows how both processes can cause a species range to expand. Introgression of an inversion that carries novel, locally adapted alleles is a particularly powerful mechanism for range expansion. The model supports the earlier proposal that introgression of an inversion triggered a large range expansion of a malaria mosquito. These results suggest a role for inversions as cassettes of genes that can accelerate adaptation by crossing species boundaries, rather than protecting genomes from introgression. PMID:25583098

  11. Balloon-borne video cassette recorders for digital data storage

    NASA Technical Reports Server (NTRS)

    Althouse, W. E.; Cook, W. R.

    1985-01-01

    A high-speed, high-capacity digital data storage system has been developed for a new balloon-borne gamma-ray telescope. The system incorporates sophisticated, yet easy to use and economical consumer products: the portable video cassette recorder (VCR) and a relatively newer item - the digital audio processor. The in-flight recording system employs eight VCRs and will provide a continuous data storage rate of 1.4 megabits/sec throughout a 40 hour balloon flight. Data storage capacity is 25 gigabytes and power consumption is only 10 watts.

  12. Balloon-borne video cassette recorders for digital data storage

    NASA Technical Reports Server (NTRS)

    Althouse, W. E.; Cook, W. R.

    1985-01-01

    A high speed, high capacity digital data storage system was developed for a new balloon-borne gamma-ray telescope. The system incorporates economical consumer products: the portable video cassette recorder (VCR) and a relatively newer item - the digital audio processor. The in-flight recording system employs eight VCRs and will provide a continuous data storage rate of 1.4 megabits/sec throughout a 40 hour balloon flight. Data storage capacity is 25 gigabytes and power consumption is only 10 watts.

  13. [Reduction of radiation dose by the use of carbon fiber cassettes].

    PubMed

    Hajek, P; Nowotny, R

    1984-03-01

    A new type of radiographic cassette, reinforced by PEEK-CFK is discussed. The amount of reduction of radiation dose by this cassette was evaluated by means of an experimental physical and clinical trial. Dose reduction may reach 30% depending on the type of examination and the organ studied. An increase of contrast of the radiographs could not be verified. This type of cassette can be recommended for routine clinical use. PMID:6423492

  14. The x-ray source application test cassette for radiation exposures at the OMEGA laser

    SciTech Connect

    Fournier, K. B.; Rekow, V.; Emig, J.; Fisher, J. H.; Newlander, C. D.; Horton, R.; Davis, J.

    2012-10-15

    We have designed a sample cassette that can be used to position up to six samples in the OMEGA laser chamber. The cassette accommodates round samples up to 38.1 mm (1.5{sup Double-Prime }) in diameter and square samples up to 27 mm on a side, any of which can be up to 12.7 mm thick. Smaller specimens are centered with spacers. The test cassette allows each sample to have a unique filter scheme, with multiple filter regions in front of each sample. This paper will present mechanical design considerations and operational aspects of the x-ray source application cassette.

  15. Staphylococcal cassette chromosome mec-like element in Macrococcus caseolyticus.

    PubMed

    Tsubakishita, Sae; Kuwahara-Arai, Kyoko; Baba, Tadashi; Hiramatsu, Keiichi

    2010-04-01

    Macrococcus is a bacterial genus that is closely related to Staphylococcus, which typically is isolated from animal skin and products. The genome analysis of multidrug-resistant Macrococcus caseolyticus strain JCSC5402, isolated from chicken, previously led to the identification of plasmid pMCCL2, which carries a transposon containing an unusual form of the Macrococcus mec gene complex (mecA(m)-mecR1(m)-mecI(m)-blaZ(m)). In M. caseolyticus strain JCSC7096, this mec transposon containing the mec gene complex (designated Tn6045 in this study) was found integrated downstream of orfX on the chromosome. Tn6045 of JCSC7096 was bracketed by the direct repeat sequences (DR) specifically recognized by cassette chromosome recombinase (CCR). A non-mecA-containing staphylococcal cassette chromosome (SCC) element, designated SCC(7096), was integrated next to the mec transposon and separated from the latter by a DR. Nested PCR experiments showed that the mec transposon not only was excised singly but also coexcised with SCC(7096) from the chromosome at the DRs. The coexcised elements formed the extrachromosomal closed circular DNA of the SCCmec-like element. SCCmec is known to be the mobile element conveying methicillin (meticillin) resistance in staphylococci. However, its origin has been unknown. Our observation revealed a potential mechanism of the generation of a new SCCmec-like element in M. caseolyticus, a commensal bacterium of food animals. PMID:20086147

  16. Retroviral vectors for the transduction of autoregulated, bidirectional expression cassettes.

    PubMed

    Unsinger, J; Kröger, A; Hauser, H; Wirth, D

    2001-11-01

    Regulated transgene expression is increasingly used in research but is also needed for certain therapies. Regulatory systems are usually composed of two expression units, one bearing the gene of interest under control of a regulatable promoter and the other, a constitutively expressed transactivator that modulates the activity of the regulatable promoter. Because the cotransfer of two independent elements is not efficient in primary cells, single transduction step vectors conferring regulatable gene expression cassettes would be helpful. We have developed retroviral vectors containing an autoregulatory bidirectional expression cassette that encodes all components necessary for regulated expression of a gene of interest. The influence of the orientation of the reporter gene with respect to the viral long terminal repeat (LTR) and the effect of transcriptionally inactive LTRs were investigated using mouse leukemia virus (MLV) and self-inactivating (SIN)-based retroviral vectors. Strict regulation was observed when the reporter was inserted in antisense orientation with respect to the LTR, whereas a sense arrangement of the reporter resulted in a loss of regulation capacity. Expression and regulation of the antisense-orientated reporter gene were homogenous in infected cell pools and investigated cell clones. Long-term observations of infected cells over a period of 30 passages revealed stable expression and regulation. These autoregulated, bidirectional retroviral vectors combine the advantages of single-step transduction with strict regulation of the gene of interest in the infected target cells. PMID:11708885

  17. Staphylococcal Cassette Chromosome mec-Like Element in Macrococcus caseolyticus▿

    PubMed Central

    Tsubakishita, Sae; Kuwahara-Arai, Kyoko; Baba, Tadashi; Hiramatsu, Keiichi

    2010-01-01

    Macrococcus is a bacterial genus that is closely related to Staphylococcus, which typically is isolated from animal skin and products. The genome analysis of multidrug-resistant Macrococcus caseolyticus strain JCSC5402, isolated from chicken, previously led to the identification of plasmid pMCCL2, which carries a transposon containing an unusual form of the Macrococcus mec gene complex (mecAm-mecR1m-mecIm-blaZm). In M. caseolyticus strain JCSC7096, this mec transposon containing the mec gene complex (designated Tn6045 in this study) was found integrated downstream of orfX on the chromosome. Tn6045 of JCSC7096 was bracketed by the direct repeat sequences (DR) specifically recognized by cassette chromosome recombinase (CCR). A non-mecA-containing staphylococcal cassette chromosome (SCC) element, designated SCC7096, was integrated next to the mec transposon and separated from the latter by a DR. Nested PCR experiments showed that the mec transposon not only was excised singly but also coexcised with SCC7096 from the chromosome at the DRs. The coexcised elements formed the extrachromosomal closed circular DNA of the SCCmec-like element. SCCmec is known to be the mobile element conveying methicillin (meticillin) resistance in staphylococci. However, its origin has been unknown. Our observation revealed a potential mechanism of the generation of a new SCCmec-like element in M. caseolyticus, a commensal bacterium of food animals. PMID:20086147

  18. Cassette Sound Filmstrip Viewers -- Evaluations of Nine Machines. An In Depth Report

    ERIC Educational Resources Information Center

    Educational Product Report, 1974

    1974-01-01

    In evaluating cassette sound filmstrip viewers, many criteria are the same as those applied to cassette recorders in Educational Product Report; v7 n5 Nov '73 and silent filmstrip viewers in Educational Product Report; v6 n5 Feb '73. These criteria cover output power; frequency response; tape speed accuracy including drift, wow, and flutter; and…

  19. Motion Pictures and Video Cassettes 1971. AV-USA Supplement 2.

    ERIC Educational Resources Information Center

    Hope, Thomas W.

    The financial status of the motion picture and of the video cassette industry in 1970 are reviewed. Based on production rates and income of these industries, trends are discovered. Figures on local origination of television programing and commercials are also included. The section on video cassettes includes the following information: the current…

  20. Spiranic BODIPYs: a ground-breaking design to improve the energy transfer in molecular cassettes.

    PubMed

    Sánchez-Carnerero, Esther M; Gartzia-Rivero, Leire; Moreno, Florencio; Maroto, Beatriz L; Agarrabeitia, Antonia R; Ortiz, María J; Bañuelos, Jorge; López-Arbeloa, Íñigo; de la Moya, Santiago

    2014-10-28

    Boosted excitation energy transfer in spiranic O-BODIPY/polyarene cassettes, when compared with the parent non-spiranic (flexible) system, is highlighted as a proof for the ability of a new structural design to improve the energy transfer in molecular cassettes. PMID:25207836

  1. Identifying Products of Recombinase-Mediated Cassette Exchange (RMCE) in Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    Homologous recombination is highly efficient when mediated between two identical target sequences by recombination enzymes such as Cre. Exploiting this, recombinase-mediated cassette exchange (RMCE) was developed for the genetic manipulation of eukaryotic cells, including those of Schizosaccharomyces pombe RMCE can be summarized in three stages: (1) A loxP-ura4(+)-loxM3 cassette is introduced into the genome using standard homologous recombination techniques to create a "base strain." (2) A Cre-expression plasmid carrying a protein tag or replacement gene flanked by loxP and loxM3 is introduced into the cell. (3) Cassette exchange between the chromosomal cassette and the plasmid cassette results in either gene tagging or gene replacement. This is selected for by loss of the marker. This protocol explains how to identify the products of the exchange events in the last stage. PMID:27140917

  2. Quantitative assessment and reduction of long-term autoradiographic background

    SciTech Connect

    Traub, R.K.; Famous, L.; Krishnan, R.; Olson, K.R.

    1990-01-01

    Quantitative autoradiography can measure distribution patterns in an animal exposed to radiolabeled compounds. A comparison of autoradiographs of rat brain containing low levels of 14C showed that a highly variable background signal had been produced. This resulted in several overexposed autoradiographs which could not be quantitatively compared. The background, believed to be produced by light emanating from the phosphor coating in the X-ray cassette, was a major impediment because it hindered correct analysis of the specimen. this article details our experiments demonstrating the sources of variance contributing to background and offers methods for its reduction. We found that placement of black polyethylene plastic between the slides and phosphor in the X-ray film cassette minimized autoradiographic background and effectively eliminated the effects caused by inherently different levels of radioactivity in the glass slides.

  3. Inducible and Reversible Lentiviral and Recombination Mediated Cassette Exchange (RMCE) Systems for Controlling Gene Expression

    PubMed Central

    Bersten, David C.; Sullivan, Adrienne E.; Li, Dian; Bhakti, Veronica; Bent, Stephen J.; Whitelaw, Murray L.

    2015-01-01

    Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function. PMID:25768837

  4. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    PubMed Central

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Leo, Rosa Di; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H. W.; Curmi, Paul M. G.; Mabbutt, Bridget C.

    2011-01-01

    Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds. PMID:21390267

  5. Identification of tumor-associated cassette exons in human cancer through EST-based computational prediction and experimental validation

    PubMed Central

    2010-01-01

    Background Many evidences report that alternative splicing, the mechanism which produces mRNAs and proteins with different structures and functions from the same gene, is altered in cancer cells. Thus, the identification and characterization of cancer-specific splice variants may give large impulse to the discovery of novel diagnostic and prognostic tumour biomarkers, as well as of new targets for more selective and effective therapies. Results We present here a genome-wide analysis of the alternative splicing pattern of human genes through a computational analysis of normal and cancer-specific ESTs from seventeen anatomical groups, using data available in AspicDB, a database resource for the analysis of alternative splicing in human. By using a statistical methodology, normal and cancer-specific genes, splice sites and cassette exons were predicted in silico. The condition association of some of the novel normal/tumoral cassette exons was experimentally verified by RT-qPCR assays in the same anatomical system where they were predicted. Remarkably, the presence in vivo of the predicted alternative transcripts, specific for the nervous system, was confirmed in patients affected by glioblastoma. Conclusion This study presents a novel computational methodology for the identification of tumor-associated transcript variants to be used as cancer molecular biomarkers, provides its experimental validation, and reports specific biomarkers for glioblastoma. PMID:20813049

  6. Delineating a conserved genetic cassette promoting outgrowth of body appendages.

    PubMed

    Lin, Congxing; Yin, Yan; Bell, Sheila M; Veith, G Michael; Chen, Hong; Huh, Sung-Ho; Ornitz, David M; Ma, Liang

    2013-01-01

    The acquisition of the external genitalia allowed mammals to cope with terrestrial-specific reproductive needs for internal fertilization, and thus it represents one of the most fundamental steps in evolution towards a life on land. How genitalia evolved remains obscure, and the key to understanding this process may lie in the developmental genetics that underpins the early establishment of the genital primordium, the genital tubercle (GT). Development of the GT is similar to that of the limb, which requires precise regulation from a distal signaling epithelium. However, whether outgrowth of the GT and limbs is mediated by common instructive signals remains unknown. In this study, we used comprehensive genetic approaches to interrogate the signaling cascade involved in GT formation in comparison with limb formation. We demonstrate that the FGF ligand responsible for GT development is FGF8 expressed in the cloacal endoderm. We further showed that forced Fgf8 expression can rescue limb and GT reduction in embryos deficient in WNT signaling activity. Our studies show that the regulation of Fgf8 by the canonical WNT signaling pathway is mediated in part by the transcription factor SP8. Sp8 mutants elicit appendage defects mirroring WNT and FGF mutants, and abolishing Sp8 attenuates ectopic appendage development caused by a gain-of-function β-catenin mutation. These observations indicate that a conserved WNT-SP8-FGF8 genetic cassette is employed by both appendages for promoting outgrowth, and suggest a deep homology shared by the limb and external genitalia. PMID:23358455

  7. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette.

    PubMed

    Dickinson, Daniel J; Pani, Ariel M; Heppert, Jennifer K; Higgins, Christopher D; Goldstein, Bob

    2015-08-01

    A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP-SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step. PMID:26044593

  8. Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

    PubMed Central

    Dickinson, Daniel J.; Pani, Ariel M.; Heppert, Jennifer K.; Higgins, Christopher D.; Goldstein, Bob

    2015-01-01

    A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP–SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step. PMID:26044593

  9. COLLECTION EFFICIENCY OF FIELD SAMPLING CASSETTES: INTERAGENCY ENERGY/ENVIRONMENT R AND D PROGRAM REPORT

    EPA Science Inventory

    Industrial hygiene particulate samples are often collected under anisokinetic sampling conditions and in crosswinds. Experiments were conducted to quantitate errors associated with sampling under these non-ideal conditions. Three types of field sampling cassetts were tested to de...

  10. Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme

    NASA Astrophysics Data System (ADS)

    Zhang, Yun-Xin

    2014-10-01

    Due to inter-subunit communication, multisubunit enzymes usually hydrolyze ATP in a concerted fashion. However, so far the principle of this process remains poorly understood. In this study, from the viewpoint of statistical mechanics, a simple model is presented. In this model, we assume that the binding of ATP will change the potential of the corresponding enzyme subunit, and the degree of this change depends on the state of its adjacent subunits. The probability of enzyme in a given state satisfies the Boltzmann's distribution. Although it looks much simple, this model can fit the recent experimental data of chaperonin TRiC/CCT well. From this model, the dominant state of TRiC/CCT can be obtained. This study provide a new way to understand biophysical processe by statistical mechanics analysis.

  11. Integron gene cassettes: a repository of novel protein folds with distinct interaction sites.

    PubMed

    Sureshan, Visaahini; Deshpande, Chandrika N; Boucher, Yan; Koenig, Jeremy E; Stokes, H W; Harrop, Stephen J; Curmi, Paul M G; Mabbutt, Bridget C

    2013-01-01

    Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites), as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+β and α/β fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions. PMID:23349695

  12. Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange.

    PubMed

    Bateman, Jack R; Lee, Anne M; Wu, C-ting

    2006-06-01

    Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs. PMID:16547094

  13. Characterization of the phd-doc and ccd Toxin-Antitoxin Cassettes from Vibrio Superintegrons

    PubMed Central

    Guérout, Anne-Marie; Iqbal, Naeem; Mine, Natacha; Ducos-Galand, Magaly; Van Melderen, Laurence

    2013-01-01

    Toxin-antitoxin (TA) systems have been reported in the genomes of most bacterial species, and their role when located on the chromosome is still debated. TA systems are particularly abundant in the massive cassette arrays associated with chromosomal superintegrons (SI). Here, we describe the characterization of two superintegron cassettes encoding putative TA systems. The first is the phd-docSI system identified in Vibrio cholerae N16961. We determined its distribution in 36 V. cholerae strains and among five V. metschnikovii strains. We show that this cassette, which is in position 72 of the V. cholerae N16961 cassette array, is functional, carries its own promoter, and is expressed from this location. Interestingly, the phd-docSI system is unable to control its own expression, most likely due to the absence of any DNA-binding domain on the antitoxin. In addition, this SI system is able to cross talk with the canonical P1 phage system. The second cassette that we characterized is the ccdVfi cassette found in the V. fischeri superintegron. We demonstrate that CcdBVfi targets DNA-gyrase, as the canonical CcBF toxin, and that ccdVfi regulates its expression in a fashion similar to the ccdF operon of the conjugative plasmid F. We also establish that this cassette is functional and expressed in its chromosomal context in V. fischeri CIP 103206T. We tested its functional interactions with the ccdABF system and found that CcdAVfi is specific for its associated CcdBVfi and cannot prevent CcdBF toxicity. Based on these results, we discuss the possible biological functions of these TA systems in superintegrons. PMID:23475970

  14. A comparison of the closed-face cassette at different orientations while measuring total particles.

    PubMed

    Cook, David M; Sleeth, Darrah K; Thiese, Matthew S; Larson, Rodney R

    2015-01-01

    The current method for sampling aerosols using the 37-mm closed-face cassette (CFC) sampler is based on the orientation of the cassette at ∼45° from horizontal. There is some concern as to whether this method is appropriate and may be underestimating exposures. An alternative orientation at ∼0° (horizontal) has been discussed. This research compared the CFC's orientation at 45° from horizontal to the proposed orientation at horizontal, 0° in a controlled laboratory setting. The particles used in this study were fused alumina oxide in four sizes, approximately 9.5 μm, 12.8 μm, 18 μm, and 44.3 μm in aerodynamic diameter. For each test, one aerosol was dispersed in a wind tunnel operating at 0.2 m/s with samplers mounted in the breathing zone of a rotating mannequin. A sampling event consisted of four pairs of samplers, placed side by side (one pair at 45° and another at 0° cassette orientation), and exposed for a period of 45 minutes. A total of 12 sampling events, 3 sample events per particle size, were conducted with a total of 94 samples collected. Mass concentration measurements were compared to assess the relationship between the sampler orientations of the cassettes. In addition, the relationship between the mass collected on the cassette filter and on the interior walls of the cassette was also assessed. The results indicated that there was no significant difference between the measured concentrations based on the orientation of the CFCs. The amount of mass collected on the interior walls of the cassettes was relatively low (<5%) compared to expected (up to 100%) wall losses for both orientations. PMID:25337937

  15. Erythromycin esterase gene ere(A) is located in a functional gene cassette in an unusual class 2 integron.

    PubMed

    Biskri, Latefa; Mazel, Didier

    2003-10-01

    The gene ere(A) of the plasmid pIP1100 is larger than originally reported and is organized as an integron gene cassette. The ere(A) gene cassette carries its own promoter and is propagated by a class 2 integron with an insertion sequence element, IS1, inserted upstream of the intI2 gene. The mobility of the ere(A) cassette has been demonstrated. PMID:14506050

  16. A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms

    NASA Technical Reports Server (NTRS)

    Hedenstierna, K. O.; Lee, Y. H.; Yang, Y.; Fox, G. E.

    1993-01-01

    A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed. The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli. The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V. proteolyticus type. Cells carrying the identifier are readily detectable by hybridization. Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E. coli 5S rRNA, and the V. proteolyticus 5S rRNA gene is not inactivated after prolonged growth. These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods. Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts.

  17. Diverse Gene Cassettes in Class 1 Integrons of Facultative Oligotrophic Bacteria of River Mahananda, West Bengal, India

    PubMed Central

    Chakraborty, Ranadhir; Kumar, Arvind; Bhowal, Suparna Saha; Mandal, Amit Kumar; Tiwary, Bipransh Kumar; Mukherjee, Shriparna

    2013-01-01

    Background In this study a large random collection (n = 2188) of facultative oligotrophic bacteria, from 90 water samples gathered in three consecutive years (2007–2009) from three different sampling sites of River Mahananda in Siliguri, West Bengal, India, were investigated for the presence of class 1 integrons and sequences of the amplification products. Methodology/Principal Findings Replica plating method was employed for determining the antibiotic resistance profile of the randomly assorted facultative oligotrophic isolates. Genomic DNA from each isolate was analyzed by PCR for the presence of class 1 integron. Amplicons were cloned and sequenced. Numerical taxonomy and 16S rRNA gene sequence analyses were done to ascertain putative genera of the class 1 integron bearing isolates. Out of 2188 isolates, 1667 (76.19%) were antibiotic-resistant comprising of both single-antibiotic resistance (SAR) and multiple-antibiotic resistant (MAR), and 521 (23.81%) were sensitive to all twelve different antibiotics used in this study. Ninety out of 2188 isolates produced amplicon(s) of varying sizes from 0.15 to 3.45 KB. Chi-square (χ2) test revealed that the possession of class 1 integron in sensitive, SAR and MAR is not equally probable at the 1% level of significance. Diverse antibiotic-resistance gene cassettes, aadA1, aadA2, aadA4, aadA5, dfrA1, dfrA5, dfrA7, dfrA12, dfrA16, dfrA17, dfrA28, dfrA30, dfr-IIe, blaIMP-9, aacA4, Ac-6′-Ib, oxa1, oxa10 and arr2 were detected in 64 isolates. The novel cassettes encoding proteins unrelated to any known antibiotic resistance gene function were identified in 26 isolates. Antibiotic-sensitive isolates have a greater propensity to carry gene cassettes unrelated to known antibiotic-resistance genes. The integron-positive isolates under the class Betaproteobacteria comprised of only two genera, Comamonas and Acidovorax of family Comamonadaceae, while isolates under class Gammaproteobacteria fell under the families

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

    PubMed

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

    2011-12-01

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

  19. Photophysical and laser properties of cassettes based on a BODIPY and rhodamine pair.

    PubMed

    Gartzia-Rivero, Leire; Yu, Haibo; Bañuelos, Jorge; López-Arbeloa, Iñigo; Costela, Angel; Garcia-Moreno, Inmaculada; Xiao, Yi

    2013-12-01

    This work deals with the synthesis and the photophysical and laser properties of new BODIPY-rhodamine cassettes. These dyads differ in their rigid and conjugated spacer group (phenyl or acetylenephenyl) and in their linking positions (meta or para). The photophysical properties of these cassettes are controlled by the formation/opening of the spirolactone ring, which, in turn, switches off/on an energy-transfer process between the chromophores. Herein, we thoroughly describe the influence of the attached spacer group, as well as the distance and orientation between the donor-acceptor pair, on the excitation energy transfer. The observed fast dynamics and efficiency suggest that the process mainly takes place "through-bond", although the "through-space" mechanism also contributes to the whole process. As a result, efficient laser emission from the rhodamine is achieved upon excitation of the BODIPY, in particular for the cassette that contains an acetylenephenyl spacer group in a para disposition. PMID:24023008

  20. Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens.

    PubMed

    Louwerse, Jeanine D; van Lier, Miranda C M; van der Steen, Dirk M; de Vlaam, Clementine M T; Hooykaas, Paul J J; Vergunst, Annette C

    2007-12-01

    Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation. PMID:17921337

  1. Stable Recombinase-Mediated Cassette Exchange in Arabidopsis Using Agrobacterium tumefaciens1

    PubMed Central

    Louwerse, Jeanine D.; van Lier, Miranda C.M.; van der Steen, Dirk M.; de Vlaam, Clementine M.T.; Hooykaas, Paul J.J.; Vergunst, Annette C.

    2007-01-01

    Site-specific integration is an attractive method for the improvement of current transformation technologies aimed at the production of stable transgenic plants. Here, we present a Cre-based targeting strategy in Arabidopsis (Arabidopsis thaliana) using recombinase-mediated cassette exchange (RMCE) of transferred DNA (T-DNA) delivered by Agrobacterium tumefaciens. The rationale for effective RMCE is the precise exchange of a genomic and a replacement cassette both flanked by two heterospecific lox sites that are incompatible with each other to prevent unwanted cassette deletion. We designed a strategy in which the coding region of a loxP/lox5171-flanked bialaphos resistance (bar) gene is exchanged for a loxP/lox5171-flanked T-DNA replacement cassette containing the neomycin phosphotransferase (nptII) coding region via loxP/loxP and lox5171/lox5171 directed recombination. The bar gene is driven by the strong 35S promoter, which is located outside the target cassette. This placement ensures preferential selection of RMCE events and not random integration events by expression of nptII from this same promoter. Using root transformation, during which Cre was provided on a cotransformed T-DNA, 50 kanamycin-resistant calli were selected. Forty-four percent contained a correctly exchanged cassette based on PCR analysis, indicating the stringency of the selection system. This was confirmed for the offspring of five analyzed events by Southern-blot analysis. In four of the five analyzed RMCE events, there were no additional T-DNA insertions or they easily segregated, resulting in high-efficiency single-copy RMCE events. Our approach enables simple and efficient selection of targeting events using the advantages of Agrobacterium-mediated transformation. PMID:17921337

  2. CMOS cassette for digital upgrade of film-based mammography systems

    NASA Astrophysics Data System (ADS)

    Baysal, Mehmet A.; Toker, Emre

    2006-03-01

    While full-field digital mammography (FFDM) technology is gaining clinical acceptance, the overwhelming majority (96%) of the installed base of mammography systems are conventional film-screen (FSM) systems. A high performance, and economical digital cassette based product to conveniently upgrade FSM systems to FFDM would accelerate the adoption of FFDM, and make the clinical and technical advantages of FFDM available to a larger population of women. The planned FFDM cassette is based on our commercial Digital Radiography (DR) cassette for 10 cm x 10 cm field-of-view spot imaging and specimen radiography, utilizing a 150 micron columnar CsI(Tl) scintillator and 48 micron active-pixel CMOS sensor modules. Unlike a Computer Radiography (CR) cassette, which requires an external digitizer, our DR cassette transfers acquired images to a display workstation within approximately 5 seconds of exposure, greatly enhancing patient flow. We will present the physical performance of our prototype system against other FFDM systems in clinical use today, using established objective criteria such as the Modulation Transfer Function (MTF), Detective Quantum Efficiency (DQE), and subjective criteria, such as a contrast-detail (CD-MAM) observer performance study. Driven by the strong demand from the computer industry, CMOS technology is one of the lowest cost, and the most readily accessible technologies available for FFDM today. Recent popular use of CMOS imagers in high-end consumer cameras have also resulted in significant advances in the imaging performance of CMOS sensors against rivaling CCD sensors. This study promises to take advantage of these unique features to develop the first CMOS based FFDM upgrade cassette.

  3. A new generic column switching system for quantitation in cassette dosing using LC/MS/MS.

    PubMed

    Ohkawa, T; Ishida, Y; Kanaoka, E; Takahashi, K; Okabe, H; Matsumoto, T; Nakamoto, S; Tamada, J; Koike, M; Yoshikawa, T

    2003-04-10

    Cassette dosing is a method in which multiple drugs are administered to a single animal at the same time, and the plasma concentrations of the individual compounds are simultaneously determined. This method enables high-throughput rapid screening for pharmacokinetic assessment of new drug candidates. An available gradient method was modified for cassette dosing analysis to attain the advantages of high sensitivity and applicability to a wide range of compounds. However, two problems arose; (1). the time-consuming optimization of mobile phases for each compound group, which limited applicability and (2). the remarkable suppression of ionization by polyethyleneglycol, which is commonly used in intravenous administration. To resolve these problems, a new column switching method was established to attain wider applicability and avoid the ionization suppression. This column switching system is very simple because the trap column and the analytical column are specified and the mobile phase is selected from only two species. Method optimization requires only the selection of the mobile phase and takes only a few hours. About 200 compounds, which were administered as about 50 cassettes, were analyzed using this column switching system. Assay validation of one cassette was carried out, and good accuracy and precision were obtained. About 90% of the compounds could be determined within 20% bias. These results showed that this new column switching system for cassette dosing is accurate enough for the screening of drug candidates and offers wide applicability for various compounds. This system was shown to be very useful for the determination of cassette dosing samples, containing multiple compounds. PMID:12667925

  4. Video Cassettes: The Systems, the Market, the Future.

    ERIC Educational Resources Information Center

    Roberts, Martin

    In its survey of the videocassette field, this book details the background, current status, problems, and potentials of the various systems designed to record and reproduce films and other audiovisual material through a conventional television set. The systems used by CBS (a miniaturized film format), Avco, Sony, Ampex (all magnetic tape formats),…

  5. A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In a previous study we developed a cassette employing a bacterial beta-recombinase acting on six recognition sequences (beta-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette...

  6. STS-37 crewmembers watch Pilot Cameron juggle cassettes on OV-104's middeck

    NASA Technical Reports Server (NTRS)

    1991-01-01

    STS-37 crewmembers watch Pilot Kenneth D. Cameron juggle cassette tapes on the middeck of Atlantis, Orbiter Vehicle (OV) 104. Laughing at Cameron's stunt are Mission Specialist (MS) Linda M. Godwin (foreground), Commander Steven R. Nagel (behind Cameron), and MS Jerry L. Ross (at floor level). Ross snacks on chocolate candy during the performance.

  7. Synthesis of Poly Linear shRNA Expression Cassettes Through Branch-PCR.

    PubMed

    Liu, Jianbing; Xi, Zhen

    2016-01-01

    A facile and universal strategy to construct the poly linear small hairpin RNA (shRNA) expression cassettes with multiple shRNA transcription templates through polymerase chain reaction with flexible branched primers (branch-PCR) is described in this protocol. Double-stranded RNA (dsRNA) is not stable enough for the study of RNA interference (RNAi) delivery in mammalian cells. Therefore, the more stable shRNA transcription template is employed to produce the endogenous transcribed dsRNA. Then, the covalent crosslinked linear shRNA expression cassettes are constructed through the branch-PCR for the long-lasting RNAi effect in this protocol. The branched primer pair is efficiently synthesized through classic click chemistry. In one step of PCR, the much more stable poly linear shRNA expression cassettes can be produced in large scale. This strategy of efficient synthesis of the poly linear gene expression cassettes can also be applied in the field for other target gene delivery. © 2016 by John Wiley & Sons, Inc. PMID:27584702

  8. Stacking multiple transgenes at a selected genomic site via repeated recombinase-mediated DNA cassette exchanges.

    PubMed

    Li, Zhongsen; Moon, Bryan P; Xing, Aiqiu; Liu, Zhan-Bin; McCardell, Richard P; Damude, Howard G; Falco, S Carl

    2010-10-01

    Recombinase-mediated DNA cassette exchange (RMCE) has been successfully used to insert transgenes at previously characterized genomic sites in plants. Following the same strategy, groups of transgenes can be stacked to the same site through multiple rounds of RMCE. A gene-silencing cassette, designed to simultaneously silence soybean (Glycine max) genes fatty acid ω-6 desaturase 2 (FAD2) and acyl-acyl carrier protein thioesterase 2 (FATB) to improve oleic acid content, was first inserted by RMCE at a precharacterized genomic site in soybean. Selected transgenic events were subsequently retransformed with the second DNA construct containing a Yarrowia lipolytica diacylglycerol acyltransferase gene (DGAT1) to increase oil content by the enhancement of triacylglycerol biosynthesis and three other genes, a Corynebacterium glutamicum dihydrodipicolinate synthetase gene (DHPS), a barley (Hordeum vulgare) high-lysine protein gene (BHL8), and a truncated soybean cysteine synthase gene (CGS), to improve the contents of the essential amino acids lysine and methionine. Molecular characterization confirmed that the second RMCE successfully stacked the four overexpression cassettes to the previously integrated FAD2-FATB gene-silencing cassette. Phenotypic analyses indicated that all the transgenes expressed expected phenotypes. PMID:20720171

  9. STS-29 MS Bagian juggles audio cassettes on Discovery's, OV-103's, middeck

    NASA Technical Reports Server (NTRS)

    1989-01-01

    On aft middeck, STS-29 Mission Specialist (MS) James P. Bagian juggles TEAC audio cassettes freefloating above foam insert as he attempts to organize them. In front of Bagian are aft middeck lockers and part of the open airlock hatch. Behind him are the starboard wall-mounted sleep restraints.

  10. Construction of heterologous gene expression cassettes for the development of recombinant Clostridium beijerinckii.

    PubMed

    Oh, Young Hoon; Eom, Gyeong Tae; Kang, Kyoung Hee; Joo, Jeong Chan; Jang, Young-Ah; Choi, Jae Woo; Song, Bong Keun; Lee, Seung Hwan; Park, Si Jae

    2016-04-01

    Gene-expression cassettes for the construction of recombinant Clostridium beijerinckii were developed as potential tools for metabolic engineering of C. beijerinckii. Gene expression cassettes containing ColE1 origin and pAMB origin along with the erythromycin resistance gene were constructed, in which promoters from Escherichia coli, Lactococcus lactis, Ralstonia eutropha, C. acetobutylicum, and C. beijerinckii are examined as potential promoters in C. beijerinckii. Zymogram analysis of the cell extracts and comparison of lipase activities of the recombinant C. beijerinckii strains expressing Pseudomonas fluorescens tliA gene suggested that the tliA gene was functionally expressed by all the examined promoters with different expression level. Also, recombinant C. beijerinckii expressing C. beijerinckii secondary alcohol dehydrogenase by the constructed expression cassettes successfully produced 2-propanol from glucose. The best promoter for TliA expression was the R. eutropha phaP promoter while that for 2-propanol production was the putative C. beijerinckii pta promoter. Gene expression cassettes developed in this study may be useful tools for the construction of recombinant C. beijerinckii strains as host strains for the valuable chemicals and fuels from renewable resources. PMID:26780375

  11. Analysis of the ISS Russian Segment Outer Surface Materials Installed on the CKK Detachable Cassette

    NASA Astrophysics Data System (ADS)

    Naumov, S. F.; Borisov, V. A.; Plotnikov, A. D.; Sokolova, S. P.; Kurilenok, A. O.; Skurat, V. E.; Leipunsky, I. O.; Pshechenkov, P. A.; Beryozkina, N. G.; Volkov, I. O.

    2009-01-01

    This report presents an analysis of the effects caused by space environmental factors (SEF) and the International Space Station's (ISS) outer environment on operational parameters of the outer surface materials of the ISS Russian Segment (RS). The tests were performed using detachable container cassettes (CKK) that serve as a part of the ISS RS contamination control system.

  12. 21 CFR 892.1870 - Radiographic film/cassette changer programmer.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Radiographic film/cassette changer programmer. 892.1870 Section 892.1870 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic...

  13. 21 CFR 892.1880 - Wall-mounted radiographic cassette holder.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Wall-mounted radiographic cassette holder. 892.1880 Section 892.1880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880...

  14. 21 CFR 892.1880 - Wall-mounted radiographic cassette holder.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Wall-mounted radiographic cassette holder. 892.1880 Section 892.1880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880...

  15. 21 CFR 892.1870 - Radiographic film/cassette changer programmer.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Radiographic film/cassette changer programmer. 892.1870 Section 892.1870 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic...

  16. 21 CFR 892.1870 - Radiographic film/cassette changer programmer.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Radiographic film/cassette changer programmer. 892.1870 Section 892.1870 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic...

  17. 21 CFR 892.1880 - Wall-mounted radiographic cassette holder.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Wall-mounted radiographic cassette holder. 892.1880 Section 892.1880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880...

  18. 21 CFR 892.1880 - Wall-mounted radiographic cassette holder.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Wall-mounted radiographic cassette holder. 892.1880 Section 892.1880 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880...

  19. 21 CFR 892.1870 - Radiographic film/cassette changer programmer.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Radiographic film/cassette changer programmer. 892.1870 Section 892.1870 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1870 Radiographic...

  20. Utilizing ARC EMCS Seedling Cassettes as Highly Versatile Miniature Growth Chambers for Model Organism Experiments

    NASA Technical Reports Server (NTRS)

    Freeman, John L.; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David; Reinsch, S.; DeSimone, Julia C.; Myers, Zachary A.

    2014-01-01

    The aim of our ground testing was to demonstrate the capability of safely putting specific model organisms into dehydrated stasis, and to later rehydrate and successfully grow them inside flight proven ARC EMCS seedling cassettes. The ARC EMCS seedling cassettes were originally developed to support seedling growth during space flight. The seeds are attached to a solid substrate, launched dry, and then rehydrated in a small volume of media on orbit to initiate the experiment. We hypothesized that the same seedling cassettes should be capable of acting as culture chambers for a wide range of organisms with minimal or no modification. The ability to safely preserve live organisms in a dehydrated state allows for on orbit experiments to be conducted at the best time for crew operations and more importantly provides a tightly controlled physiologically relevant growth experiment with specific environmental parameters. Thus, we performed a series of ground tests that involved growing the organisms, preparing them for dehydration on gridded Polyether Sulfone (PES) membranes, dry storage at ambient temperatures for varying periods of time, followed by rehydration. Inside the culture cassettes, the PES membranes were mounted above blotters containing dehydrated growth media. These were mounted on stainless steel bases and sealed with plastic covers that have permeable membrane covered ports for gas exchange. The results showed we were able to demonstrate acceptable normal growth of C.elegans (nematodes), E.coli (bacteria), S.cerevisiae (yeast), Polytrichum (moss) spores and protonemata, C.thalictroides (fern), D.discoideum (amoeba), and H.dujardini (tardigrades). All organisms showed acceptable growth and rehydration in both petri dishes and culture cassettes initially, and after various time lengths of dehydration. At the end of on orbit ISS European Modular Cultivation System experiments the cassettes could be frozen at ultra-low temperatures, refrigerated, or chemically

  1. Evaluating the accuracy of technicians and pharmacists in checking unit dose medication cassettes.

    PubMed

    Ambrose, Peter J; Saya, Frank G; Lovett, Larry T; Tan, Sandy; Adams, Dale W; Shane, Rita

    2002-06-15

    The accuracy rates of board-registered pharmacy technicians and pharmacists in checking unit dose medication cassettes in the inpatient setting at two separate institutions were examined. Cedars-Sinai Medical Center and Long Beach Memorial Medical Center, both in Los Angeles county, petitioned the California State Board of Pharmacy to approve a waiver of the California Code of Regulations to conduct an experimental program to compare the accuracy of unit dose medication cassettes checked by pharmacists with that of cassettes checked by trained, certified pharmacy technicians. The study consisted of three parts: assessing pharmacist baseline checking accuracy (Phase I), developing a technician-training program and certifying technicians who completed the didactic and practical training (Phase II), and evaluating the accuracy of certified technicians checking unit dose medication cassettes as a daily function (Phase III). Twenty-nine pharmacists and 41 technicians (3 of whom were pharmacy interns) participated in the study. Of the technicians, all 41 successfully completed the didactic and practical training, 39 successfully completed the audits and became certified checkers, and 2 (including 1 of the interns) did not complete the certification audits because they were reassigned to another work area or had resigned. In Phase II, the observed accuracy rate and its lower confidence limit exceeded the predetermined minimum requirement of 99.8% for a certified checker. The mean accuracy rates for technicians were identical at the two institutions (p = 1.0). The difference in mean accuracy rates between pharmacists (99.52%; 95% confidence interval [CI] 99.44-99.58%) and technicians, (99.89%; 95% CI 99.87-99.90%) was significant (p < 0.0001). Inpatient technicians who had been trained and certified in a closely supervised program that incorporated quality assurance mechanisms could safely and accurately check unit dose medication cassettes filled by other technicians

  2. Use of cassette dosing to enhance the throughput of rat brain microdialysis studies.

    PubMed

    Deshmukh, Gauri; Sun, Kefeng; Liederer, Bianca M; Ding, Xiao; Liu, Xingrong

    2015-07-01

    This study was designed to increase the throughput of rat brain microdialysis studies by administration of compounds as a cassette as opposed to discrete study. Eight compounds (carbamazepine, citalopram, desmethylclozapine, diphenhydramine, gabapentin, metoclopramide, naltrexone, and stavudine) were selected and administered as an intravenous bolus dose at 0.5-3.3 mg/kg each followed by an intravenous infusion at 1 mg/kg per hour for 6 hours in rats in a cassette or discrete dosing. The dialysate, plasma, brain, and cerebrospinal fluid were collected and analyzed using liquid chromatography-tandem mass spectrometry. The microdialysis probe recovery was determined by an in vitro gain method. The recovery between the cassette and discrete dosing was similar, with an average of 1.0 ± 0.10-fold difference. The stavudine interstitial fluid (ISF) concentration, as measured by brain microdialysis, was below the low limit of quantitation and was excluded from the analyses. The ratios of ISF concentration to unbound plasma concentration were within 2-fold for six of the remaining seven compounds, with an average of 0.92 ± 0.51-fold difference between the cassette and discrete methods. The ratios of ISF concentration to unbound brain concentration, as measured by the brain homogenate method, were also similar, with a 1.1 ± 0.7-fold difference. In addition, the ratios of ISF to cerebrospinal fluid concentrations were similar, with a 1.5 ± 0.6-fold difference. The results from this study support the use of a cassette dosing approach to enhance the throughput of rat brain microdialysis studies in drug discovery. PMID:25943358

  3. Ground Testing of the EMCS Seed Cassette for Biocompatibility with the Cellular Slime Mold, Dictyostelium Discoideum

    NASA Technical Reports Server (NTRS)

    Hanely, Julia C.; Reinsch, Sigrid; Myers, Zachary A.; Freeman, John; Steele, Marianne K.; Sun, Gwo-Shing; Heathcote, David G.

    2014-01-01

    The European Modular Cultivation System, EMCS, was developed by ESA for plant experiments. To expand the use of flight verified hardware for various model organisms, we performed ground experiments to determine whether ARC EMCS Seed Cassettes could be adapted for use with cellular slime mold for future space flight experiments. Dictyostelium is a cellular slime mold that can exist both as a single-celled independent organism and as a part of a multicellular colony which functions as a unit (pseudoplasmodium). Under certain stress conditions, individual amoebae will aggregate to form multicellular structures. Developmental pathways are very similar to those found in Eukaryotic organisms, making this a uniquely interesting organism for use in genetic studies. Dictyostelium has been used as a genetic model organism for prior space flight experiments. Due to the formation of spores that are resistant to unfavorable conditions such as desiccation, Dictyostelium is also a good candidate for use in the EMCS Seed Cassettes. The growth substratum in the cassettes is a gridded polyether sulfone (PES) membrane. A blotter beneath the PES membranes contains dried growth medium. The goals of this study were to (1) verify that Dictyostelium are capable of normal growth and development on PES membranes, (2) develop a method for dehydration of Dictyostelium spores with successful recovery and development after rehydration, and (3) successful mock rehydration experiments in cassettes. Our results show normal developmental progression in two strains of Dictyostelium discoideum on PES membranes with a bacterial food source. We have successfully performed a mock rehydration of spores with developmental progression from aggregation to slug formation, and production of morphologically normal spores within 9 days of rehydration. Our results indicate that experiments on the ISS using the slime mold, Dictyostelium discoideum could potentially be performed in the flight verified hardware of

  4. An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids

    PubMed Central

    Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R.; Malamud, Daniel; Corstjens, Paul L. A. M.

    2010-01-01

    A self-contained, integrated, disposable, sample-to-answer, polycarbonate microfluidic cassette for nucleic acid—based detection of pathogens at the point of care was designed, constructed, and tested. The cassette comprises on-chip sample lysis, nucleic acid isolation, enzymatic amplification (polymerase chain reaction and, when needed, reverse transcription), amplicon labeling, and detection. On-chip pouches and valves facilitate fluid flow control. All the liquids and dry reagents needed for the various reactions are pre-stored in the cassette. The liquid reagents are stored in flexible pouches formed on the chip surface. Dry (RT-)PCR reagents are pre-stored in the thermal cycling, reaction chamber. The process operations include sample introduction; lysis of cells and viruses; solid-phase extraction, concentration, and purification of nucleic acids from the lysate; elution of the nucleic acids into a thermal cycling chamber and mixing with pre-stored (RT-)PCR dry reagents; thermal cycling; and detection. The PCR amplicons are labeled with digoxigenin and biotin and transmitted onto a lateral flow strip, where the target analytes bind to a test line consisting of immobilized avidin-D. The immobilized nucleic acids are labeled with up-converting phosphor (UCP) reporter particles. The operation of the cassette is automatically controlled by an analyzer that provides pouch and valve actuation with electrical motors and heating for the thermal cycling. The functionality of the device is demonstrated by detecting the presence of bacterial B.Cereus, viral armored RNA HIV, and HIV I virus in saliva samples. The cassette and actuator described here can be used to detect other diseases as well as the presence of bacterial and viral pathogens in the water supply and other fluids. PMID:20401537

  5. Rising Cellular Immune Response after Injection of pVax/iutA: A Genetic DNA Cassette as Candidate Vaccine against Urinary Tract Infection

    PubMed Central

    BAKHTIARI, Ronak; AHMADIAN, Shahin; FALLAH MEHRABADI, Jalil

    2016-01-01

    Background: Uropathogenic Escherichia coli (UPEC) are major bacterial agent of Urinary Tract Infection (UTI). This infection is more prevalent among women because approximately half of all women will experience a UTI in their life-time and near a quarter of them will have a recurrent infection within 6–12 months. IutA protein has a major role during UPEC pathogenesis and consequently infection. Therefore, the aim of current study was assessment of IutA protein roles as a potential candidate antigen based for vaccine designing. Methods: This survey was conducted during 2014–2015 at the University of Tehran, Iran. Chromosomal DNA extracted from E. coli 35218 and iutA gene amplified by PCR. The amplicon cloned to pVax.1 eukaryotic expression vector and recombinant vector confirmed by sequencing. The iutA gene expression in genetic cassette of pVax/iutA was evaluated in COS7 cell line by RT-PCR. Then, injected to mouse model, which divided to three groups: injected with pVax vector, PBS and pVax/iutA cassette respectively in two stages (d 1 and 14). One week after the second injection, bleeding from immunized mouse was performed and IFN-gamma was measured. Results: The mice immunized with pVax/iutA showed increased interferon-γ responses significantly higher than two non-immunized groups (P<0.05). Conclusion: Cellular immune response has a main protective role against UTI. Raising this kind of immune response is important to preventing of recurrent infection. Moreover, the current DNA cassette will be valuable for more trying to prepare a new vaccine against UTI. PMID:27516995

  6. "Gene-swap knock-in" cassette in mice to study allelic differences in human genes.

    PubMed

    Nebert, D W; Dalton, T P; Stuart, G W; Carvan, M J

    2000-01-01

    Genetic differences in environmental toxicity and cancer susceptibility among individuals in a human population often reflect polymorphisms in the genes encoding drug-metabolizing enzymes (DMEs), drug transporters, and receptors that control DME levels. This field of study is called "ecogenetics", and a subset of this field--concerning genetic variability in response to drugs--is termed "pharmacogenetics". Although human-mouse differences might be 3- to perhaps 10-fold, human interindividual differences can be as great as 20-fold or more than 40-fold. It would be helpful, therefore, to study toxicokinetics/pharmacokinetics of particular environmental agents and drugs in mice containing these "high-" and "low-extreme" human alleles. We hope to use transgenic "knock-in" technology in order to insert human alleles in place of the orthologous mouse gene. However, the knock-in of each gene has normally been a separate event requiring the following: (a) construction of the targeting vector, (b) transfection into embryonic stem (ES) cells, (c) generation of a targeted mouse having germline transmission of the construct, and (d) backcross breeding of the knock-in mouse (at least 6-8 times) to produce a suitable genetically homogeneous background (i.e., to decrease "experimental noise"). These experiments require 1 1/2 to 2 years to complete, making this very powerful technology inefficient for routine applications. If, on the other hand, the initial knock-in targeting vector might include sequences that would allow the knocked-in gene to be exchanged (quickly and repeatedly) for one new allele after another, then testing distinctly different human polymorphic alleles in transgenic mice could be accomplished in a few months instead of several years. This "gene-swapping" technique will soon be done by zygotic injection of a "human allele cassette" into the sperm or fertilized ovum of the parental knock-in mouse inbred strain or by the cloning of whole mice from cumulus

  7. Diversity of gene cassettes and the abundance of the class 1 integron-integrase gene in sediment polluted by metals.

    PubMed

    Oliveira-Pinto, Clarisse; Costa, Patrícia S; Reis, Mariana P; Chartone-Souza, Edmar; Nascimento, Andréa M A

    2016-05-01

    The integron-gene cassette system has typically been associated with antibiotic-resistant pathogens. However, the diversity of gene cassettes and the abundance of class 1 integrons outside of the clinical context are not fully explored. Primers targeting the conserved segments of attC recombination sites were used to amplify gene cassettes from the sediment of the Mina stream, which exhibited a higher degree of stress to metal pollution in the dry season than the rainy season. Of the 143 total analyzed sequences, 101 had no matches to proteins in the database, where cassette open reading frames could be identified by homology with database entries. There was a predominance of sequences encoding essential cellular functions. Each season that was sampled yielded a specific pool of gene cassettes. Real-time PCR revealed that 8.5 and 41.6 % of bacterial cells potentially harbored a class 1 integron in the rainy and dry seasons, respectively. In summary, our findings demonstrate that most of the gene cassettes have no ascribable function and, apparently, historically metal-contaminated sediment favors the maintenance of bacteria containing the intI1 gene. Thus, the diversity of gene cassettes is far from being fully explored deserving further attention. PMID:26961777

  8. Light without substrate amendment: the bacterial luciferase gene cassette as a mammalian bioreporter

    NASA Astrophysics Data System (ADS)

    Close, Dan M.; Xu, Tingting; Smartt, Abby E.; Jegier, Pat; Ripp, Steven A.; Sayler, Gary S.

    2011-06-01

    Bioluminescent production represents a facile method for bioreporter detection in mammalian tissues. The lack of endogenous bioluminescent reactions in these tissues allows for high signal to noise ratios even at low signal strength compared to fluorescent signal detection. While the luciferase enzymes commonly employed for bioluminescent detection are those from class Insecta (firefly and click beetle luciferases), these are handicapped in that they require concurrent administration of a luciferin compound to elicit a bioluminescent signal. The bacterial luciferase (lux) gene cassette offers the advantages common to other bioluminescent proteins, but is simultaneously capable of synthesizing its own luciferin substrates using endogenously available cellular compounds. The longstanding shortcoming of the lux cassette has been its recalcitrance to function in the mammalian cellular environment. This paper will present an overview of the work completed to date to overcome this limitation and provide examples of mammalian lux-based bioreporter technologies that could provide the framework for advanced, biomedically relevant real-time sensor development.

  9. Identification of a Novel Cassette Array in Integronbearing Helicobacter Pylori Strains Isolated from Iranian Patients.

    PubMed

    Goudarzi, Mehdi; Seyedjavadi, Sima Sadat; Fazeli, Maryam; Roshani, Maryam; Azad, Mehdi; Heidary, Mohsen; Navidinia, Masoumeh; Goudarzi, Hossein

    2016-01-01

    Helicobacter pylori as the second most common cause of gastric cancer in the world infects approximately half of the developed countries population and 80% of the population living in developing countries. Integrons as genetic reservoirs play major roles in dissemination of antimicrobial resistance genes. To the best of our knowledge, this is the first study to report carriage of class 1 and 2 integrons and associated gene cassettes in H. pylori isolates from Iran. This crosssectional study was conducted in Tehran among 110 patients with H. pylori infection. Antimicrobial susceptibility testing (AST) for H. pylori strains were assessed by the micro broth dilution method. Class 1 and 2 integrons were detected using PCR. In order to determine gene cassettes, amplified fragments were subjected to DNA sequencing of both amplicon strands. The prevalence of resistance to clarithromycin, metronidazole, clarithromycin, tetracycline, amoxicillin, rifampin, and levofloxacin were 68.2% (n=75), 25.5% (n=28), 24.5% (n=27), 19.1% (n=21), 18.2% (n=20) and 16.4% (n=18), respectively. Frequency of multidrug resistance among H. pylori isolates was 12.7%. Class 2 integron was detected in 50 (45.5%) and class 1 integron in 10 (9.1%) H. pylori isolates. The most predominant gene cassette arrays in class 2 integron bearing H. pylori were included sateraaadA1, dfrA1sat2aadA1, blaoxa2 and, aadB whereas common gene cassette arrays in class 1 integron were aadBaadA1cmlA6, aacA4, blaoxa2, and catB3. The high frequency of class 2 integron and multidrug resistance in the present study should be considered as a warning for clinicians that continuous surveillance is necessary to prevent the further spread of resistant isolates. PMID:27509968

  10. Linear friction weld process monitoring of fixture cassette deformations using empirical mode decomposition

    NASA Astrophysics Data System (ADS)

    Bakker, O. J.; Gibson, C.; Wilson, P.; Lohse, N.; Popov, A. A.

    2015-10-01

    Due to its inherent advantages, linear friction welding is a solid-state joining process of increasing importance to the aerospace, automotive, medical and power generation equipment industries. Tangential oscillations and forge stroke during the burn-off phase of the joining process introduce essential dynamic forces, which can also be detrimental to the welding process. Since burn-off is a critical phase in the manufacturing stage, process monitoring is fundamental for quality and stability control purposes. This study aims to improve workholding stability through the analysis of fixture cassette deformations. Methods and procedures for process monitoring are developed and implemented in a fail-or-pass assessment system for fixture cassette deformations during the burn-off phase. Additionally, the de-noised signals are compared to results from previous production runs. The observed deformations as a consequence of the forces acting on the fixture cassette are measured directly during the welding process. Data on the linear friction-welding machine are acquired and de-noised using empirical mode decomposition, before the burn-off phase is extracted. This approach enables a direct, objective comparison of the signal features with trends from previous successful welds. The capacity of the whole process monitoring system is validated and demonstrated through the analysis of a large number of signals obtained from welding experiments.

  11. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research. PMID:19255730

  12. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy

    PubMed Central

    Powell, Sara Kathleen; Rivera-Soto, Ricardo; Gray, Steven James

    2015-01-01

    Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy. PMID:25636961

  13. Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee (Apis mellifera)

    PubMed Central

    Schulte, Christina; Theilenberg, Eva; Müller-Borg, Marion; Gempe, Tanja; Beye, Martin

    2014-01-01

    Honeybees (Apis mellifera), which are important pollinators of plants, display remarkable individual behaviors that collectively contribute to the organization of a complex society. Advances in dissecting the complex processes of honeybee behavior have been limited in the recent past due to a lack of genetic manipulation tools. These tools are difficult to apply in honeybees because the unit of reproduction is the colony, and many interesting phenotypes are developmentally specified at later stages. Here, we report highly efficient integration and expression of piggyBac-derived cassettes in the honeybee. We demonstrate that 27 and 20% of queens stably transmitted two different expression cassettes to their offspring, which is a 6- to 30-fold increase in efficiency compared with those generally reported in other insect species. This high efficiency implies that an average beekeeping facility with a limited number of colonies can apply this tool. We demonstrated that the cassette stably and efficiently expressed marker genes in progeny under either an artificial or an endogenous promoter. This evidence of efficient expression encourages the use of this system to inhibit gene functions through RNAi in specific tissues and developmental stages by using various promoters. We also showed that the transgenic marker could be used to select transgenic offspring to be employed to facilitate the building of transgenic colonies via the haploid males. We present here the first to our knowledge genetic engineering tool that will efficiently allow for the systematic detection and better understanding of processes underlying the biology of honeybees. PMID:24821811

  14. Modulation of Gene Expression by Polymer Nanocapsule Delivery of DNA Cassettes Encoding Small RNAs.

    PubMed

    Yan, Ming; Wen, Jing; Liang, Min; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2015-01-01

    Small RNAs, including siRNAs, gRNAs and miRNAs, modulate gene expression and serve as potential therapies for human diseases. Delivery to target cells remains the fundamental limitation for use of these RNAs in humans. To address this challenge, we have developed a nanocapsule delivery technology that encapsulates small DNA molecules encoding RNAs into a small (30 nm) polymer nanocapsule. For proof of concept, we transduced DNA expression cassettes for three small RNAs. In one application, the DNA cassette encodes an shRNA transcriptional unit that downregulates CCR5 and protects from HIV-1 infection. The DNA cassette nanocapsules were further engineered for timed release of the DNA cargo for prolonged knockdown of CCR5. Secondly, the nanocapsules provide an efficient means for delivery of gRNAs in the CRISPR/Cas9 system to mutate integrated HIV-1. Finally, delivery of microRNA-125b to mobilized human CD34+ cells enhances survival and expansion of the CD34+ cells in culture. PMID:26035832

  15. Enhanced performance of methamphetamine lateral flow cassettes using an electronic lateral flow reader

    PubMed Central

    Smith, Jerome P.; Sammons, Deborah L.; Robertson, Shirley A.; Snawder, John E.

    2015-01-01

    Surface contamination from methamphetamine in meth labs continues to be a problem. We had previously developed a lateral flow assay cassette for field detection of methamphetamine contamination that is commercially available and has been used by a number of groups to assess contamination. This cassette uses the complete disappearance of the test line as an end point for detection of 50 ng/100 cm2 of methamphetamine contamination for surface sampling with cotton swabs. In the present study, we further evaluate the response of the cassettes using an electronic lateral flow reader to measure the intensities of the test and control lines. The cassettes were capable of detecting 0.25 ng/ml for calibration solutions. For 100 cm2 ceramic tiles that were spiked with methamphetamine and wiped with cotton tipped wooden swabs wetted in assay/sampling buffer, 1 ng/tile was detected using the reader. Semi-quantitative results can be produced over the range 0–10 ng/ml for calibration solutions and 0–25 ng/tile for spiked tiles using either a 4-parameter logistic fit of test line intensity versus concentration or spiked mass or the ratio of the control line to the test line intensity fit to concentration or spiked mass. Recovery from the tiles was determined to be about 30% using the fitted curves. Comparison of the control line to the test line was also examined as a possible visual detection end point and it was found that the control line became more intense than the test line at 0.5 to 1 ng/ml for calibration solutions or 1 to 2 ng/tile for spiked tiles. Thus the lateral flow cassettes for methamphetamine have the potential to produce more sensitive semi-quantitative results if an electronic lateral flow reader is used and can be more sensitive for detection if the comparison of the control line to the test line is used as the visual end point. PMID:25379615

  16. Ubiquitin promoter-terminator cassette promotes genetically stable expression of the taste-modifying protein miraculin in transgenic lettuce.

    PubMed

    Hirai, Tadayoshi; Shohael, Abdullah Mohammad; Kim, You-Wang; Yano, Megumu; Ezura, Hiroshi

    2011-12-01

    Lettuce is a commercially important leafy vegetable that is cultivated worldwide, and it is also a target crop for plant factories. In this study, lettuce was selected as an alternative platform for recombinant miraculin production because of its fast growth, agronomic value, and wide availability. The taste-modifying protein miraculin is a glycoprotein extracted from the red berries of the West African native shrub Richadella dulcifica. Because of its limited natural availability, many attempts have been made to produce this protein in suitable alternative hosts. We produced transgenic lettuce with miraculin gene driven either by the ubiquitin promoter/terminator cassette from lettuce or a 35S promoter/nos terminator cassette. Miraculin gene expression and miraculin accumulation in both cassettes were compared by quantitative real-time PCR analysis, Western blotting, and enzyme-linked immunosorbent assay. The expression level of the miraculin gene and protein in transgenic lettuce was higher and more genetically stable in the ubiquitin promoter/terminator cassette than in the 35S promoter/nos terminator cassette. These results demonstrated that the ubiquitin promoter/terminator cassette is an efficient platform for the genetically stable expression of the miraculin protein in lettuce and hence this platform is of benefit for recombinant miraculin production on a commercial scale. PMID:21830129

  17. Ambient Influenza and Avian Influenza Virus during Dust Storm Days and Background Days

    PubMed Central

    Chen, Pei-Shih; Tsai, Feng Ta; Lin, Chien Kun; Yang, Chun-Yuh; Chan, Chang-Chuan; Young, Chea-Yuan; Lee, Chien-Hung

    2010-01-01

    Background The spread of influenza and highly pathogenic avian influenza (H5N1) presents a significant threat to human health. Avian influenza outbreaks in downwind areas of Asian dust storms (ADS) suggest that viruses might be transported by dust storms. Objectives We developed a technique to measure ambient influenza and avian influenza viruses. We then used this technique to measure concentrations of these viruses on ADS days and background days, and to assess the relationships between ambient influenza and avian influenza viruses, and air pollutants. Methods A high-volume air sampler was used in parallel with a filter cassette to evaluate spiked samples and unspiked samples. Then, air samples were monitored during ADS seasons using a filter cassette coupled with a real-time quantitative polymerase chain reaction (qPCR) assay. Air samples were monitored during ADS season (1 January to 31 May 2006). Results We successfully quantified ambient influenza virus using the filtration/real-time qPCR method during ADS days and background days. To our knowledge, this is the first report describing the concentration of influenza virus in ambient air. In both the spiked and unspiked samples, the concentration of influenza virus sampled using the filter cassette was higher than that using the high-volume sampler. The concentration of ambient influenza A virus was significantly higher during the ADS days than during the background days. Conclusions Our data imply the possibility of long-range transport of influenza virus. PMID:20435545

  18. A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis.

    PubMed

    Lin, Zhiwei; Deng, Bin; Jiao, Zhihua; Wu, Bingbing; Xu, Xin; Yu, Dongyou; Li, Weifen

    2013-11-01

    There are some drawbacks for MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodology and non-strictly controlled expression system. In our study, the modifications on mazF-cassette are carried out, such as using mini Zeocin resistance gene as positive-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodology, without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Besides, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes. PMID:23911571

  19. Evaluation of retroviral vector design in defined chromosomal loci by Flp-mediated cassette replacement.

    PubMed

    Verhoeyen, E; Hauser, H; Wirth, D

    2001-05-20

    Successful retroviral vector construction is still empirical. Test systems for vector efficiency are based on statistical comparison of numerous infectants with single proviral integrates, since their expression depends on the chromosomal surroundings. More reliable data would be obtained if different vector constructs were studied in an identical chromosomal context. Here, we demonstrate the use of a new method, in which chromosomal sites are provirally tagged in such a way that they can be targeted with other expression cassettes. The original tagging integrate is replaced in one step by the targeting element. This permits a reliable comparison of different retroviral vector configurations, eliminating the influence of neighboring chromosomal elements. We compared different retroviral vector types for coexpression of two genes: a vector containing an internal promoter and a vector with an internal ribosome entry site (IRES) element. In contrast to bicistronic retroviral vectors, dual-promoter proviruses exhibited rapid inactivation of the long terminal repeat (LTR)-driven gene expression. Targeted exchange of the dual-promoter provirus with a bicistronic retroviral cassette resulted in gain of expression stability. The reverse experiment confirmed this promoter interaction phenomenon since initial expression stability from a single-promoter bicistronic provirus was lost by targeted exchange with a dual-promoter cassette. In addition, targeting exchange of the dual-promoter provirus, replacing the LTR with an artificial (Tet) promoter restored expression stability. These observations, valid for various integration sites, prove the strong interaction between the LTR and the internal promoter. Our results have implications for retroviral vector design and suggest that retroviral coexpression of two genes is more predictable in the bicistronic configuration. PMID:11387058

  20. Novel cassette array in a class 1 integron in clinical isolates of Acinetobacter baumannii from central Iran.

    PubMed

    Japoni-Nejad, Alireza; Farshad, Shohreh; van Belkum, Alex; Ghaznavi-Rad, Ehsanollah

    2013-12-01

    Antibiotic resistance in Acinetobacter baumannii is a major problem in the hospital and outbreaks caused by this organism have been reported frequently. The present study aimed at determining the antibiotic susceptibility patterns, the prevalence of different classes of integrons and the characterization of integron class 1 gene cassettes in Iranian A. baumannii isolates. A total of 63 non-duplicate A. baumannii isolates were collected from clinical and environmental specimens in the Vali-Asr hospital in the central province of Iran (March to September, 2011). The antimicrobial susceptibility for 15 antibiotics which are used conventionally was determined by disk diffusion. The presence of different integron classes was investigated by PCR and the size of gene cassettes in class 1 integrons was then determined by PCR as well. Moreover, integron cassette arrays of isolates were delineated by RFLP and sequencing amplicons with different lengths. Of 63 isolates 62 (98.4%) carried a class 1 integron. The prevalence of IntI2 was 15.9% and the length of the amplicons ranged from 500 bp to 3 kb. Sequencing of integrons of class 1 revealed the presence of many resistance genes (aadA, aacA, aacC, dfrA, bla(GES) and bla(IMP)). We identified a completely new gene cassette which contained aacA7-qacF-aadA5-bla(IMP), this cassette has not been reported previously in A. baumannii. PMID:24161711

  1. Oral Spirochetes Implicated in Dental Diseases Are Widespread in Normal Human Subjects and Carry Extremely Diverse Integron Gene Cassettes

    PubMed Central

    Wu, Yu-Wei; Rho, Mina; Doak, Thomas G.

    2012-01-01

    The NIH Human Microbiome Project (HMP) has produced several hundred metagenomic data sets, allowing studies of the many functional elements in human-associated microbial communities. Here, we survey the distribution of oral spirochetes implicated in dental diseases in normal human individuals, using recombination sites associated with the chromosomal integron in Treponema genomes, taking advantage of the multiple copies of the integron recombination sites (repeats) in the genomes, and using a targeted assembly approach that we have developed. We find that integron-containing Treponema species are present in ∼80% of the normal human subjects included in the HMP. Further, we are able to de novo assemble the integron gene cassettes using our constrained assembly approach, which employs a unique application of the de Bruijn graph assembly information; most of these cassette genes were not assembled in whole-metagenome assemblies and could not be identified by mapping sequencing reads onto the known reference Treponema genomes due to the dynamic nature of integron gene cassettes. Our study significantly enriches the gene pool known to be carried by Treponema chromosomal integrons, totaling 826 (598 97% nonredundant) genes. We characterize the functions of these gene cassettes: many of these genes have unknown functions. The integron gene cassette arrays found in the human microbiome are extraordinarily dynamic, with different microbial communities sharing only a small number of common genes. PMID:22635997

  2. Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

    PubMed

    Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

    2016-02-01

    Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence. PMID:26443821

  3. A large family of Dscam genes with tandemly arrayed 5' cassettes in Chelicerata.

    PubMed

    Yue, Yuan; Meng, Yijun; Ma, Hongru; Hou, Shouqing; Cao, Guozheng; Hong, Weiling; Shi, Yang; Guo, Pengjuan; Liu, Baoping; Shi, Feng; Yang, Yun; Jin, Yongfeng

    2016-01-01

    Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7-8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5' variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity. PMID:27080167

  4. Fully Automated Data Collection Using PAM and the Development of PAM/SPACE Reversible Cassettes

    NASA Astrophysics Data System (ADS)

    Hiraki, Masahiko; Watanabe, Shokei; Chavas, Leonard M. G.; Yamada, Yusuke; Matsugaki, Naohiro; Igarashi, Noriyuki; Wakatsuki, Soichi; Fujihashi, Masahiro; Miki, Kunio; Baba, Seiki; Ueno, Go; Yamamoto, Masaki; Suzuki, Mamoru; Nakagawa, Atsushi; Watanabe, Nobuhisa; Tanaka, Isao

    2010-06-01

    To remotely control and automatically collect data in high-throughput X-ray data collection experiments, the Structural Biology Research Center at the Photon Factory (PF) developed and installed sample exchange robots PAM (PF Automated Mounting system) at PF macromolecular crystallography beamlines; BL-5A, BL-17A, AR-NW12A and AR-NE3A. We developed and installed software that manages the flow of the automated X-ray experiments; sample exchanges, loop-centering and X-ray diffraction data collection. The fully automated data collection function has been available since February 2009. To identify sample cassettes, PAM employs a two-dimensional bar code reader. New beamlines, BL-1A at the Photon Factory and BL32XU at SPring-8, are currently under construction as part of Targeted Proteins Research Program (TPRP) by the Ministry of Education, Culture, Sports, Science and Technology of Japan. However, different robots, PAM and SPACE (SPring-8 Precise Automatic Cryo-sample Exchanger), will be installed at BL-1A and BL32XU, respectively. For the convenience of the users of both facilities, pins and cassettes for PAM and SPACE are developed as part of the TPRP.

  5. A large family of Dscam genes with tandemly arrayed 5′ cassettes in Chelicerata

    PubMed Central

    Yue, Yuan; Meng, Yijun; Ma, Hongru; Hou, Shouqing; Cao, Guozheng; Hong, Weiling; Shi, Yang; Guo, Pengjuan; Liu, Baoping; Shi, Feng; Yang, Yun; Jin, Yongfeng

    2016-01-01

    Drosophila Dscam1 (Down Syndrome Cell Adhesion Molecules) and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the extraordinary isoform diversity from a single genomic locus. Dscam1 encodes 38,016 distinct isoforms via mutually exclusive splicing in D. melanogaster, while the vertebrate clustered Pcdhs utilize alternative promoters to generate isoform diversity. Here we reveal a shortened Dscam gene family with tandemly arrayed 5′ cassettes in Chelicerata. These cassette repeats generally comprise two or four exons, corresponding to variable Immunoglobulin 7 (Ig7) or Ig7–8 domains of Drosophila Dscam1. Furthermore, extraordinary isoform diversity has been generated through a combination of alternating promoter and alternative splicing. These sDscams have a high sequence similarity with Drosophila Dscam1, and share striking organizational resemblance to the 5′ variable regions of vertebrate clustered Pcdhs. Hence, our findings have important implications for understanding the functional similarities between Drosophila Dscam1 and vertebrate Pcdhs, and may provide further mechanistic insights into the regulation of isoform diversity. PMID:27080167

  6. Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.

    PubMed

    Siehl, Daniel L; Tao, Yumin; Albert, Henrik; Dong, Yuxia; Heckert, Matthew; Madrigal, Alfredo; Lincoln-Cabatu, Brishette; Lu, Jian; Fenwick, Tamara; Bermudez, Ericka; Sandoval, Marian; Horn, Caroline; Green, Jerry M; Hale, Theresa; Pagano, Peggy; Clark, Jenna; Udranszky, Ingrid A; Rizzo, Nancy; Bourett, Timothy; Howard, Richard J; Johnson, David H; Vogt, Mark; Akinsola, Goke; Castle, Linda A

    2014-11-01

    With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione. PMID:25192697

  7. Use of cassette-electrode microbial fuel cell for wastewater treatment.

    PubMed

    Miyahara, Morio; Hashimoto, Kazuhito; Watanabe, Kazuya

    2013-02-01

    Cassette-electrode microbial fuel cells (CE-MFCs) have been developed for the conversion of biomass wastes into electric energy. The present study modified CE-MFC for its application to wastewater treatment and examined its utility in a long-term (240 days) experiment to treat a synthetic wastewater, containing starch, yeast extract, peptone, plant oil, and a detergent (approximately 500 mg of total chemical oxygen demand [COD] per liter). A test MFC reactor (1 l in capacity) was equipped with 10 cassette electrodes with total anode and cathode projection areas of 1440 cm(2), and the operation was initiated by inoculating with rice paddy-field soil. It was demonstrated that CE-MFC achieved COD removal rates of 80% at hydraulic-retention times of 6 h or greater, and electricity was generated at a maximum power density of 150 mW m(-2) and Coulombic efficiency of 20%. Microbial communities established on anodes of CEs were analyzed by pyrosequencing of PCR-amplified 16S rRNA gene fragments, showing that Geobacter, Clostridium, and Geothrix were abundantly detected in anode biofilms. These results demonstrate the utility of CE-MFC for wastewater treatment, in which Geobacter and Geothrix would be involved in the electricity generation. PMID:23041137

  8. Sewage treatment plant serves as a hot-spot reservoir of integrons and gene cassettes.

    PubMed

    Ma, Liping; Zhang, Xu-Xiang; Zhao, Fuzheng; Wu, Bing; Cheng, Shupei; Yang, Liuyan

    2013-04-01

    This study investigated the occurrence and abundance of class 1 integrons and related antibiotic resistance genes (ARGs) in a sewage treatment plant (STP) of China. Totally, 189 bacterial strains were isolated from influent, activated sludge and effluent, and 40 isolates contained the integons with a complete structure. The intl1-carrying isolates were found to harbor two types of gene cassettes: dfr17-aadA5 and aadA2, conferring resistances to trimethoprim and streptomycin, which were further confirmed by antimicrobial susceptibility analysis. Many other gene cassettes were carried on integron, including qnrVC1, catB-8-blaoxa-10-aadA1-aac(6'), aadB-aacA29b, aadA2, aac(6')-1b, aadA6 and aadA12, which were detected using DNA cloning. Quantitative real time PCR showed that over 99% of the integrons was eliminated in activated sludge process, but average copy number of integrons in given bacterial cells was increased by 56% in treated sewage. Besides integrons, other mobile gene elements (MGEs) were present in the STP with high abundance. MGEs and the associated ARGs may be wide-spread in STPs, which constitute a potential hot spot for selection of antibiotic resistant bacteria and horizontal transfer of ARGs. PMID:24620610

  9. Identification and analysis of integrons and cassette arrays in bacterial genomes.

    PubMed

    Cury, Jean; Jové, Thomas; Touchon, Marie; Néron, Bertrand; Rocha, Eduardo Pc

    2016-06-01

    Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program - IntegronFinder - to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome. PMID:27130947

  10. Identification and analysis of integrons and cassette arrays in bacterial genomes

    PubMed Central

    Cury, Jean; Jové, Thomas; Touchon, Marie; Néron, Bertrand; Rocha, Eduardo PC

    2016-01-01

    Integrons recombine gene arrays and favor the spread of antibiotic resistance. Their broader roles in bacterial adaptation remain mysterious, partly due to lack of computational tools. We made a program – IntegronFinder – to identify integrons with high accuracy and sensitivity. IntegronFinder is available as a standalone program and as a web application. It searches for attC sites using covariance models, for integron-integrases using HMM profiles, and for other features (promoters, attI site) using pattern matching. We searched for integrons, integron-integrases lacking attC sites, and clusters of attC sites lacking a neighboring integron-integrase in bacterial genomes. All these elements are especially frequent in genomes of intermediate size. They are missing in some key phyla, such as α-Proteobacteria, which might reflect selection against cell lineages that acquire integrons. The similarity between attC sites is proportional to the number of cassettes in the integron, and is particularly low in clusters of attC sites lacking integron-integrases. The latter are unexpectedly abundant in genomes lacking integron-integrases or their remains, and have a large novel pool of cassettes lacking homologs in the databases. They might represent an evolutionary step between the acquisition of genes within integrons and their stabilization in the new genome. PMID:27130947

  11. Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes.

    PubMed

    Xu, R; Janson, C G; Mastakov, M; Lawlor, P; Young, D; Mouravlev, A; Fitzsimons, H; Choi, K L; Ma, H; Dragunow, M; Leone, P; Chen, Q; Dicker, B; During, M J

    2001-09-01

    This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette. PMID:11571569

  12. Evaluation of D-1 tape and cassette characteristics: Moisture content of Sony and Ampex D-1 tapes when delivered

    NASA Technical Reports Server (NTRS)

    Ashton, Gary

    1993-01-01

    Commercial D-1 cassette tapes and their associated recorders were designed to operate in broadcast studios and record in accordance with the International Radio Consultative Committee (CCIR) 607 digital video standards. The D-1 recorder resulted in the Society of Motion Picture and Television Engineers (SMPTE) standards 224 to 228 and is the first digital video recorder to be standardized for the broadcast industry. The D-1 cassette and associated media are currently marketed for broadcast use. The recorder was redesigned for data applications and is in the early stages of being evaluated. The digital data formats used are specified in MIL-STD-2179 and the American National Standards Institute (ANSI) X3.175-190 standard. In early 1990, the National Media Laboratory (NML) was asked to study the effects of time, temperature, and relative humidity on commercial D-1 cassettes. The environmental range to be studied was the one selected for the Advanced Tactical Air Reconnaissance System (ATARS) program. Several discussions between NML personnel, ATARS representatives, recorder contractors, and other interested parties were held to decide upon the experimental plan to be implemented. Review meetings were held periodically during the course of the experiment. The experiments were designed to determine the dimensional stability of the media and cassette since this is one of the major limiting factors of helical recorders when the media or recorders are subjected to non-broadcasting environments. Measurements were also made to characterize each sample of cassettes to give preliminary information on which purchase specifications could be developed. The actual tests performed on the cassettes and media before and after aging fall into the general categories listed.

  13. Evaluation of D-1 tape and cassette characteristics: Moisture content of Sony and Ampex D-1 tapes when delivered

    NASA Astrophysics Data System (ADS)

    Ashton, Gary

    Commercial D-1 cassette tapes and their associated recorders were designed to operate in broadcast studios and record in accordance with the International Radio Consultative Committee (CCIR) 607 digital video standards. The D-1 recorder resulted in the Society of Motion Picture and Television Engineers (SMPTE) standards 224 to 228 and is the first digital video recorder to be standardized for the broadcast industry. The D-1 cassette and associated media are currently marketed for broadcast use. The recorder was redesigned for data applications and is in the early stages of being evaluated. The digital data formats used are specified in MIL-STD-2179 and the American National Standards Institute (ANSI) X3.175-190 standard. In early 1990, the National Media Laboratory (NML) was asked to study the effects of time, temperature, and relative humidity on commercial D-1 cassettes. The environmental range to be studied was the one selected for the Advanced Tactical Air Reconnaissance System (ATARS) program. Several discussions between NML personnel, ATARS representatives, recorder contractors, and other interested parties were held to decide upon the experimental plan to be implemented. Review meetings were held periodically during the course of the experiment. The experiments were designed to determine the dimensional stability of the media and cassette since this is one of the major limiting factors of helical recorders when the media or recorders are subjected to non-broadcasting environments. Measurements were also made to characterize each sample of cassettes to give preliminary information on which purchase specifications could be developed. The actual tests performed on the cassettes and media before and after aging fall into the general categories listed.

  14. Identification of a Novel Transposon (Tn6072) and a Truncated Staphylococcal Cassette Chromosome mec Element in Methicillin-Resistant Staphylococcus aureus ST239▿ †

    PubMed Central

    Chen, Liang; Mediavilla, José R.; Smyth, Davida S.; Chavda, Kalyan D.; Ionescu, Ramona; Roberts, Richard B.; Robinson, D. Ashley; Kreiswirth, Barry N.

    2010-01-01

    A novel composite transposon (Tn6072) resembling staphylococcal cassette chromosome mercury (SCCHg) was identified in a collection of sequence type (ST) 239 methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates from Romanian hospitals. Tn6072 is homologous to the 5′ region of SCCHg found in staphylococcal cassette chromosome mec (SCCmec) type III prototype strain 85/2082 but lacks the characteristic mer operon. SCCHg has previously been reported to integrate downstream of orfX, at the same chromosomal location as SCCmec. Tn6072, by contrast, is demarcated by two IS431 elements, flanked by 8-bp direct repeats, and inserted upstream of the origin of replication, within an open reading frame homologous to SAR2700 of S. aureus strain MRSA252. Analysis of a geographically and temporally diverse collection of 111 strains from the ST239 clonal group uncovered 11 additional strains harboring Tn6072, demonstrating a lineage-specific insertion pattern. Complete sequence analysis of the SCCmec regions of two representative Romanian strains (BK16704, BK16691) revealed two additional novel structures derived from a type III SCCmec background. BK16704 possesses an SCCmec 3A.1.4 structure, with an IS256 insertion downstream of the right chromosomal junction. In contrast, the SCCmec element of BK16691 is truncated downstream of the mec gene complex, with a 24-kb deletion encompassing the right chromosomal junction and an inverted downstream IS256 element. This structure, tentatively named “ψSCCmec16691,” confers methicillin resistance but lacks most of the J1/J2 region, including the ccr gene complex. Taken together, these findings provide evidence for the continuing evolution of SCC elements, as well as the ST239 clonal group. PMID:20479198

  15. The chromosome 3q26 OncCassette: A multigenic driver of human cancer.

    PubMed

    Fields, Alan P; Justilien, Verline; Murray, Nicole R

    2016-01-01

    define a "3q26 OncCassette" that mediates 3q26 CNG-driven tumorigenesis. Genomic analysis indicates that the 3q26 OncCassette also operates in other major tumor types that exhibit frequent 3q26 CNGs, including head and neck squamous cell carcinoma (HNSCC), ovarian serous cancer and cervical cancer. Finally, we discuss how the 3q26 OncCassette represents a tractable target for development of novel therapeutic intervention strategies that hold promise for improving treatment of 3q26-driven cancers. PMID:26754874

  16. Background sources at PEP

    SciTech Connect

    Lynch, H.; Schwitters, R.F.; Toner, W.T.

    1988-01-01

    Important sources of background for PEP experiments are studied. Background particles originate from high-energy electrons and positrons which have been lost from stable orbits, ..gamma..-rays emitted by the primary beams through bremsstrahlung in the residual gas, and synchrotron radiation x-rays. The effect of these processes on the beam lifetime are calculated and estimates of background rates at the interaction region are given. Recommendations for the PEP design, aimed at minimizing background are presented. 7 figs., 4 tabs.

  17. Copiers and Duplicators for Audio Tape Cassettes: Criteria for Selection, Laboratory Test Findings, Manufacturers' Data Reports. EPIE Report: Number 84e.

    ERIC Educational Resources Information Center

    Educational Products Information Exchange Inst., Stony Brook, NY.

    Because needs for cassette duplication equipment differ, the user must match his requirements to the capabilities of manufactured products, and the essential product characteristics. To help educators in choosing cassette duplicators, EPIE has gathered data from manufacturers and tested 11 units. The first section of the report explains the…

  18. Building Background Knowledge

    ERIC Educational Resources Information Center

    Neuman, Susan B.; Kaefer, Tanya; Pinkham, Ashley

    2014-01-01

    This article make a case for the importance of background knowledge in children's comprehension. It suggests that differences in background knowledge may account for differences in understanding text for low- and middle-income children. It then describes strategies for building background knowledge in the age of common core standards.

  19. Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes.

    PubMed

    Norris, Adam D; Kim, Hyun-Min; Colaiácovo, Mónica P; Calarco, John A

    2015-10-01

    Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4-5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments. PMID:26232410

  20. Efficient Genome Editing in Caenorhabditis elegans with a Toolkit of Dual-Marker Selection Cassettes

    PubMed Central

    Norris, Adam D.; Kim, Hyun-Min; Colaiácovo, Mónica P.; Calarco, John A.

    2015-01-01

    Use of the CRISPR/Cas9 RNA-guided endonuclease complex has recently enabled the generation of double-strand breaks virtually anywhere in the C. elegans genome. Here, we present an improved strategy that makes all steps in the genome editing process more efficient. We have created a toolkit of template-mediated repair cassettes that contain an antibiotic resistance gene to select for worms carrying the repair template and a fluorescent visual marker that facilitates identification of bona fide recombinant animals. Homozygous animals can be identified as early as 4–5 days post-injection, and minimal genotyping by PCR is required. We demonstrate that our toolkit of dual-marker vectors can generate targeted disruptions, deletions, and endogenous tagging with fluorescent proteins and epitopes. This strategy should be useful for a wide variety of additional applications and will provide researchers with increased flexibility when designing genome editing experiments. PMID:26232410

  1. Site-specific cassette exchange systems in the Aedes aegypti mosquito and the Plutella xylostella moth.

    PubMed

    Haghighat-Khah, Roya Elaine; Scaife, Sarah; Martins, Sara; St John, Oliver; Matzen, Kelly Jean; Morrison, Neil; Alphey, Luke

    2015-01-01

    Genetically engineered insects are being evaluated as potential tools to decrease the economic and public health burden of mosquitoes and agricultural pest insects. Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms. We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes. A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process. Complete iRMCE was achieved in two important insect pests, Aedes aegypti and the diamondback moth, Plutella xylostella, demonstrating the transferability of the system across a wide phylogenetic range of insect pests. PMID:25830287

  2. Site-Specific Cassette Exchange Systems in the Aedes aegypti Mosquito and the Plutella xylostella Moth

    PubMed Central

    Haghighat-Khah, Roya Elaine; Scaife, Sarah; Martins, Sara; St John, Oliver; Matzen, Kelly Jean; Morrison, Neil; Alphey, Luke

    2015-01-01

    Genetically engineered insects are being evaluated as potential tools to decrease the economic and public health burden of mosquitoes and agricultural pest insects. Here we describe a new tool for the reliable and targeted genome manipulation of pest insects for research and field release using recombinase mediated cassette exchange (RMCE) mechanisms. We successfully demonstrated the established ΦC31-RMCE method in the yellow fever mosquito, Aedes aegypti, which is the first report of RMCE in mosquitoes. A new variant of this RMCE system, called iRMCE, combines the ΦC31-att integration system and Cre or FLP-mediated excision to remove extraneous sequences introduced as part of the site-specific integration process. Complete iRMCE was achieved in two important insect pests, Aedes aegypti and the diamondback moth, Plutella xylostella, demonstrating the transferability of the system across a wide phylogenetic range of insect pests. PMID:25830287

  3. Generation of a Mouse Full-length Balancer with Versatile Cassette-shuttling Selection Strategy

    PubMed Central

    Ye, Zhisheng; Sun, Lei; Li, Rongbo; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2016-01-01

    Balancer chromosomes are important tools for a variety of genetic manipulations in lower model organisms, owing to their ability to suppress recombination. In mouse, however, such effort has not been accomplished, mostly due to the size of the chromosomes and the complexity of multiple step chromosomal engineering. We developed an effective and versatile cassette-shuttling selection (CASS) strategy involving only two selection markers to achieve the sequential production of multiple large inversions along the chromosome. Using this strategy, we successfully generated the first full-length balancer in mice and showed that Balancer 17M-GFP can efficiently suppress recombination. Our study has not only generated a useful genetic resource, but also provided a strategy for constructing mammalian balancer chromosomes. PMID:27489495

  4. Recognition of alternatively spliced cassette exons based on a hybrid model.

    PubMed

    Zhang, Xiaokang; Peng, Qinke; Li, Liang; Li, Xintong

    2016-03-11

    Alternative splicing (AS) is an important mechanism of gene regulation that contributes to protein diversity. It is of great significance to recognize different kinds of AS accurately so as to understand the mechanism of gene regulation. Many in silico methods have been applied to detecting AS with vast features, but the result is far from satisfactory. In this paper, we used the features proven to be useful in recognizing AS in previous literature and proposed a hybrid method combining Gene Expression Programming (GEP) and Random Forests (RF) to classify the constitutive exons and cassette exons which is the most common AS phenomenon. GEP will firstly make prediction to the samples of strong signal, and the other samples of weak signal will be distinguished with a more complex classifier based on RF. The experiment result indicates that this method can highly improve the recognition level in this issue. PMID:26869516

  5. Bacterial resistance evolution by recruitment of super-integron gene cassettes.

    PubMed

    Rowe-Magnus, Dean A; Guerout, Anne-Marie; Mazel, Didier

    2002-03-01

    The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram-negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super-integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae, a species known to harbour a SI. We show that MRIs can randomly recruit genes directly from the cache of SI cassettes. By applying a selective constraint for the development of antibiotic resistance, we demonstrate bacterial resistance evolution through the recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase gene from the V. cholerae SI and its precise insertion into the MRI. The resulting resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events. PMID:11952913

  6. The lysis cassette of DLP12 defective prophage is regulated by RpoE.

    PubMed

    Rueggeberg, Karl-Gustav; Toba, Faustino A; Bird, Jeremy G; Franck, Nathan; Thompson, Mitchell G; Hay, Anthony G

    2015-08-01

    Expression of the lysis cassette (essD, ybcT, rzpD/rzoD) from the defective lambdoid prophage at the 12th minute of Escherichia coli's genome (DLP12) is required in some strains for proper curli expression and biofilm formation. Regulating production of the lytic enzymes encoded by these genes is critical for maintaining cell wall integrity. In lambdoid phages, late-gene regulation is mediated by the vegetative sigma factor RpoD and the lambda antiterminator Qλ. We previously demonstrated that DLP12 contains a Q-like protein (QDLP12) that positively regulates transcription of the lysis cassette, but the sigma factor responsible for this transcription initiation remained to be elucidated. In silico analysis of essDp revealed the presence of a putative - 35 and - 10 sigma site recognized by the extracytoplasmic stress response sigma factor, RpoE. In this work, we report that RpoE overexpression promoted transcription from essDp in vivo, and in vitro using purified RNAP. We demonstrate that the - 35 region is important for RpoE binding in vitro and that this region is also important for QDLP12-mediated transcription of essDp in vivo. A bacterial two-hybrid assay indicated that QDLP12 and RpoE physically interact in vivo, consistent with what is seen for Qλ and RpoD. We propose that RpoE regulates transcription of the DLP12 lysis genes through interaction with QDLP12 and that proper expression is dependent on an intact - 35 sigma region in essDp. This work provides evidence that the unique Q-dependent regulatory mechanism of lambdoid phages has been co-opted by E. coli harbouring defective DLP12 and has been integrated into the tightly controlled RpoE regulon. PMID:25998262

  7. Endotoxin deposits on the inner surfaces of closed-face cassettes during bioaerosol sampling: a field investigation at composting facilities.

    PubMed

    Duquenne, Philippe; Simon, Xavier; Demange, Valérie; Harper, Martin; Wild, Pascal

    2015-05-01

    A set of 270 bioaerosol samples was taken from 15 composting facilities using polystyrene closed-face filter cassettes (CFCs). The objective was to measure the quantity of endotoxin deposits on the inner surfaces of the cassettes (sometimes referred to as 'wall deposits'). The results show that endotoxins are deposited on the inner surfaces of the CFCs through sampling and/or handling of samples. The quantity of endotoxins measured on inner surfaces range between 0.05 (the limit of detection of the method) and 3100 endotoxin units per cassette. The deposits can represent a large and variable percentage of the endotoxins sampled. More than a third of the samples presented a percentage of inner surface deposits >40% of the total quantity of endotoxins collected (filter + inner surfaces). Omitting these inner surface deposits in the analytical process lead to measurement errors relative to sampling all particles entering the CFC sampler, corresponding to a developing consensus on matching the inhalable particulate sampling convention. The result would be underestimated exposures and could affect the decision as to whether or not a result is acceptable in comparison to airborne concentration limits defined in terms of the inhalability convention. The results of this study suggest including the endotoxins deposited on the inner surfaces of CFCs during analysis. Further researches are necessary to investigate endotoxin deposits on the inner cassette surfaces in other working sectors. PMID:25535181

  8. Identification of antibiotic resistance cassettes in class 1 integrons in Aeromonas spp. strains isolated from fresh fish (Cyprinus carpio L.).

    PubMed

    Sarria-Guzmán, Yohanna; López-Ramírez, María Patricia; Chávez-Romero, Yosef; Ruiz-Romero, Erick; Dendooven, Luc; Bello-López, Juan Manuel

    2014-05-01

    Forty-six Aeromonas spp. strains were isolated from fresh fish and investigated for their antimicrobial susceptibility, detection of Class 1 integrons by PCR, and arrangement of gene cassettes. Selected isolates were further characterized by enterobacterial repetitive intergenic consensus-PCR. Twenty isolates were found to carry Class 1 integrons. Amplification of the variable regions of the integrons revealed diverse bands ranging in size from 150 to 1,958 pb. Sequence analysis of the variable regions revealed the presence of several gene cassettes, such as adenylyl transferases (aadA2 and aadA5), dihydrofolate reductases (dfrA17 and dfrA1), chloramphenicol acetyl transferase (catB3), β-lactamase (oxa2), lincosamide nucleotidil transferase (linF), aminoglycoside-modifying enzyme (apha15), and oxacillinase (bla OXA-10). Two open reading frames with an unknown function were identified as orfC and orfD. The aadA2 cassette was the most common integron found in this study. Interestingly, five integrons were detected in the plasmids that might be involved in the transfer of resistance genes to other bacteria. This is a first report of cassette encoding for lincosamides (linF) resistance in Aeromonas spp. Implications on the incidence of integrons in isolates of Aeromonas spp. from fresh fish for human consumption, and its possible consequences to human health are discussed. PMID:24370627

  9. Detection of E. coli O157:H7 with a reporter phage containing the luxCDABE cassette

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteriophage and reporter phage are used for typing and/or detection of pathogens. The temperate tailed phage fV10 has been utilized for phage-typing E. coli O157:H7. By modifying fV10 to transduce kanamycin resistance and the a luxCDABE cassette, we developed a reporter bacteriophage (fV10-lux) p...

  10. Consumer Education Resources Catalog. 1980 Supplement. 16mm Films, Multi Media Kits, Video Cassettes, Simulations & Games, Printed Material.

    ERIC Educational Resources Information Center

    Jones, Sandra

    This supplement to the Consumer Education Resources Catalog (see note) lists teaching-learning resources available for preview at the Michigan Consumer Education Center. A subject index to multi-media identifies titles of films, video cassettes, multi-media kits, and games under seven specific subjects. These are (1) Factors Affecting Consumer…

  11. Consumer Education Resources Catalog: 16mm Films, Multi Media Kits, Video Cassettes, Simulations & Games, Printed Materials. 1978 Edition.

    ERIC Educational Resources Information Center

    Jones, Sandra; Neal, Kathy

    This consumer education resources catalog provides an annotated guide to 16mm films, multi-media kits, video cassettes, simulations and games, and printed materials related to consumer education available from Michigan Department of Education's Regional Education Media Centers. The first major section lists available media by specific subject…

  12. Characterization of integrons and novel cassette arrays in bacteria from clinical isloates in China, 2000-2014

    PubMed Central

    Xia, Wenying; Xu, Ting; Qin, Tingting; Li, Pengpeng; Liu, Yun; Kang, Haiquan; Gu, Bing; Ma, Ping

    2016-01-01

    Abstract Rapid dissemination of antibiotic resistance genes among bacterial isolates is an increasing problem in China. Integron, a conserved DNA sequence, which is carried on episomal genetic structures, plays a very important role in development of antibiotic resistance. This systematic analysis was based on MEDLINE and EMBASE databases. We summarized the distribution and proportion of different types of gene cassette arrays of integrons (including class 1, 2, 3 and atypical class 1 integron) from clinical bacteria isolates in China. Fifty-six literatures were included in this study. Most of the strains were Gram-negative bacteria (94.1%, 7,364/7,822) while only 5.9% strains were Gram-positive bacteria. Class 1 integrons were detected in 54.2% (3956/7295) Gram-negative strains. aadA2 was the most popular gene cassette array detected from 60 Gram-positive bacteria while dfrA17-aadA5 were detected in 426 Gram-negative bacteria. This study identified 12 novel gene cassette arrays which have not been previously found in any species. All the novel gene cassette arrays were detected from Gram-negative bacteria. A regional characteristic of distribution of integrons was presented in this study. The results highlight a need for continuous surveillance of integrons and provide a guide for future research on integron-mediated bacteria resistance.

  13. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

    PubMed

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah

    2016-09-10

    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption. PMID:27411901

  14. The Cosmic Background Explorer.

    ERIC Educational Resources Information Center

    Gulkis, Samuel; And Others

    1990-01-01

    Outlines the Cosmic Background Explorer (COBE) mission to measure celestial radiation. Describes the instruments used and experiments involving differential microwave radiometers, and a far infrared absolute spectrophotometer. (YP)

  15. Backgrounds in Language.

    ERIC Educational Resources Information Center

    Maxwell, John C.; Long, Barbara K.

    "Backgrounds in Language," a field-tested inservice course designed for use by groups of 15 or 25 language arts teachers, provides the subject matter background teachers need to make informed decisions about what curriculum materials to use in what way, at what time, and with which students. The course is comprised of eight 2-hour sessions,…

  16. Correlators in nontrivial backgrounds

    SciTech Connect

    Mello Koch, Robert de; Ives, Norman; Stephanou, Michael

    2009-01-15

    Operators in N=4 super Yang-Mills theory with an R-charge of O(N{sup 2}) are dual to backgrounds which are asymtotically AdS{sub 5}xS{sup 5}. In this article we develop efficient techniques that allow the computation of correlation functions in these backgrounds. We find that (i) contractions between fields in the string words and fields in the operator creating the background are the field theory accounting of the new geometry, (ii) correlation functions of probes in these backgrounds are given by the free field theory contractions but with rescaled propagators and (iii) in these backgrounds there are no open string excitations with their special end point interactions; we have only closed string excitations.

  17. Comparison of a wipe method with and without a rinse to recover wall losses in closed face 37-mm cassettes used for sampling lead dust particulates

    PubMed Central

    Ceballos, Diana; King, Bradley; Beaucham, Catherine; Brueck, Scott E.

    2015-01-01

    Closed-face 37-millimeter (mm) polystyrene cassettes are often used for exposure monitoring of metal particulates. Several methods have been proposed to account for the wall loss in air sampling cassettes, including rinsing, wiping, within-cassette dissolution, and an internal capsule fused to the filter that could be digested with the filter. Until internal capsules replace filters, other methods for assessing wall losses may be considered. To determine if rinsing and wiping or wiping alone is adequate to determine wall losses on cassettes, we collected 54 full-shift area air samples at a battery recycling facility. We collected six replicate samples at three locations within the facility for 3 consecutive days. The wall losses of three replicate cassettes from each day-location were analyzed following a rinse and two consecutive wipes. The wall losses of the other three replicates from each day-location were analyzed following two consecutive wipes only. Mixed-cellulose ester membrane filter, rinse, and wipes were analyzed separately following NIOSH Method 7303. We found an average of 29% (range: 8%–54%) recovered lead from the cassette walls for all samples. We also found that rinsing prior to wiping the interior cassette walls did not substantially improve recovery of wall losses compared to wiping alone. A rinse plus one wipe recovered on average 23% (range: 13%–33%) of the lead, while one wipe alone recovered on average 21% (range: 16%–22%). Similarly we determined that a second wipe did not provide substantial additional recovery of lead (average: 4%, range: 0.4%–19%) compared to the first wipe disregarding the rinse (average: 18%, range: 4%–39%). We concluded that when an internal capsule is not used, wall losses of lead dust in air sampling cassettes can be adequately recovered by wiping the internal wall surfaces of the cassette with a single wipe. PMID:26125330

  18. Background Underground at WIPP

    NASA Astrophysics Data System (ADS)

    Esch, Ernst-Ingo; Hime, A.; Bowles, T. J.

    2001-04-01

    Recent interest to establish a dedicated underground laboratory in the United States prompted an experimental program at to quantify the enviromental backgrounds underground at the Waste Isolation Pilot Plant (WIPP) in Carlsbad, New Mexico. An outline of this program is provided along with recent experimental data on the cosmic ray muon flux at the 650 meter level of WIPP. The implications of the cosmic ray muon and fast neutron background at WIPP will be discussed in the context of new generation, low background experiments envisioned in the future.

  19. The cosmic neutrino background

    NASA Technical Reports Server (NTRS)

    Dar, Arnon

    1991-01-01

    The cosmic neutrino background is expected to consist of relic neutrinos from the big bang, of neutrinos produced during nuclear burning in stars, of neutrinos released by gravitational stellar collapse, and of neutrinos produced by cosmic ray interactions with matter and radiation in the interstellar and intergalactic medium. Formation of baryonic dark matter in the early universe, matter-antimatter annihilation in a baryonic symmetric universe, and dark matter annihilation could have also contributed significantly to the cosmic neutrino background. The purpose of this paper is to review the properties of these cosmic neutrino backgrounds, the indirect evidence for their existence, and the prospects for their detection.

  20. Adaptive background model

    NASA Astrophysics Data System (ADS)

    Lu, Xiaochun; Xiao, Yijun; Chai, Zhi; Wang, Bangping

    2007-11-01

    An adaptive background model aiming at outdoor vehicle detection is presented in this paper. This model is an improved model of PICA (pixel intensity classification algorithm), it classifies pixels into K-distributions by color similarity, and then a hypothesis that the background pixel color appears in image sequence with a high frequency is used to evaluate all the distributions to determine which presents the current background color. As experiments show, the model presented in this paper is a robust, adaptive and flexible model, which can deal with situations like camera motions, lighting changes and so on.

  1. Design, Construction and Cloning of Truncated ORF2 and tPAsp-PADRE-Truncated ORF2 Gene Cassette From Hepatitis E Virus in the pVAX1 Expression Vector

    PubMed Central

    Farshadpour, Fatemeh; Makvandi, Manoochehr; Taherkhani, Reza

    2015-01-01

    Background: Hepatitis E Virus (HEV) is the causative agent of enterically transmitted acute hepatitis and has high mortality rate of up to 30% among pregnant women. Therefore, development of a novel vaccine is a desirable goal. Objectives: The aim of this study was to construct tPAsp-PADRE-truncated open reading frame 2 (ORF2) and truncated ORF2 DNA plasmid, which can assist future studies with the preparation of an effective vaccine against Hepatitis E Virus. Materials and Methods: A synthetic codon-optimized gene cassette encoding tPAsp-PADRE-truncated ORF2 protein was designed, constructed and analyzed by some bioinformatics software. Furthermore, a codon-optimized truncated ORF2 gene was amplified by the polymerase chain reaction (PCR), with a specific primer from the previous construct. The constructs were sub-cloned in the pVAX1 expression vector and finally expressed in eukaryotic cells. Results: Sequence analysis and bioinformatics studies of the codon-optimized gene cassette revealed that codon adaptation index (CAI), GC content, and frequency of optimal codon usage (Fop) value were improved, and performance of the secretory signal was confirmed. Cloning and sub-cloning of the tPAsp-PADRE-truncated ORF2 gene cassette and truncated ORF2 gene were confirmed by colony PCR, restriction enzymes digestion and DNA sequencing of the recombinant plasmids pVAX-tPAsp-PADRE-truncated ORF2 (aa 112-660) and pVAX-truncated ORF2 (aa 112-660). The expression of truncated ORF2 protein in eukaryotic cells was approved by an Immunofluorescence assay (IFA) and the reverse transcriptase polymerase chain reaction (RT-PCR) method. Conclusions: The results of this study demonstrated that the tPAsp-PADRE-truncated ORF2 gene cassette and the truncated ORF2 gene in recombinant plasmids are successfully expressed in eukaryotic cells. The immunogenicity of the two recombinant plasmids with different formulations will be evaluated as a novel DNA vaccine in future investigations. PMID

  2. The GLAST Background Model

    SciTech Connect

    Ormes, J.F.; Atwood, W.; Burnett, T.; Grove, E.; Longo, F.; McEnery, J.; Mizuno, T.; Ritz, S.; /NASA, Goddard

    2007-10-17

    In order to estimate the ability of the GLAST/LAT to reject unwanted background of charged particles, optimize the on-board processing, size the required telemetry and optimize the GLAST orbit, we developed a detailed model of the background particles that would affect the LAT. In addition to the well-known components of the cosmic radiation, we included splash and reentrant components of protons, electrons (e+ and e-) from 10 MeV and beyond as well as the albedo gamma rays produced by cosmic ray interactions with the atmosphere. We made estimates of the irreducible background components produced by positrons and hadrons interacting in the multilayered micrometeorite shield and spacecraft surrounding the LAT and note that because the orbital debris has increased, the shielding required and hence the background are larger than were present in EGRET. Improvements to the model are currently being made to include the east-west effect.

  3. The GLAST Background Model

    SciTech Connect

    Ormes, J. F.; Atwood, W.; Burnett, T.; Grove, E.; Longo, F.; McEnery, J.; Ritz, S.; Mizuno, T.

    2007-07-12

    In order to estimate the ability of the GLAST/LAT to reject unwanted background of charged particles, optimize the on-board processing, size the required telemetry and optimize the GLAST orbit, we developed a detailed model of the background particles that would affect the LAT. In addition to the well-known components of the cosmic radiation, we included splash and reentrant components of protons, electrons (e+ and e-) from 10 MeV and beyond as well as the albedo gamma rays produced by cosmic ray interactions with the atmosphere. We made estimates of the irreducible background components produced by positrons and hadrons interacting in the multilayered micrometeorite shield and spacecraft surrounding the LAT and note that because the orbital debris has increased, the shielding required and hence the background are larger than were present in EGRET. Improvements to the model are currently being made to include the east-west effect.

  4. Plug-and-Play Genetic Access to Drosophila Cell Types Using Exchangeable Exon Cassettes

    PubMed Central

    Diao, Fengqiu; Ironfield, Holly; Luan, Haojiang; Diao, Feici; Shropshire, William C.; Ewer, John; Marr, Elizabeth; Potter, Christopher J.; Landgraf, Matthias; White, Benjamin H.

    2015-01-01

    Summary Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here we introduce a simple, versatile method for achieving cell type-specific expression of transgenes that leverages the untapped potential of “coding introns” (i.e. introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted “plug-and-play” cassettes (called “Trojan exons”) that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system. PMID:25732830

  5. Energy use of televisions and video cassette recorders in the U.S.

    SciTech Connect

    Meier, Alan; Rosen, Karen

    1999-03-01

    In an effort to more accurately determine nationwide energy consumption, the U.S. Department of Energy has recently commissioned studies with the goal of improving its understanding of the energy use of appliances in the miscellaneous end-use category. This study presents an estimate of the residential energy consumption of two of the most common domestic appliances in the miscellaneous end-use category: color televisions (TVs) and video cassette recorders (VCRs). The authors used a bottom-up approach in estimating national TV and VCR energy consumption. First, they obtained estimates of stock and usage from national surveys, while TV and VCR power measurements and other data were recorded at repair and retail shops. Industry-supplied shipment and sales distributions were then used to minimize bias in the power measurement samples. To estimate national TV and VCR energy consumption values, ranges of power draw and mode usage were created to represent situations in homes with more than one unit. Average energy use values for homes with one unit, two units, etc. were calculated and summed to provide estimates of total national TV and VCR energy consumption.

  6. The effects on hearing from portable cassette players: A follow-up study

    NASA Astrophysics Data System (ADS)

    Hellström, P. A.

    1991-12-01

    The high-frequency hearing loss (3-8 kHz) in 230 male and 238 female subjects (age 16) was compared concerning their habits of listening to portable cassette players (PCPs). In the group that listened to PCPs daily, we found fewer subjects with high-frequency hearing loss compared to the group that never listened to PCPs. In a temporary threshold shift (TTS) study, three groups of subjects with different listening habits to music were exposed to {1}/{3} octave band noise (2 kHz). These groups participated in a TTS study on another occasion, on which they listened to music from PCPs at a high but comfortable level for 1 hour. Together with the music exposure, the sound pressure level ( SPL) was measured in the external auditory canal with a probe-provided mini-microphone (MMP). There was no significant difference in TTS between groups from the noise exposure. There were significant differences in listening levels and in the resulting TTS between groups. The noise exposure resulted in significantly higher TTS than the music exposure at comparable exposure levels.

  7. Transduction of staphylococcal cassette chromosome mec elements between strains of Staphylococcus aureus.

    PubMed

    Scharn, Caitlyn R; Tenover, Fred C; Goering, Richard V

    2013-11-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known public health concern. However, the means by which methicillin resistance genes are transferred among staphylococci in nature remains unknown. Older scientific literature suggests transduction as a means of mecA transfer, but the optimal conditions are reported to require plasmids and potentially a lysogenic phage. These reports preceded discovery of the staphylococcal cassette chromosome mec (SCCmec) elements. We undertook studies to confirm and clarify the conditions promoting transduction of SCCmec in S. aureus populations using well-characterized donor and recipient strains primarily of the USA300 lineage. Both bacteriophages 80α and 29 were capable of transducing SCCmec type IV and SCCmec type I to recipient strains of S. aureus. Pulsed-field gel electrophoresis and mec-associated dru typing were used to confirm the identity of the transductants. Transfer of mecA via transduction occurred at low frequency and required extended selection times for mecA gene expression and the presence of a penicillinase plasmid in the recipient. However, interference with the process by clavulanic acid and the necessity of lysogeny with 11 in the recipient or the presence of a small (4-kb) tetracycline resistance plasmid, as previously reported, were not confirmed. SCCmec transduction was occasionally associated with substantial deletions or truncation of SCCmec and the arginine catabolic metabolic element in USA300 recipients. Overall, these data clarify the conditions required for SCCmec transduction and document that rearrangements may occur during the process. PMID:23939891

  8. False positives observed on the Seratec® PSA SemiQuant Cassette Test with condom lubricants.

    PubMed

    Bitner, Sara E

    2012-11-01

    In the course of the validation of a new component of the prostate-specific antigen (PSA) SemiQuant Cassette Test marketed by Seratec(®) , a false-positive reaction was observed when testing samples collected from the surface of unused, lubricated condoms. A variety of personal lubricants and condoms were tested to determine the frequency of the false positive, as well as its potential source. Samples were extracted in both water and the manufacturer-provided buffer, and the test was performed according to the manufacturer's suggested protocol. The false positive was observed intermittently, but occurred consistently with samples containing nonoxynol-9, a strong detergent utilized as a spermicide. The reaction may be attributable to the combination of latex and nonoxynol-9. Because of the unreliability of the test to confirm the presence of PSA in samples collected from condoms, the PSA cassette is an unsuitable method for confirming the presence of seminal fluid in condoms. PMID:22494324

  9. A Novel Multiplex Real-Time PCR Assay for Rapid Typing of Major Staphylococcal Cassette Chromosome mec Elements

    PubMed Central

    Francois, Patrice; Renzi, Gesuele; Pittet, Didier; Bento, Manuela; Lew, Daniel; Harbarth, Stephan; Vaudaux, Pierre; Schrenzel, Jacques

    2004-01-01

    We describe a novel procedure for rapid typing of the staphylococcal cassette chromosome mec element, a molecular marker allowing discrimination between community- and hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) strains. Oligonucleotides targeting the recombinase genes were type specific and used to type a collection of 399 MRSA isolates recovered during patient screening at admission. This novel assay constitutes a valuable tool for evaluating the molecular epidemiology of MRSA and adjusting infection control strategies against MRSA. PMID:15243102

  10. Construction of Synthetic Promoter-Based Expression Cassettes for the Production of Cadaverine in Recombinant Corynebacterium glutamicum.

    PubMed

    Oh, Young Hoon; Choi, Jae Woo; Kim, Eun Young; Song, Bong Keun; Jeong, Ki Jun; Park, Kyungmoon; Kim, Il-Kwon; Woo, Han Min; Lee, Seung Hwan; Park, Si Jae

    2015-08-01

    Corynebacterium glutamicum is an important microorganism in the biochemical industry for the production of various platform chemicals. However, despite its importance, a limited number of studies have been conducted on how to constitute gene expression cassettes in engineered C. glutamicum to obtain desired amounts of the target products. Therefore, in this study, six expression cassettes for the expression of the second lysine decarboxylase of Escherichia coli, LdcC, were constructed using six synthetic promoters with different strengths and were examined in C. glutamicum for the production of cadaverine. Among six expression cassettes, the expression of the E. coli ldcC gene under the PH30 promoter supported the highest production of cadaverine in flask and fed-batch cultivations. A fed-batch fermentation of recombinant C. glutamicum expressing E. coli ldcC gene under the PH30 promoter resulted in the production of 40.91 g/L of cadaverine in 64 h. This report is expected to contribute toward developing engineered C. glutamicum strains to have desired features. PMID:26047931

  11. Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels

    PubMed Central

    Dronadula, Nagadhara; Du, Liang; Flynn, Rowan; Buckler, Joshua; Kho, Jordan; Jiang, Zhilong; Tanaka, Shinji; Dichek, David A.

    2010-01-01

    The success of gene therapy hinges on achievement of adequate transgene expression. To ensure high transgene expression, many gene-therapy vectors include highly active virus-derived transcriptional elements. Other vectors include tissue-specific eukaryotic transcriptional elements, intended to limit transgene expression to specific cell types, avoid toxicity, and prevent immune responses. Unfortunately, tissue specificity is often accompanied by lower transgene expression. Here we use eukaryotic (murine) transcriptional elements and a virus-derived posttranscriptional element to build cassettes designed to express a potentially therapeutic gene (interleukin-10) in large vessel endothelial cells (EC) at levels as high as obtained with the CMV immediate-early promoter, while retaining EC-specificity. The cassettes were tested by incorporation into helper-dependent adenoviral vectors, and transduction into bovine aortic EC in vitro and rabbit carotid EC in vivo. The murine endothelin-1 promoter showed EC-specificity, but expressed only 3% as much IL-10 mRNA as CMV. Inclusion of precisely 4 copies of an EC-specific enhancer and a posttranscriptional regulatory element increased IL-10 expression to a level at or above the CMV promoter in vivo, while retaining—and possibly enhancing—EC specificity, as measured in vitro. The cassette reported here will likely be useful for maximizing transgene expression in large vessel EC, while minimizing systemic effects. PMID:21179172

  12. An automated system to mount cryo-cooled protein crystals on a synchrotron beam line, using compact sample cassettes and a small-scale robot

    PubMed Central

    Cohen, Aina E.; Ellis, Paul J.; Miller, Mitchell D.; Deacon, Ashley M.; Phizackerley, R. Paul

    2014-01-01

    An automated system for mounting and dismounting pre-frozen crystals has been implemented at the Stanford Synchrotron Radiation Laboratory (SSRL). It is based on a small industrial robot and compact cylindrical cassettes, each holding up to 96 crystals mounted on Hampton Research sample pins. For easy shipping and storage, the cassette fits inside several popular dry-shippers and long-term storage Dewars. A dispensing Dewar holds up to three cassettes in liquid nitrogen adjacent to the beam line goniometer. The robot uses a permanent magnet tool to extract samples from, and insert samples into a cassette, and a cryo-tong tool to transfer them to and from the beam line goniometer. The system is simple, with few moving parts, reliable in operation and convenient to use. PMID:24899734

  13. The Cosmic Background Explorer

    NASA Technical Reports Server (NTRS)

    Gulkis, Samuel; Lubin, Philip M.; Meyer, Stephan S.; Silverberg, Robert F.

    1990-01-01

    The Cosmic Background Explorer (CBE), NASA's cosmological satellite which will observe a radiative relic of the big bang, is discussed. The major questions connected to the big bang theory which may be clarified using the CBE are reviewed. The satellite instruments and experiments are described, including the Differential Microwave Radiometer, which measures the difference between microwave radiation emitted from two points on the sky, the Far-Infrared Absolute Spectrophotometer, which compares the spectrum of radiation from the sky at wavelengths from 100 microns to one cm with that from an internal blackbody, and the Diffuse Infrared Background Experiment, which searches for the radiation from the earliest generation of stars.

  14. China: Background Notes Series.

    ERIC Educational Resources Information Center

    Reams, Joanne Reppert

    Concise background information on the People's Republic of China is provided. The publication begins with a profile of the country, outlining the people, geography, economy, and membership in international organizations. The bulk of the document then discusses in more detail China's people, geography, history, government, education, economy, and…

  15. Nonthermal cosmic neutrino background

    NASA Astrophysics Data System (ADS)

    Chen, Mu-Chun; Ratz, Michael; Trautner, Andreas

    2015-12-01

    We point out that, for Dirac neutrinos, in addition to the standard thermal cosmic neutrino background (C ν B ), there could also exist a nonthermal neutrino background with comparable number density. As the right-handed components are essentially decoupled from the thermal bath of standard model particles, relic neutrinos with a nonthermal distribution may exist until today. The relic density of the nonthermal (nt) background can be constrained by the usual observational bounds on the effective number of massless degrees of freedom Neff and can be as large as nν nt≲0.5 nγ. In particular, Neff can be larger than 3.046 in the absence of any exotic states. Nonthermal relic neutrinos constitute an irreducible contribution to the detection of the C ν B and, hence, may be discovered by future experiments such as PTOLEMY. We also present a scenario of chaotic inflation in which a nonthermal background can naturally be generated by inflationary preheating. The nonthermal relic neutrinos, thus, may constitute a novel window into the very early Universe.

  16. Berkeley Low Background Facility

    NASA Astrophysics Data System (ADS)

    Thomas, K. J.; Smith, A. R.; Poon, A. W. P.; Chan, Y. D.; Norman, E. B.; Lesko, K. T.

    2015-08-01

    The Berkeley Low Background Facility (BLBF) at Lawrence Berkeley National Laboratory (LBNL) in Berkeley, California provides low background gamma spectroscopy services to a wide array of experiments and projects. The analysis of samples takes place within two unique facilities; locally within a carefully-constructed, low background laboratory on the surface at LBNL and at the Sanford Underground Research Facility (SURF) in Lead, SD. These facilities provide a variety of gamma spectroscopy services to low background experiments primarily in the form of passive material screening for primordial radioisotopes (U, Th, K) or common cosmogenic/anthropogenic products; active screening via neutron activation analysis for U,Th, and K as well as a variety of stable isotopes; and neutron flux/beam characterization measurements through the use of monitors. A general overview of the facilities, services, and sensitivities will be presented. Recent activities and upgrades will also be described including an overview of the recently installed counting system at SURF (recently relocated from Oroville, CA in 2014), the installation of a second underground counting station at SURF in 2015, and future plans. The BLBF is open to any users for counting services or collaboration on a wide variety of experiments and projects.

  17. Berkeley Low Background Facility

    SciTech Connect

    Thomas, K. J.; Norman, E. B.; Smith, A. R.; Poon, A. W. P.; Chan, Y. D.; Lesko, K. T.

    2015-08-17

    The Berkeley Low Background Facility (BLBF) at Lawrence Berkeley National Laboratory (LBNL) in Berkeley, California provides low background gamma spectroscopy services to a wide array of experiments and projects. The analysis of samples takes place within two unique facilities; locally within a carefully-constructed, low background laboratory on the surface at LBNL and at the Sanford Underground Research Facility (SURF) in Lead, SD. These facilities provide a variety of gamma spectroscopy services to low background experiments primarily in the form of passive material screening for primordial radioisotopes (U, Th, K) or common cosmogenic/anthropogenic products; active screening via neutron activation analysis for U,Th, and K as well as a variety of stable isotopes; and neutron flux/beam characterization measurements through the use of monitors. A general overview of the facilities, services, and sensitivities will be presented. Recent activities and upgrades will also be described including an overview of the recently installed counting system at SURF (recently relocated from Oroville, CA in 2014), the installation of a second underground counting station at SURF in 2015, and future plans. The BLBF is open to any users for counting services or collaboration on a wide variety of experiments and projects.

  18. Foregrounding the Background.

    ERIC Educational Resources Information Center

    Robbins, Bruce

    1998-01-01

    Argues that when introductory activities to the classics begin with background information, it can upstage or confine the life of the story, and shows little faith in the students as readers or in the literature itself. Suggests sometimes letting the literature begin, and then helping students make sense of it. Discusses examples from "To Kill a…

  19. Monitored background radiometer

    NASA Technical Reports Server (NTRS)

    Ruel, C.; Larouche, M.; Donato, M.

    1986-01-01

    The infrared (IR) testing of the Olympus thermal model has provided a capability to perform cost effective thermal balance testing of satellites and satellite components. A high-accuracy monitored background radiometer was developed for the measurement of absorbed radiation heat flux encountered during IR thermal vacuum testing of spacecraft. The design, development, and calibration of this radiometer is described.

  20. The Lophotrochozoan TGF-β signalling cassette - diversification and conservation in a key signalling pathway.

    PubMed

    Kenny, Nathan J; Namigai, Erica K O; Dearden, Peter K; Hui, Jerome H L; Grande, Cristina; Shimeld, Sebastian M

    2014-01-01

    TGF-β signalling plays a key role in the patterning of metazoan body plans and growth. It is widely regarded as a 'module' capable of co-option into novel functions. The TGF-β pathway arose in the Metazoan lineage, and while it is generally regarded as well conserved across evolutionary time, its components have been largely studied in the Ecdysozoa and Deuterostomia. The recent discovery of the Nodal molecule in molluscs has underlined the necessity of untangling this signalling network in lophotrochozoans in order to truly comprehend the evolution, conservation and diversification of this key pathway. Three novel genome resources, the mollusc Patella vulgata, annelid Pomatoceros lamarcki and rotifer Brachionus plicatilis, along with other publicly available data, were searched for the presence of TGF-β pathway genes. Bayesian and Maximum Likelihood analyses, along with some consideration of conserved domain structure, was used to confirm gene identity. Analysis revealed conservation of key components within the canonical pathway, allied with extensive diversification of TGF-β ligands and partial loss of genes encoding pathway inhibitors in some lophotrochozoan lineages. We fully describe the TGF-β signalling cassette of a range of lophotrochozoans, allowing firm inference to be drawn as to the ancestral state of this pathway in this Superphylum. The TGF-β signalling cascade's reputation as being highly conserved across the Metazoa is reinforced. Diversification within the activin-like complement, as well as potential wide loss of regulatory steps in some Phyla, hint at specific evolutionary implications for aspects of this cascade's functionality in this Superphylum. PMID:25690968

  1. Comprehensive evaluation of formulation factors for ocular penetration of fluoroquinolones in rabbits using cassette dosing technique

    PubMed Central

    Sharma, Charu; Biswas, Nihar R; Ojha, Shreesh; Velpandian, Thirumurthy

    2016-01-01

    Objective Corneal permeability of drugs is an important factor used to assess the efficacy of topical preparations. Transcorneal penetration of drugs from aqueous formulation is governed by various physiological, physiochemical, and formulation factors. In the present study, we investigated the effect of formulation factors like concentration, pH, and volume of instillation across the cornea using cassette dosing technique for ophthalmic fluoroquinolones (FQs). Materials and methods Sterile cocktail formulations were prepared using four congeneric ophthalmic FQs (ofloxacin, sparfloxacin, pefloxacin mesylate, and gatifloxacin) at concentrations of 0.025%, 0.5%, and 0.1%. Each formulation was adjusted to different pH ranges (4.5, 7.0, and 8.0) and assessed for transcorneal penetration in vivo in rabbit’s cornea (n=4 eyes) at three different volumes (12.5, 25, and 50 μL). Aqueous humor was aspirated through paracentesis after applying local anesthesia at 0, 5, 15, 30, 60, 120, and 240 minutes postdosing. The biosamples collected from a total of 27 groups were analyzed using liquid chromatography–tandem mass spectroscopy to determine transcorneal permeability of all four FQs individually. Results Increase in concentration showed an increase in penetration up to 0.05%; thereafter, the effect of concentration was found to be dependent on volume of instillation as we observed a decrease in transcorneal penetration. The highest transcorneal penetration of all FQs was observed at pH 7.0 at concentration 0.05% followed by 0.025% at pH 4.5. Lastly, increasing the volume of instillation from 12.5 to 50 μL showed a significant fall in transcorneal penetration. Conclusion The study concludes that formulation factors showed discernible effect on transcorneal permeation; therefore, due emphasis should be given on drug development and design of ophthalmic formulation. PMID:26955263

  2. Alternatively spliced proline-rich cassettes link WNK1 to aldosterone action

    PubMed Central

    Roy, Ankita; Al-Qusairi, Lama; Donnelly, Bridget F.; Ronzaud, Caroline; Marciszyn, Allison L.; Gong, Fan; Chang, Y.P. Christy; Butterworth, Michael B.; Pastor-Soler, Núria M.; Hallows, Kenneth R.; Staub, Olivier; Subramanya, Arohan R.

    2015-01-01

    The thiazide-sensitive NaCl cotransporter (NCC) is important for renal salt handling and blood-pressure homeostasis. The canonical NCC-activating pathway consists of With-No-Lysine (WNK) kinases and their downstream effector kinases SPAK and OSR1, which phosphorylate NCC directly. The upstream mechanisms that connect physiological stimuli to this system remain obscure. Here, we have shown that aldosterone activates SPAK/OSR1 via WNK1. We identified 2 alternatively spliced exons embedded within a proline-rich region of WNK1 that contain PY motifs, which bind the E3 ubiquitin ligase NEDD4-2. PY motif–containing WNK1 isoforms were expressed in human kidney, and these isoforms were efficiently degraded by the ubiquitin proteasome system, an effect reversed by the aldosterone-induced kinase SGK1. In gene-edited cells, WNK1 deficiency negated regulatory effects of NEDD4-2 and SGK1 on NCC, suggesting that WNK1 mediates aldosterone-dependent activity of the WNK/SPAK/OSR1 pathway. Aldosterone infusion increased proline-rich WNK1 isoform abundance in WT mice but did not alter WNK1 abundance in hypertensive Nedd4-2 KO mice, which exhibit high baseline WNK1 and SPAK/OSR1 activity toward NCC. Conversely, hypotensive Sgk1 KO mice exhibited low WNK1 expression and activity. Together, our findings indicate that the proline-rich exons are modular cassettes that convert WNK1 into a NEDD4-2 substrate, thereby linking aldosterone and other NEDD4-2–suppressing antinatriuretic hormones to NCC phosphorylation status. PMID:26241057

  3. Sampling efficiency of modified 37-mm sampling cassettes using computational fluid dynamics.

    PubMed

    Anthony, T Renée; Sleeth, Darrah; Volckens, John

    2016-01-01

    In the U.S., most industrial hygiene practitioners continue to rely on the closed-face cassette (CFC) to assess worker exposures to hazardous dusts, primarily because ease of use, cost, and familiarity. However, mass concentrations measured with this classic sampler underestimate exposures to larger particles throughout the inhalable particulate mass (IPM) size range (up to aerodynamic diameters of 100 μm). To investigate whether the current 37-mm inlet cap can be redesigned to better meet the IPM sampling criterion, computational fluid dynamics (CFD) models were developed, and particle sampling efficiencies associated with various modifications to the CFC inlet cap were determined. Simulations of fluid flow (standard k-epsilon turbulent model) and particle transport (laminar trajectories, 1-116 μm) were conducted using sampling flow rates of 10 L min(-1) in slow moving air (0.2 m s(-1)) in the facing-the-wind orientation. Combinations of seven inlet shapes and three inlet diameters were evaluated as candidates to replace the current 37-mm inlet cap. For a given inlet geometry, differences in sampler efficiency between inlet diameters averaged less than 1% for particles through 100 μm, but the largest opening was found to increase the efficiency for the 116 μm particles by 14% for the flat inlet cap. A substantial reduction in sampler efficiency was identified for sampler inlets with side walls extending beyond the dimension of the external lip of the current 37-mm CFC. The inlet cap based on the 37-mm CFC dimensions with an expanded 15-mm entry provided the best agreement with facing-the-wind human aspiration efficiency. The sampler efficiency was increased with a flat entry or with a thin central lip adjacent to the new enlarged entry. This work provides a substantial body of sampling efficiency estimates as a function of particle size and inlet geometry for personal aerosol samplers. PMID:26513395

  4. Background illumination simulator

    SciTech Connect

    Towry, E.R.

    1992-05-12

    This patent describes a testing apparatus for testing and evaluating the performance of laser seeking warheads for missiles, under simulated weather conditions. It comprises support means for supporting a warhead seeker; laser means for generating a laser beam and for directing a laser beam towards the seeker; a diffusion screen interposed between the seeker support means and the laser means for diffusing the laser beam; a collimating lens interposed between the diffusion screen and the seeker support means for collimating the diffused laser beam and for directing the collimated laser beam onto a warhead seeker, supported in the seeker support; background illuminator means for illuminating the seeker support and a seeker disposed therein, supported for movement into and out of an operating position between the diffusion means and the collimating lens for providing background lighting in simulation of weather lighting conditions; and control means for controlling the intensity of the light provided by the illuminator means to simulate various weather conditions.

  5. Some background about satellites

    NASA Technical Reports Server (NTRS)

    Burns, Joseph A.

    1986-01-01

    Four tables of planetary and satellite data are presented which list satellite discoveries, planetary parameters, satellite orbits, and satellite physical properties respectively. A scheme for classifying the satellites is provided and it is noted that most known moons fall into three general classes: regular satellites, collisional shards, and irregular satellites. Satellite processes are outlined with attention given to origins, dynamical and thermal evolution, surface processes, and composition and cratering. Background material is provided for each family of satellites.

  6. Comparison of Lead and Tin Concentrations in Air at a Solder Manufacturer from the Closed-Face 37-mm Cassette With and Without a Custom Cellulose-Acetate Cassette Insert

    PubMed Central

    Lee, Eun Gyung; Chisholm, William P.; Burns, Dru A.; Nelson, John H.; Kashon, Michael L.; Harper, Martin

    2015-01-01

    A polyvinyl chloride (PVC) cassette insert with PVC filter (ACCU-CAP) in a 37-mm closed-face cassette (CFC) was designed for gravimetric analysis. A customized version of the ACCU-CAP, also to be used in the CFC, was manufactured from an acid-digestible cellulose-acetate cassette insert joined to a mixed cellulose ester (MCE) filter for wet chemical analysis. The aim of this study was to compare metal particle concentrations as sampled by the customized insert (CI) in a CFC sampler with the traditional sampling method using only a MCE filter in the CFC. Thirty-nine personal and 13 area samples were taken using paired filter-based CFC and the CI in CFC samplers at a solder manufacturing plant. The CI was removed from its CFC, and digested and analyzed as a whole. The MCE filter from the typical CFC was removed for analysis and then the interior of the cassette was wiped with Ghost Wipe for a separate analysis. The MCE filter only, Ghost Wipe, and CI were separately dissolved in heated nitric acid for ICP-MS analysis. Overall, the geometric mean concentration of the filter-only (FO) samples was considerably lower than that of the CI samples, by 53% for lead and 32% for tin. However, if the FO analysis was added to the corresponding Ghost Wipe analysis, i.e., filter+interior wipe (FW), the geometric mean concentrations of the FW results were similar to those of the CI results (by 113% for lead and 98% for tin). For both lead and tin the comparison of (log-transformed) metal concentrations between the FW and CI results showed no statistically significant difference (p-value = 0.3009 for lead and 0.800 for tin), while the comparison between the FO and CI results shows statistically significant differences (all p-values < 0.05). In conclusion, incorporating the sampler internal non-filter deposits by wiping or use of an internal filter capsule gave higher results than analyzing only the filter. Close agreement between the two methods of including non-filter deposits is

  7. Comparison of lead and tin concentrations in air at a solder manufacturer from the closed-face 37-mm cassette with and without a custom cellulose-acetate cassette insert.

    PubMed

    Lee, Eun Gyung; Chisholm, William P; Burns, Dru A; Nelson, John H; Kashon, Michael L; Harper, Martin

    2014-01-01

    A polyvinyl chloride (PVC) cassette insert with PVC filter (ACCU-CAP) in a 37-mm closed-face cassette (CFC) was designed for gravimetric analysis. A customized version of the ACCU-CAP, also to be used in the CFC, was manufactured from an acid-digestible cellulose-acetate cassette insert joined to a mixed cellulose ester (MCE) filter for wet chemical analysis. The aim of this study was to compare metal particle concentrations as sampled by the customized insert (CI) in a CFC sampler with the traditional sampling method using only a MCE filter in the CFC. Thirty-nine personal and 13 area samples were taken using paired filter-based CFC and the CI in CFC samplers at a solder manufacturing plant. The CI was removed from its CFC, and digested and analyzed as a whole. The MCE filter from the typical CFC was removed for analysis and then the interior of the cassette was wiped with Ghost Wipe for a separate analysis. The MCE filter only, Ghost Wipe, and CI were separately dissolved in heated nitric acid for ICP-MS analysis. Overall, the geometric mean concentration of the filter-only (FO) samples was considerably lower than that of the CI samples, by 53% for lead and 32% for tin. However, if the FO analysis was added to the corresponding Ghost Wipe analysis, i.e., filter+interior wipe (FW), the geometric mean concentrations of the FW results were similar to those of the CI results (by 113% for lead and 98% for tin). For both lead and tin the comparison of (log-transformed) metal concentrations between the FW and CI results showed no statistically significant difference (p-value = 0.3009 for lead and 0.800 for tin), while the comparison between the FO and CI results shows statistically significant differences (all p-values < 0.05). In conclusion, incorporating the sampler internal non-filter deposits by wiping or use of an internal filter capsule gave higher results than analyzing only the filter. Close agreement between the two methods of including non-filter deposits is

  8. Changes in expression of splice cassettes of NMDA receptor GluN1 subunits within the frontal lobe and memory in mice during aging.

    PubMed

    Das, Siba R; Magnusson, Kathy R

    2011-09-12

    Age-related decline in memory has been associated with changes in mRNA and protein expression of different NMDA receptor subunits. The NMDA receptor GluN1 subunit appears to be necessary and sufficient for receptor function. There is evidence that the mRNA expressions of some splice forms of the subunit are influenced by aging and/or behavioral testing experience in old mice. The present study explored the relationships between behavioral testing experience and protein expression of different GluN1 subunit isoforms in the prefrontal/frontal cortex of the brain during aging. Aged C57BL/6 mice with behavioral testing experience showed declines in performance in both spatial working and reference memory tasks. Protein expression of GluN1 C-terminal cassettes C2 and C2', but not the C1 or N1 cassettes, was observed to decline with increasing age, regardless of experience. In middle-age animals, higher expressions of the GluN1 subunit and C2' cassette proteins were associated with good reference memory on initial search. Aged animals with a higher protein expression of GluN1 subunits containing C1 cassettes and the whole population of GluN1 subunits exhibited a closer proximity to the former platform location within the final phase of probe trials. However, the old mice with high expression of the C1 cassette did not show an accurate search during this phase. The old mice with lower expression of the C1 cassette protein more closely mimicked the performances of the young and middle-aged mice. These results indicate that there was heterogeneity in the effect of aging on the expression of the GluN1 subunits containing different splice cassettes. It also suggests that the GluN1 subunit might be most important for good reference memory during middle age, but this relationship may not be maintained into old age. PMID:21443909

  9. The Backgrounds Data Center

    NASA Technical Reports Server (NTRS)

    Snyder, W. A.; Gursky, H.; Heckathorn, H. M.; Lucke, R. L.; Berg, S. L.; Dombrowski, E. G.; Kessel, R. A.

    1993-01-01

    The Strategic Defense Initiative Organization has created data centers for midcourse, plumes, and backgrounds phenomenologies. The Backgrounds Data Center (BDC) has been designated as the prime archive for data collected by SDIO programs. The BDC maintains a Summary Catalog that contains 'metadata,' that is, information about data, such as when the data were obtained, what the spectral range of the data is, and what region of the Earth or sky was observed. Queries to this catalog result in a listing of all data sets (from all experiments in the Summary Catalog) that satisfy the specified criteria. Thus, the user can identify different experiments that made similar observations and order them from the BDC for analysis. On-site users can use the Science Analysis Facility (SAFE for this purpose. For some programs, the BDC maintains a Program Catalog, which can classify data in as many ways as desired (rather than just by position, time, and spectral range as in the Summary Catalog). For example, data sets could be tagged with such diverse parameters as solar illumination angle, signal level, or the value of a particular spectral ratio, as long as these quantities can be read from the digital record or calculated from it by the ingest program. All unclassified catalogs and unclassified data will be remotely accessible.

  10. Backgrounds Data Center

    NASA Astrophysics Data System (ADS)

    Snyder, William A.; Gursky, Herbert; Heckathorn, Harry M.; Lucke, Bob L.; Dorland, Bryan N.; Kessel, R. A.; Berg, S. L.; Dombrowski, E. G.

    1994-09-01

    The Backgrounds Data Center (BDC) is the designated archive for backgrounds data collected by Ballistic Missile Defense Organization (BMDO) programs, some of which include ultraviolet sensors. Currently, the BDC holds ultraviolet data from the IBSS, UVPI, UVLIM, and FUVCAM sensors. The BDC will also be the prime archive for Midcourse Space Experiment (MSX) data and is prepared to negotiate with program managers to handle other datasets. The purpose of the BDC is to make data accessible to users and to assist them in analyzing it. The BDC maintains the Science Catalog Information Exchange System (SCIES) allowing remote users to log in, read or post notices about current programs, search the catalogs for datasets of interest, and submit orders for data. On-site facilities are also available for the analysis of data, and consist of VMS and UNIX workstations with access to software analysis packages such as IDL, IRAF, and Khoros. Either on-site or remotely, users can employ the BDC-developed graphical user interface called the Visual Interface for Space and Terrestrial Analysis (VISTA) to generate catalog queries and to display and analyze data. SCIES and VISTA permit nearly complete access to BDC services and capabilities without the need to be physically present at the data center.

  11. Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

    SciTech Connect

    Deshpande, Chandrika N.; Harrop, Stephen J.; Boucher, Yan; Hassan, Karl A.; Di Leo, Rosa; Xu, Xiaohui; Cui, Hong; Savchenko, Alexei; Chang, Changsoo; Labbate, Maurizio; Paulsen, Ian T.; Stokes, H.W.; Curmi, Paul M.G.; Mabbutt, Bridget C.

    2012-02-15

    The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. We report the 1.8 {angstrom} crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.

  12. UMP kinase from Streptococcus pneumoniae: evidence for co-operative ATP binding and allosteric regulation

    PubMed Central

    2004-01-01

    UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl2 or MgCl2, the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (Km=0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S0.5=9.4±0.7 mM and n=1.9±0.1 in the presence of MgCl2). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient. The kcat (175±13 s−1 in the presence of MgCl2) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP. PMID:15324307

  13. Partial Excision of the Chromosomal Cassette Containing the Methicillin Resistance Determinant Results in Methicillin-Susceptible Staphylococcus aureus

    PubMed Central

    Donnio, Pierre-Yves; Oliveira, Duarte C.; Faria, Nuno A.; Wilhelm, Nathalie; Le Coustumier, Alain; de Lencastre, Herminia

    2005-01-01

    We report a detailed characterization of methicillin-susceptible Staphylococcus aureus isolates from five French hospitals negative for both the mecA and the ccrAB loci but positive for the IS431::pUB110::IS431::dcs structure, present in some Staphylococcus cassette chromosome mec (SCCmec) types. The presence of SCCmec-associated elements suggests that this unusual resistant phenotype is due to a partial excision of SCCmec from epidemic methicillin-resistant S. aureus. The hypothesis of a genetic relatedness is strengthened by common sequence and spa types and similar susceptibility patterns. PMID:16081974

  14. The cosmic background explorer

    SciTech Connect

    Gulkis, G. ); Lubin, P.M. ); Meyer, S.S. ); Silverberg, R.F.

    1990-01-01

    Late last year the National Aeronautics and Space Administration launched its first satellite dedicated to the study of phenomena related to the origins of the universe. The satellite, called the Cosmic Background Explorer (COBE), carries three complementary detectors that will make fundamental measurements of the celestial radiation. Part of that radiation is believed to have originated in processes that occurred at the very dawn of the universe. By measuring the remnant radiation at wavelengths from one micrometer to one centimeter across the entire sky, scientists hope to be able to solve many mysteries regarding the origin and evolution of the early universe. Unfortunately, these radiative relics of the early universe are weak and veiled by local astrophysical and terrestrial sources of radiation. The wavelengths of the various cosmic components may also overlap, thereby making the understanding of the diffuse celestial radiation a challenge. Nevertheless, the COBE instruments, with their full-sky coverage, high sensitivity to a wide range of wavelengths and freedom from interference from the earth's atmosphere, will constitute for astrophysicists an observatory of unprecedented sensitivity and scope. The interesting cosmic signals will then be separated from one another and from noncosmic radiation sources by a comprehensive analysis of the data.

  15. Genetical background of intelligence.

    PubMed

    Junkiert-Czarnecka, Anna; Haus, Olga

    2016-01-01

    Intelligence as an ability to reason, think abstractly and adapt effectively to the environment is a subject of research in the field of psychology, neurobiology, and in the last twenty years genetics as well. Genetical testing of twins carried out from XX century indicated heritebility of intelligence, therefore confirmed an influence of genetic factor on cognitive processes. Studies on genetic background of intelligence focus on dopaminergic (DRD2, DRD4, COMT, SLC6A3, DAT1, CCKAR) and adrenergic system (ADRB2, CHRM2) genes as well as, neutrofins (BDNF) and oxidative stress genes (LTF, PRNP). Positive effect of investigated gene polymorphism was indicated by variation c.957C>T DRD2 gene (if in polymorphic site is thymine), polymorphism c.472G>A COMT gene (presence of adenine) and also gene ADRB2 c.46A->G (guanine), CHRM2 (thymine in place c.1890A>T) and BDNF (guanine in place c.472G>A) Obtained results indicate that intelligence is a feature dependent not only on genetic but also an environmental factor. PMID:27333929

  16. Maritime infrared background clutter

    NASA Astrophysics Data System (ADS)

    Schwering, Piet B. W.

    1996-06-01

    The detection of small targets in maritime infrared surveillance is hampered by the presence of clutter. Sea surface structure, reflection and emission changes related to incident angle variations and surface effects are standard features governing the clutter behavior. Also special effects as sun glint and horizon effects play an important role for clutter. In order to optimize the detection process, quantitative clutter estimates are of use for filter settings. We have recorded a large amount of infrared backgrounds in the last few years, during common NATO trials. A large amount of different meteorological conditions took place during the various experiments. A first set of these data have been analyzed to obtain statistical data that represent the infrared scene. We have derived vertical temperature profiles, vertical fluctuation profiles, horizontal correlation coefficients and temporal correlation functions. In this paper we present the first analysis of these data. We are in the process of obtaining a condensed database of information to regenerate clutter images from bulk meteo parameters, and clutter parameters. The clutter and meteo parameters have been used to simulate various infrared scenes. Examples of this simulation process are shown in the presentation. The simulated images are statistically similar to the original images that were used to derive the parameters. A description of the image- generation is presented. Future expansions of the model are discussed.

  17. Biological aerosol background characterization

    NASA Astrophysics Data System (ADS)

    Blatny, Janet; Fountain, Augustus W., III

    2011-05-01

    To provide useful information during military operations, or as part of other security situations, a biological aerosol detector has to respond within seconds or minutes to an attack by virulent biological agents, and with low false alarms. Within this time frame, measuring virulence of a known microorganism is extremely difficult, especially if the microorganism is of unknown antigenic or nucleic acid properties. Measuring "live" characteristics of an organism directly is not generally an option, yet only viable organisms are potentially infectious. Fluorescence based instruments have been designed to optically determine if aerosol particles have viability characteristics. Still, such commercially available biological aerosol detection equipment needs to be improved for their use in military and civil applications. Air has an endogenous population of microorganisms that may interfere with alarm software technologies. To design robust algorithms, a comprehensive knowledge of the airborne biological background content is essential. For this reason, there is a need to study ambient live bacterial populations in as many locations as possible. Doing so will permit collection of data to define diverse biological characteristics that in turn can be used to fine tune alarm algorithms. To avoid false alarms, improving software technologies for biological detectors is a crucial feature requiring considerations of various parameters that can be applied to suppress alarm triggers. This NATO Task Group will aim for developing reference methods for monitoring biological aerosol characteristics to improve alarm algorithms for biological detection. Additionally, they will focus on developing reference standard methodology for monitoring biological aerosol characteristics to reduce false alarm rates.

  18. Methicillin-Resistant Staphylococcus aureus Isolated From Various Types of Hospital Infections in Pediatrics: Panton-Valentine Leukocidin, Staphylococcal Chromosomal Cassette mec SCCmec Phenotypes and Antibiotic Resistance Properties

    PubMed Central

    Dormanesh, Banafshe; Siroosbakhat, Soheila; Khodaverdi Darian, Ebrahim; Afsharkhas, Ladan

    2015-01-01

    Background: Staphylococcus aureus has long been considered as a major pathogen of hospital infections. Objectives: The present investigation was carried out to study the distribution of Staphylococcal Chromosomal Cassette mec (SCCmec) types, Panton-Valentine Leukocidin (PVL) gene and antibiotic resistance properties of Methicillin Resistant Staphylococcus aureus (MRSA) strains isolated from various types of infections found in Iranian pediatric patients. Patients and Methods: Two-hundred and fifty-five clinical specimens were collected from four major provinces of Iran. Samples were cultured and the MRSA strains were subjected to Polymerase Chain Reaction (PCR). The patterns of antibiotic resistance were determined using the disk diffusion method. Results: Seventy-four out of 255 (29.01%) clinical samples were positive for MRSA. Of the 74 MRSA strains, 47 (63.51%) were PVL positive. The clinical samples of respiratory tract infections (36.36%), those from the Shiraz province (37.87%) and samples collected during the summer season (56.48%) were the most commonly infected samples. The most commonly detected antibiotic resistance genes were tetK (89.18%), mecA (71.62%), msrA (56.75%) and tetM (54.05%). Methicillin Resistant Staphylococcus aureus had the highest levels of resistance against penicillin (100%), tetracycline (98.64%), ampicillin (93.24%) and oxacillin (93.24%). The most commonly detected SCCmec types in the MRSA strains were type V (18.91%) and III (17.56%). Conclusions: Regular surveillance of hospital-associated infections and monitoring of the antibiotic sensitivity patterns are required to reduce the prevalence of MRSA. We recommend initial management of children affected by MRSA with imipenem, lincomycin and cephalothin prescriptions. PMID:26862375

  19. A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia.

    PubMed

    Ostedgaard, Lynda S; Rokhlina, Tatiana; Karp, Philip H; Lashmit, Philip; Afione, Sandra; Schmidt, Michael; Zabner, Joseph; Stinski, Mark F; Chiorini, Jay A; Welsh, Michael J

    2005-02-22

    Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds approximately 4,900-5,000 bp. To partially circumvent this size constraint, we previously developed a CF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTRDeltaR). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate/early (CMVie) enhancer/promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTRDeltaR transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl(-) transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer. PMID:15703296

  20. A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia

    PubMed Central

    Ostedgaard, Lynda S.; Rokhlina, Tatiana; Karp, Philip H.; Lashmit, Philip; Afione, Sandra; Schmidt, Michael; Zabner, Joseph; Stinski, Mark F.; Chiorini, Jay A.; Welsh, Michael J.

    2005-01-01

    Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds ≈4,900–5,000 bp. To partially circumvent this size constraint, we previously developed a CF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTRΔR). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate/early (CMVie) enhancer/promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTRΔR transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl– transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer. PMID:15703296

  1. Design and construction of two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast.

    PubMed

    Abdemami, Baharak; Shokrgozar, Mohammad Ali; Shahreza, Hossein Khanahmad; Ghavami, Mehdi

    2011-01-01

    Collagens are the most abundant proteins in the human body. Their main function is to provide structural and mechanical support for the tissues, but they are also involved in a number of other biological functions including cell attachment, migration and differentiation. Collagens and gelatins are widely used in pharmaceutical and medical applications. Every year, more than 50,000 tons of collagen and gelatin are used in medical applications. These materials may have some viral and prion impurity and/or stimulate allergic response in human body. Therefore, scientists have produced human collagen in recombinant systems. In this study we have constructed two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast. Total RNA was extracted from human skin fibroblast cell line, and cDNA synthesis was done by oligo dt. Then gene fragments were amplified from the cDNA with the necessary changes by Polymerase Chain Reaction (PCR). Finally they were cloned in yeast vector pPICZαA containing regulatory sequences for expressing and secreting the polypeptide product. Two yeast shuttle vectors containing human COL1A1 and COL1A2 expression cassettes were created. Final constructs were confirmed by enzymatic digestion, PCR of desired fragment and sequencing. The yeast shuttle vectors containing human COL1A1 and COL1A2 can be transferred into the yeast in the later stages to determine the scale of expression. PMID:23407617

  2. Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette[W][OPEN

    PubMed Central

    Siehl, Daniel L.; Tao, Yumin; Albert, Henrik; Dong, Yuxia; Heckert, Matthew; Madrigal, Alfredo; Lincoln-Cabatu, Brishette; Lu, Jian; Fenwick, Tamara; Bermudez, Ericka; Sandoval, Marian; Horn, Caroline; Green, Jerry M.; Hale, Theresa; Pagano, Peggy; Clark, Jenna; Udranszky, Ingrid A.; Rizzo, Nancy; Bourett, Timothy; Howard, Richard J.; Johnson, David H.; Vogt, Mark; Akinsola, Goke; Castle, Linda A.

    2014-01-01

    With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione. PMID:25192697

  3. Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors.

    PubMed

    Ramachandra, Chrishan J A; Shahbazi, Mohammad; Kwang, Timothy W X; Choudhury, Yukti; Bak, Xiao Ying; Yang, Jing; Wang, Shu

    2011-09-01

    Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome. PMID:21685448

  4. A vector system for efficient and economical switching of a ura4(+) module to three commonly used antibiotic marker cassettes in Schizosaccharomyces pombe.

    PubMed

    Chen, Yinghui; Chen, Lihua; An, Ke; Wang, Yamei; Jin, Quanwen

    2015-11-01

    We describe here the development of a set of plasmid vectors that allow simple, efficient and economical switching of a ura4(+) module in existing Schizosaccharomyces pombe strains to any of the three routinely used antibiotic marker cassettes, kanMX6, hphMX6 and natMX6. In principle, the applications of this system can also be extended to switching ura4(+) for additional MX6 module-based cassettes, such as bleMX6, as long as the antibiotic marker has been cloned into an ura4(+) module-switching vector. We illustrate the application of this set of vectors in exchange of the ura4(+) marker in existing strains with three antibiotic marker cassettes with high efficiency. PMID:26343233

  5. Background sources in optical communications

    NASA Technical Reports Server (NTRS)

    Vilnrotter, V. A.

    1983-01-01

    The characterization and measurement of background radiation relevant to optical communications system performance is addressed. The necessary optical receiver parameters are described, and radiometric concepts required for the calculation of collected background power are developed. The most important components of optical background power are discussed, and their contribution to the total collected background power in various communications scenarios is examined.

  6. A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line

    PubMed Central

    2012-01-01

    Background Although chronic myeloid leukemia (CML) treatment has improved since the introduction of imatinib mesylate (IM), cases of resistance have been reported. This resistance has been associated with the emergence of multidrug resistance (MDR) phenotype, as a BCR-ABL independent mechanism. The classic pathway studied in MDR promotion is ATP-binding cassette (ABC) family transporters expression, but other mechanisms that drive drug resistance are largely unknown. To better understand IM therapy relapse due to the rise of MDR, we compared the proteomic profiles of K562 and Lucena (K562/VCR) cells. Results The use of 2-DE coupled with a MS approach resulted in the identification of 36 differentially expressed proteins. Differential mRNA levels of leucine-rich PPR motif-containing (LRPPRC) protein, minichromosome maintenance complex component 7 (MCM7) and ATP-binding cassette sub-family B (MDR/TAP) member 1 (ABCB1) were capable of defining samples from CML patients as responsive or resistant to therapy. Conclusions Through the data presented in this work, we show the relevance of MDR to IM therapy. In addition, our proteomic approach identified candidate actors involved in resistance, which could lead to additional information on BCR-ABL-independent molecular mechanisms. PMID:22458888

  7. WBC27, an adenosine tri-phosphate-binding cassette protein, controls pollen wall formation and patterning in Arabidopsis.

    PubMed

    Dou, Xiao-Ying; Yang, Ke-Zhen; Zhang, Yi; Wang, Wei; Liu, Xiao-Lei; Chen, Li-Qun; Zhang, Xue-Qin; Ye, De

    2011-01-01

    In flowering plants, the exine components are derived from tapetum. Despite its importance to sexual plant reproduction, little is known about the translocation of exine materials from tapetum to developing microspores. Here we report functional characterization of the arabidopsis WBC27 gene. WBC27 encodes an adenosine tri-phosphate binding cassette (ABC) transporter and is expressed preferentially in tapetum. Mutation of WBC27 disrupted the exine formation. The wbc27 mutant microspores began to degenerate once released from tetrads and most of the microspores collapsed at the uninucleate stage. Only a small number of wbc27-1 microspores could develop into tricellular pollen grains. These survival pollen grains lacked exine and germinated in the anther before anthesis. All of these results suggest that the ABC transporter, WBC27 plays important roles in the formation of arabidopsis exine, possibly by translocation of lipidic precursors of sporopollenin from tapetum to developing microspores. PMID:21205178

  8. Functions of the conserved thrombospondin carboxy-terminal cassette in cell-extracellular matrix interactions and signaling.

    PubMed

    Adams, Josephine C

    2004-06-01

    Thrombospondins (TSPs) are extracellular, multidomain, calcium-binding glycoproteins that function at cell surfaces, in extracellular matrix (ECM) and as bridging molecules in cell-cell interactions. TSPs are multifunctional and modulate cell behavior during development, wound-healing, immune response, tumor growth and in the homeostasis of adult tissues. TSPs are assembled as oligomers that are composed of homologous polypeptides. In all the TSP polypeptides, the most highly-conserved region is the carboxyl-region, which contains a characteristic set of domains comprising EGF domains, TSP type 3 repeats and a globular carboxy-terminal domain. This large region is termed here the thrombospondin carboxy-terminal cassette (TSP-CTC). The strong conservation of the TSP-CTC suggests that it may mediate ancestral functions that are shared by all TSPs. This review summarizes the current knowledge of the TSP-CTC and areas of future interest. PMID:15094125

  9. Integration of a contactless conductivity detector into a commercial capillary cassette. Detection of inorganic cations and catecholamines.

    PubMed

    Vuorinen, Pasi S; Jussila, Matti; Sirén, Heli; Palonen, Sami; Riekkola, Marja-Liisa

    2003-03-21

    A contactless conductivity detector integrated into the capillary cassette of Agilent (3D)CE equipment is described. The detector is user-friendly, compact and easily modified. The UV detector of