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1

ATP-binding cassette systems in Burkholderia pseudomallei and Burkholderia mallei  

Microsoft Academic Search

BACKGROUND: ATP binding cassette (ABC) systems are responsible for the import and export of a wide variety of molecules across cell membranes and comprise one of largest protein superfamilies found in prokarya, eukarya and archea. ABC systems play important roles in bacterial lifestyle, virulence and survival. In this study, an inventory of the ABC systems of Burkholderia pseudomallei strain K96243

David N Harland; Elie Dassa; Richard W Titball; Katherine A Brown; Helen S Atkins

2007-01-01

2

ATP-Binding Cassette Efflux Transporters in Human Placenta  

PubMed Central

Pregnant women are often complicated with diseases including viral or bacterial infections, epilepsy, hypertension, or pregnancy-induced conditions such as depression and gestational diabetes that require treatment with medication. In addition, substance abuse during pregnancy remains a major public health problem. Many drugs used by pregnant women are off label without the necessary dose, efficacy, and safety data required for rational dosing regimens of these drugs. Thus, a major concern arising from the widespread use of drugs by pregnant women is the transfer of drugs across the placental barrier, leading to potential toxicity to the developing fetus. Knowledge regarding the ATP-binding cassette (ABC) efflux transporters, which play an important role in drug transfer across the placental barrier, is absolutely critical for optimizing the therapeutic strategy to treat the mother while protecting the fetus during pregnancy. Such transporters include P-glycoprotein (P-gp, gene symbol ABCB1), the breast cancer resistance protein (BCRP, gene symbol ABCG2), and the multidrug resistance proteins (MRPs, gene symbol ABCCs). In this review, we summarize the current knowledge with respect to developmental expression and regulation, membrane localization, functional significance, and genetic polymorphisms of these ABC transporters in the placenta and their relevance to fetal drug exposure and toxicity. PMID:21118087

Ni, Zhanglin; Mao, Qingcheng

2010-01-01

3

ATP binding cassette G8 T400K polymorphism may affect the risk of gallstone disease among Chinese males  

Microsoft Academic Search

BackgroundSupersaturation of bile with cholesterol is a primary step in the formation of cholesterol gallstones. ATP binding cassette (ABC) G5 and G8 play an important role in regulating sterol absorption and secretion. To investigate a possible association between transporter gene polymorphism and gallstone formation, we examined five common polymorphisms in the ABCG5 (Q604E) and ABCG8 (D19H, Y54C, T400K, A632V) genes

Yong Wang; Zhao-Yan Jiang; Jian Fei; Lin Xin; Qu Cai; Zhi-Hong Jiang; Zheng-Gang Zhu; Tian-Quan Han; Sheng-Dao Zhang

2007-01-01

4

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

Microsoft Academic Search

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

2010-01-01

5

The ATP-binding cassette transporter ABCA2 as a mediator of intracellular trafficking  

Microsoft Academic Search

ATP-binding cassette (ABC) transporters are a family of proteins that translocate molecules across cellular membranes. Substrates can include lipids, cholesterol and drugs. Mutations in ABC transporter genes can cause human pathologies and drug resistance phenotypes in cancer cells. ABCA2, the second member the A sub-family to be identified, was found at high levels in ovarian carcinoma cells resistant to the

J. T. Mack; V. Beljanski; K. D. Tew; D. M. Townsend

2006-01-01

6

ATP-binding cassette transporter ABCA4: Molecular properties and role in vision and macular degeneration  

Microsoft Academic Search

ABCA4, also known as ABCR or the rim protein, is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters\\u000a expressed in vertebrate rod and cone photoreceptor cells and localized to outer segment disk membranes. ABCA4 is organized\\u000a in two tandem halves, each consisting of a transmembrane segment followed successively by a large exocytoplasmic domain, a\\u000a multispanning membrane

Robert S. Molday

2007-01-01

7

Secretion of Secondary Metabolites by ATP-Binding Cassette Transporters in Plant Cell Suspension Cultures1  

Microsoft Academic Search

The substrate specificity of the yeast (Saccharomyces cerevisiae) pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporters is extended to plant secondary metabolites of the tropane alka- loid family. Functional analysis of yeast PDR5 genes in transgenic tobacco (Nicotiana tabacum L. cv Bright- Yellow 2 (BY-2)) cell lines suggest that PDR genes can be used to stimulate the secretion of secondary

Alain Goossens; Suvi T. Hakkinen; Kirsi-Marja Oksman-Caldentey; Dirk Inze ´

8

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

PubMed

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

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

2002-10-01

9

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

PubMed Central

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

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

2008-01-01

10

Characterization and classification of ATP-binding cassette transporter ABCA3 mutants in fatal surfactant deficiency.  

PubMed

The ATP-binding cassette transporter ABCA3 is expressed predominantly at the limiting membrane of the lamellar bodies in lung alveolar type II cells. Recent study has shown that mutation of the ABCA3 gene causes fatal surfactant deficiency in newborns. In this study, we investigated in HEK293 cells the intracellular localization and N-glycosylation of the ABCA3 mutants so far identified in fatal surfactant deficiency patients. Green fluorescent protein-tagged L101P, L982P, L1553P, Q1591P, and Ins1518fs/ter1519 mutant proteins remained localized in the endoplasmic reticulum, and processing of oligosaccharide was impaired, whereas wild-type and N568D, G1221S, and L1580P mutant ABCA3 proteins trafficked to the LAMP3-positive intracellular vesicle, accompanied by processing of oligosaccharide from high mannose type to complex type. Vanadate-induced nucleotide trapping and ATP-binding analyses showed that ATP hydrolysis activity was dramatically decreased in the N568D, G1221S, and L1580P mutants, accompanied by a moderate decrease in ATP binding in N568D and L1580P mutants but not in the G1221S mutant, compared with the wild-type ABCA3 protein. In addition, mutational analyses of the Gly-1221 residue in the 11th transmembrane segment and the Leu-1580 residue in the cytoplasmic tail, and homology modeling of nucleotide binding domain 2 demonstrate the significance of these residues for ATP hydrolysis and suggest a mechanism for impaired ATP hydrolysis in G1221S and L1580P mutants. Thus, surfactant deficiency because of ABCA3 gene mutation may be classified into two categories as follows: abnormal intracellular localization (type I) and normal intracellular localization with decreased ATP binding and/or ATP hydrolysis of the ABCA3 protein (type II). These distinct pathophysiologies may reflect both the severity and effective therapy for surfactant deficiency. PMID:16959783

Matsumura, Yoshihiro; Ban, Nobuhiro; Ueda, Kazumitsu; Inagaki, Nobuya

2006-11-10

11

Molecular Disruption of the Power Stroke in the ATP-binding Cassette Transport Protein MsbA*  

PubMed Central

ATP-binding cassette transporters affect drug pharmacokinetics and are associated with inherited human diseases and impaired chemotherapeutic treatment of cancers and microbial infections. Current alternating access models for ATP-binding cassette exporter activity suggest that ATP binding at the two cytosolic nucleotide-binding domains provides a power stroke for the conformational switch of the two membrane domains from the inward-facing conformation to the outward-facing conformation. In outward-facing crystal structures of the bacterial homodimeric ATP-binding cassette transporters MsbA from Gram-negative bacteria and Sav1866 from Staphylococcus aureus, two transmembrane helices (3 and 4) in the membrane domains have their cytoplasmic extensions in close proximity, forming a tetrahelix bundle interface. In biochemical experiments on MsbA from Escherichia coli, we show for the first time that a robust network of inter-monomer interactions in the tetrahelix bundle is crucial for the transmission of nucleotide-dependent conformational changes to the extracellular side of the membrane domains. Our observations are the first to suggest that modulation of tetrahelix bundle interactions in ATP-binding cassette exporters might offer a potent strategy to alter their transport activity. PMID:23306205

Doshi, Rupak; Ali, Anam; Shi, Wilma; Freeman, Elizabeth V.; Fagg, Lisa A.; van Veen, Hendrik W.

2013-01-01

12

The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis  

PubMed Central

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters. PMID:22724033

Lawler, Karen; Self, Tim J.; Dyall, Sabrina D.; Kerr, Ian D.

2012-01-01

13

LEM1, an ATP-binding-cassette transporter, selectively modulates the biological potency of steroid hormones.  

PubMed Central

The rat glucocorticoid receptor confers hormone-dependent transcriptional enhancement when expressed in yeast, thereby enabling the genetic identification of nonreceptor proteins that function in the hormone signal-transduction pathway. We isolated a yeast mutant, lem1, with increased sensitivity to dexamethasone and triamcinolone acetonide; responsiveness to a third agonist, deoxycorticosterone, is unaffected. Cloning of wild-type LEM1 revealed a putative transport protein of the ATP-binding cassette family. Dexamethasone accumulation is increased in lem1 cells, suggesting that wild-type LEM1 decreases dexamethasone potency by exporting this ligand. LEM1 appears to affect certain steroids and not others. We propose that transporters like LEM1 can selectively modulate the intracellular levels of steroid hormones. Differential activities of such transporters in mammalian cells might regulate hormone availability and thereby hormone signaling in a cell-type specific manner. Images Fig. 3 PMID:7753868

Kralli, A; Bohen, S P; Yamamoto, K R

1995-01-01

14

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

PubMed

The ATP-binding cassette subfamily B member 1 (ABCB1) is an efflux transporter that excretes xenobiotics and waste matter. High expression of ABCB1 induced by forskolin (FSK) and rifampicin (RIF) in the bovine blastocysts reportedly improves the cellular quality. In the present study, interferon-?, similar to FSK and RIF, was highly potent in inducing the expression of ABCB1 in the bovine blastocysts but did not exhibit an additive effect with FSK and RIF. Bovine blastocysts stimulated by the combined treatment with FSK, RIF, and interferon-? to express high levels of ABCB1 displayed better freezing resistance as indicated by higher cell numbers in post thawing cultures. On transfer to recipients, such embryos established pregnancies with significantly higher frequencies in repeat breeder cows rather than normal ones. PMID:24411494

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

2014-03-15

15

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

PubMed

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/pxa2? 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

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

2014-08-29

16

Candida Drug Resistance Protein 1, a Major Multidrug ATP Binding Cassette Transporter of Candida albicans, Translocates Fluorescent Phospholipids in a  

E-print Network

ABSTRACT: Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump of azole resistance in the pathogenic yeast Candida albicans. This is especially clear in fluconazoleCandida Drug Resistance Protein 1, a Major Multidrug ATP Binding Cassette Transporter of Candida

Menon, Anant K.

17

ATP binding cassette transporter A-I and HDL metabolism: Effects of fatty acids  

PubMed Central

Ample evidence indicates that dietary fatty acids alters the plasma levels of HDL-C. However, the mechanisms underlying the effects of fatty acids still remain elusive. Recent advances in our understanding of ATP binding cassette transporter A1 (ABCA1) function and regulation have provided a valuable insight into the mechanisms by which fatty acids may affect plasma HDL-C levels. ABCA1 mediates the assembly of phospholipids and free cholesterol with apolipoprotein A-I, which is a critical step for HDL biogenesis. Studies have shown that unsaturated fatty acids, but not saturated fatty acids, repress the expression of ABCA1 in vitro. Although information on mechanisms for the fatty acid-mediated regulation of ABCA1 expression is still limited and controversial, recent evidence suggests that unsaturated fatty acids inhibit the expression of ABCA1 at the transcriptional and posttranscriptional levels. The transcriptional repression of ABCA1 expression by unsaturated fatty acids is likely LXR-dependent. Evidence also suggests that histone deacetylation may play a role in the repression. Posttranscriptionally, unsaturated fatty acids may facilitate ABCA1 protein degradation, which may involve phosphorylation of ABCA1 by protein kinases. Further studies are warranted to better understand the role of dietary fatty acids in HDL metabolism and their effects on cardiovascular health. PMID:21684139

Lee, Jiyoung; Park, Youngki; Koo, Sung I.

2011-01-01

18

Fructose Uptake in Bifidobacterium longum NCC2705 Is Mediated by an ATP-binding Cassette Transporter*  

PubMed Central

Recently, a putative ATP-binding cassette (ABC) transport system was identified in Bifidobacterium longum NCC2705 that is highly up-regulated during growth on fructose as the sole carbon source. Cloning and expression of the corresponding ORFs (bl0033–0036) result in efficient fructose uptake by bacteria. Sequence analysis reveals high similarity to typical ABC transport systems and suggests that these genes are organized as an operon. Expression of FruE is induced by fructose, ribose, or xylose and is able to bind these sugars with fructose as the preferred substrate. Our data suggest that BL0033–0036 constitute a high affinity fructose-specific ABC transporter of B. longum NCC2705. We thus suggest to rename the coding genes to fruEKFG and the corresponding proteins to FruE (sugar-binding protein), FruK (ATPase subunit), FruF, and FruG (membrane permeases). Furthermore, protein-protein interactions between the components of the transporter complex were determined by GST pulldown and Western blot analysis. This revealed interactions between the membrane subunits FruF and FruG with FruE, which in vivo is located on the external side of the membrane, and with the cytoplasmatic ATPase FruK. This is in line with the proposed model for bacterial ABC sugar transporters. PMID:22102285

Wei, Xiao; Guo, Yanhong; Shao, Changlin; Sun, Zhongke; Zhurina, Daria; Liu, Dawei; Liu, Wei; Zou, Dayang; Jiang, Zheng; Wang, Xuesong; Zhao, Jiangli; Shang, Wei; Li, Xuelian; Liao, Xiangru; Huang, Liuyu; Riedel, Christian U.; Yuan, Jing

2012-01-01

19

The ATP-Binding Cassette Transporter ABCB19 Regulates Postembryonic Organ Separation in Arabidopsis  

PubMed Central

The phytohormone auxin plays a critical role in plant development, including embryogenesis, organogenesis, tropism, apical dominance and in cell growth, division, and expansion. In these processes, the concentration gradient of auxin, which is established by polar auxin transport mediated by PIN-FORMED (PIN) proteins and several ATP-binding cassette/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) transporters, is a crucial signal. Here, we characterized the function of ABCB19 in the control of Arabidopsis organ boundary development. We identified a new abcb19 allele, abcb19-5, which showed stem-cauline leaf and stem-pedicel fusion defects. By virtue of the DII-VENUS marker, the auxin level was found to be increased at the organ boundary region in the inflorescence apex. The expression of CUP-SHAPED COTYLEDON2 (CUC2) was decreased, while no obvious change in the expression of CUC3 was observed, in abcb19. In addition, the fusion defects were greatly enhanced in cuc3 abcb19-5, which was reminiscent of cuc2 cuc3. We also found that some other organ boundary genes, such as LOF1/2 were down-regulated in abcb19. Together, these results reveal a new aspect of auxin transporter ABCB19 function, which is largely dependent on the positive regulation of organ boundary genes CUC2 and LOFs at the postembryonic organ boundary. PMID:23560110

Mo, Huixian; Qian, Litao; Cao, Ying; Cui, Sujuan; Li, Xia; Ma, Ligeng

2013-01-01

20

Novel roles for ATP-binding cassette G transporters in lipid redistribution in Toxoplasma.  

PubMed

Toxoplasma is a protozoan parasite proficiently adapted to thrive in a parasitophorous vacuole (PV) formed in the cytoplasm of a large variety of mammalian cells. As an actively dividing organism, the parasite must adjust the lipid composition of its membranes to preserve organelle vitality and expand the size of the PV membrane to accommodate growing progeny. We showed that Toxoplasma takes up host lipids and can expel major lipids in an ATP-dependent process. In order to provide detailed mechanistic insights into lipid trafficking phenomena relevant to Toxoplasma biology, we characterized six parasite ATP-binding cassette (ABC) G family transporters and investigated their potential contribution to lipid homeostatic processes. All these transporters are expressed in the parasite and five of them are upregulated upon exposure to sterols. Four ABCG are localized to secretory organelles and the plasma membrane, and promote cholesterol and phospholipid efflux, reflecting the importance in exportation of large amounts of lipids into the PV. Interestingly, one ABCG that is associated with vesicles in the PV and the plasma membrane acts as a cholesterol importer. This last finding expands our current view on the role of some ABCG transporters in eukaryotic sterol influx. PMID:20487267

Ehrenman, Karen; Sehgal, Alfica; Lige, Bao; Stedman, Timothy T; Joiner, Keith A; Coppens, Isabelle

2010-06-01

21

A Plant Plasma Membrane ATP Binding Cassette–Type Transporter Is Involved in Antifungal Terpenoid Secretion  

PubMed Central

ATP binding cassette (ABC) transporters, which are found in all species, are known mainly for their ability to confer drug resistance. To date, most of the ABC transporters characterized in plants have been localized in the vacuolar membrane and are considered to be involved in the intracellular sequestration of cytotoxins. Working on the assumption that certain ABC transporters might be involved in defense metabolite secretion and their expression might be regulated by the concentration of these metabolites, we treated a Nicotiana plumbaginifolia cell culture with sclareolide, a close analog of sclareol, an antifungal diterpene produced at the leaf surface of Nicotiana spp; this resulted in the appearance of a 160-kD plasma membrane protein, which was partially sequenced. The corresponding cDNA (NpABC1) was cloned and shown to encode an ABC transporter. In vitro and in situ immunodetection showed NpABC1 to be localized in the plasma membrane. Under normal conditions, expression was found in the leaf epidermis. In cell culture and in leaf tissues, NpABC1 expression was strongly enhanced by sclareolide and sclareol. In parallel with NpABC1 induction, cells acquired the ability to excrete a labeled synthetic sclareolide derivative. These data suggest that NpABC1 is involved in the secretion of a secondary metabolite that plays a role in plant defense. PMID:11340184

Jasi?ski, Michal; Stukkens, Yvan; Degand, Hervé; Purnelle, Bénédicte; Marchand-Brynaert, Jacqueline; Boutry, Marc

2001-01-01

22

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

PubMed Central

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 ?M) significantly decreases the resistance to mitoxantrone (MX), SN38 and doxorubicin in HEK293 and H460 cells overexpressing the ABCG2 transporter. In addition, masitinib (2.5 ?M) 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 ?M) 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

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

23

Novel roles for ATP-binding cassette G transporters in lipid redistribution in Toxoplasma  

PubMed Central

Summary Toxoplasma is a protozoan parasite proficiently adapted to thrive in a parasitophorous vacuole (PV) formed in the cytoplasm of a large variety of mammalian cells. As an actively dividing organism, the parasite must adjust the lipid composition of its membranes to preserve organelle vitality and expand the size of the PV membrane to accommodate growing progeny. We showed that Toxoplasma takes up host lipids and can expel major lipids in an ATP-dependent process. In order to provide detailed mechanistic insights into lipid trafficking phenomena relevant to Toxoplasma biology, we characterized six parasite ATP-binding cassette (ABC) G family transporters and investigated their potential contribution to lipid homeostatic processes. All these transporters are expressed in the parasite and five of them are upregulated upon exposure to sterols. Four ABCG are localized to secretory organelles and the plasma membrane, and promote cholesterol and phospholipid efflux, reflecting the importance in exportation of large amounts of lipids into the PV. Interestingly, one ABCG that is associated with vesicles in the PV and the plasma membrane acts as a cholesterol importer. This last finding expands our current view on the role of some ABCG transporters in eukaryotic sterol influx. PMID:20487267

Ehrenman, Karen; Sehgal, Alfica; Lige, Bao; Stedman, Timothy T.; Joiner, Keith A.; Coppens, Isabelle

2014-01-01

24

A Conserved Mitochondrial ATP-binding Cassette Transporter Exports Glutathione Polysulfide for Cytosolic Metal Cofactor Assembly*?  

PubMed Central

An ATP-binding cassette transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe2+ alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondrion-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol. PMID:25006243

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

2014-01-01

25

The livestock photosensitizer, phytoporphyrin (phylloerythrin), is a substrate of the ATP-binding cassette transporter ABCG2  

Microsoft Academic Search

Hepatogenous photosensitization occurs in livestock following damage to the liver or biliary apparatus that results in impaired excretion of phytoporphyrin (phylloerythrin), a photosensitizer. Based on earlier observations that porphyrin-based photosensitizers are substrates of the ATP-binding cassette transporter ABCG2, we examined the ability of the hepatic transporters ABCB1 (P-glycoprotein) and ABCG2 to transport phytoporphyrin. Transport of phytoporphyrin was blocked by the

Robert W. Robey; Patricia A. Fetsch; Orsolya Polgar; Michael Dean; Susan E. Bates

2006-01-01

26

ATP binding cassette transporter retina genotypes and age related macular degeneration: an analysis on exudative non-familial Japanese patients  

Microsoft Academic Search

AIMTo determine whether mutations in the Stargardt’s disease gene, ATP binding cassette transporter retina (ABCR) affect the occurrence of age related macular degeneration (AMD) in Japanese non-familial patients.METHODS80 unrelated Japanese patients with AMD (67 males and 13 females; mean age, 67.2 years) diagnosed by indocyanine green angiography and 100 age matched control subjects were studied. Among the AMD patients, 70

Sachiko Kuroiwa; Hidenobu Kojima; Takanobu Kikuchi; Nagahisa Yoshimura

1999-01-01

27

The rod photoreceptor ATP-binding cassette transporter gene, ABCR, and retinal disease: from monogenic to multifactorial  

Microsoft Academic Search

The ABCR gene encodes a rod photoreceptor specific ATP-binding cassette transporter. Mutations in ABCR are associated with at least four inherited retinal dystrophies: Stargardt disease, Fundus Flavimaculatus, cone-rod dystrophy, and retinitis pigmentosa. A statistically significant increase in heterozygous ABCR alterations has been identified in patients with age-related macular degeneration (AMD). A pedigree is described which manifests both Stargardt disease and

Noah F Shroyer; Richard Alan Lewis; Rando Allikmets; Nanda Singh; Michael Dean; Mark Leppert; James R Lupski

1999-01-01

28

ATP binding cassette transporter G1 (ABCG1) is an intracellular sterol transporter  

PubMed Central

Four members of the mammalian ATP binding cassette (ABC) transporter G subfamily are thought to be involved in transmembrane (TM) transport of sterols. The residues responsible for this transport are unknown. The mechanism of action of ABCG1 is controversial and it has been proposed to act at the plasma membrane to facilitate the efflux of cellular sterols to exogenous high-density lipoprotein (HDL). Here we show that ABCG1 function is dependent on localization to intracellular endosomes. Importantly, localization to the endosome pathway distinguishes ABCG1 and/or ABCG4 from all other mammalian members of this superfamily, including other sterol transporters. We have identified critical residues within the TM domains of ABCG1 that are both essential for sterol transport and conserved in some other members of the ABCG subfamily and/or the insulin-induced gene 2 (INSIG-2). Our conclusions are based on studies in which (i) biotinylation of peritoneal macrophages showed that endogenous ABCG1 is intracellular and undetectable at the cell surface, (ii) a chimeric protein containing the TM of ABCG1 and the cytoplasmic domains of the nonsterol transporter ABCG2 is both targeted to endosomes and functional, and (iii) ABCG1 colocalizes with multiple proteins that mark late endosomes and recycling endosomes. Mutagenesis studies identify critical residues in the TM domains that are important for ABCG1 to alter sterol efflux, induce sterol regulatory element binding protein-2 (SREBP-2) processing, and selectively attenuate the oxysterol-mediated repression of SREBP-2 processing. Our data demonstrate that ABCG1 is an intracellular sterol transporter that localizes to endocytic vesicles to facilitate the redistribution of specific intracellular sterols away from the endoplasmic reticulum (ER). PMID:22095132

Tarling, Elizabeth J.; Edwards, Peter A.

2011-01-01

29

Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state.  

PubMed

Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors' knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3-6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters. PMID:24920594

Choudhury, Hassanul G; Tong, Zhen; Mathavan, Indran; Li, Yanyan; Iwata, So; Zirah, Séverine; Rebuffat, Sylvie; van Veen, Hendrik W; Beis, Konstantinos

2014-06-24

30

An ATP-Binding Cassette Transporter GhWBC1 from Elongating Cotton Fibers1  

PubMed Central

We have isolated a cDNA (GhWBC1) from cotton (Gossypium hirsutum) that encodes an ATP-binding cassette transporter of the WBC (white/brown complex) subfamily. Members of this subfamily are half-sized transporters and are reported to mediate lipid and drug excretion in human (Homo sapiens). GhWBC1 is highly expressed in developing fiber cells, but transcripts were also detectable in other tissues except roots. The transcript level peaked in rapidly expanding fibers from 5 to 9 DPA and then decreased. The GhWBC1 expression was weak in fiber cells of an li (ligon-lintless) mutant, which is defective in fiber cell elongation. These data indicate that GhWBC1 gene expression correlates with cotton fiber elongation. Transient expression of enhanced green fluorescence protein-GhWBC1 fusion protein in onion (Allium cepa) epidermal cells revealed plasma membrane localization. The GhWBC1 cDNA driven by a constitutive 35S promoter was introduced into Arabidopsis. About 13% of the transformants produced short siliques (SSs), whereas others had normal siliques (long siliques [LSs]). In siliques of SS lines, most embryos were severely shriveled, and only several seeds per silique could be found at maturity. The transgene expression level was higher in SS lines than in LS lines. Expression of AtWBC11, the closest homolog of GhWBC1 in Arabidopsis, was not altered in either SS or LS transgenic plants examined. These data suggest that GhWBC1 interferes with substance translocation that is required for Arabidopsis seed and silique development. Characterization of Arabidopsis WBC members, particularly AtWBC11, will help to dissect the role of GhWBC1 in cotton fiber development and elongation. PMID:12972649

Zhu, Yong-Qing; Xu, Ke-Xiang; Luo, Bin; Wang, Jia-Wei; Chen, Xiao-Ya

2003-01-01

31

ATP binding cassette modulators control abscisic acid-regulated slow anion channels in guard cells  

PubMed Central

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells. PMID:10368184

Leonhardt, N; Vavasseur, A; Forestier, C

1999-01-01

32

Expression profiling of ATP-binding cassette transporters in childhood T-cell acute lymphoblastic leukemia.  

PubMed

A major issue in the treatment of T-cell acute lymphoblastic leukemia (T-ALL) is resistance to chemotherapeutic drugs. Multidrug resistance can be caused by ATP-binding cassette (ABC) transporters. The majority of these proteins have not yet been examined in T-ALL. Using a newly developed microarray for the simultaneous quantification of 38 ABC transporter genes, we observed a consistent overexpression of ABCA2/ABCA3 in clinical samples of ALL. Therefore, we analyzed the association of these two genes with drug resistance. Treatment of CCRF-CEM and Jurkat cells with methotrexate, vinblastine, or doxorubicin led to an induction of ABCA3 expression, whereas a significant increase of ABCA2 expression was only observed in Jurkat cells. To study the causal relationship of ABCA2/A3 overexpression with drug resistance, we applied RNA interference (RNAi) technology. RNAi specific for ABCA2 or ABCA3 led to a partial decrease of expression in these two ABC transporters. Upon cotreatment of RNAi for ABCA2 with methotrexate and vinblastine, a partial decrease of ABCA2 expression as well as a simultaneous increase of ABCA3 expression was observed. Vice versa, ABCA3 RNAi plus drugs decreased ABCA3 and increased ABCA2 expression. This indicates that down-regulation of one ABC transporter was compensated by the up-regulation of the other. Application of RNAi for both ABCA2 and ABCA3 resulted in a more efficient reduction of the expression of both transporters. As a consequence, a significant sensitization of cells to cytostatic drugs was achieved. In conclusion, ABCA2 and ABCA3 are expressed in many T-ALL and contribute to drug resistance. PMID:16928819

Efferth, Thomas; Gillet, Jean-Pierre; Sauerbrey, Axel; Zintl, Felix; Bertholet, Vincent; de Longueville, Françoise; Remacle, Jose; Steinbach, Daniel

2006-08-01

33

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

PubMed Central

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 ?B (NF-?B) 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-?B signaling. Decreased transporter efflux function in microglia may lead to the retention of toxic chemicals and aberrant cell-cell communication during neuroinflammation. PMID:22942241

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

2012-01-01

34

ATP-Binding Cassette Transporter 1 Attenuates Ovalbumin-Induced Neutrophilic Airway Inflammation.  

PubMed

Apolipoprotein A-I (apoA-I) is an important component of high-density lipoprotein particles that mediates reverse cholesterol transport out of cells by interacting with the ATP-binding cassette transporter 1 (ABCA1). apoA-I has also been shown to attenuate neutrophilic airway inflammation in experimental ovalbumin (OVA)-induced asthma by reducing the expression of granulocyte colony-stimulating factor (G-CSF). Here, we hypothesized that overexpression of the ABCA1 transporter might similarly attenuate OVA-induced neutrophilic airway inflammation. Tie2-human ABCA1 (hABCA1) mice expressing human ABCA1 under the control of the Tie2 promoter, which is primarily expressed by vascular endothelial cells, but can also be expressed by macrophages, received daily intranasal OVA challenges, 5 d/wk for 5 weeks. OVA-challenged Tie2-hABCA1 mice had significant reductions in total bronchoalveolar lavage fluid (BALF) cells that reflected a decrease in neutrophils, as well as reductions in peribronchial inflammation, OVA-specific IgE levels, and airway epithelial thickness. The reduced airway neutrophilia in OVA-challenged Tie2-hABCA1 mice was associated with significant decreases in G-CSF protein levels in pulmonary vascular endothelial cells, alveolar macrophages, and BALF. Intranasal administration of recombinant murine G-CSF to OVA-challenged Tie2-hABCA1 mice for 5 days increased BALF neutrophils to a level comparable to that of OVA-challenged wild-type mice. We conclude that ABCA1 suppresses OVA-induced airway neutrophilia by reducing G-CSF production by vascular endothelial cells and alveolar macrophages. These findings suggest that ABCA1 expressed by vascular endothelial cells and alveolar macrophages may play important roles in attenuating the severity of neutrophilic airway inflammation in asthma. PMID:24813055

Dai, Cuilian; Yao, Xianglan; Vaisman, Boris; Brenner, Todd; Meyer, Katharine S; Gao, Meixia; Keeran, Karen J; Nugent, Gayle Z; Qu, Xuan; Yu, Zu-Xi; Dagur, Pradeep K; McCoy, J Philip; Remaley, Alan T; Levine, Stewart J

2014-11-01

35

The Structure, Function, and Origin of the Microcin H47 ATP-Binding Cassette Exporter Indicate Its Relatedness to That of Colicin V  

Microsoft Academic Search

Microcin H47, a gene-encoded peptide antibiotic produced by a natural Escherichia coli strain, was shown to be secreted by a three-component ATP-binding cassette exporter which was revealed to be strongly related to that of colicin V. The results of sequence and gene fusion analyses, as well as heterologous complementation assays, are presented. For gram-negative bacteria, several three-component ATP- binding cassette

MARIA F. AZPIROZ; ELIANA RODRIGUEZ; MAGELA LAVINA

2001-01-01

36

The Conformational Coupling and Translocation Mechanism of Vitamin B12 ATP-Binding Cassette Transporter BtuCD  

PubMed Central

ATP-binding cassette transporter BtuCD mediating vitamin B12 uptake in Escherichia coli couples the energy of ATP hydrolysis to the translocation of vitamin B12 across the membrane into the cell. Elastic normal mode analysis of BtuCD demonstrates that the simultaneous substrate trapping at periplasmic cavity and ATP binding at the ATP-binding cassette (BtuD) dimer proceeds readily along the lowest energy pathway. The transport power stroke is attributed to ATP-hydrolysis-induced opening of the nucleotide-binding domain dimer, which is coupled to conformational rearrangement of transmembrane domain (BtuC) helices leading to the closing at the periplasmic side and opening at the cytoplasmic gate. Simultaneous hydrolysis of two ATP is supported by the fact that antisymmetric movement of BtuD dimer implying alternating hydrolysis cannot induce effective conformational change of the translocation pathway. A plausible mechanism of translocation cycle is proposed in which the possible effect of the association of periplasmic binding protein BtuF to the transporter is also considered. PMID:17951296

Weng, Jingwei; Ma, Jianpeng; Fan, Kangnian; Wang, Wenning

2008-01-01

37

ATP-binding cassette superfamily transporter gene expression in human soft tissue sarcomas.  

PubMed

The phenomenon of multidrug resistance (MDR) in various malignant neoplasms has been reported as being caused by one or multiple expressions of ATP-binding cassette (ABC) superfamily protein, including P-glycoprotein/multidrug resistance (MDR) 1 and the MDR protein (MRP) family. However, their expression levels and distribution within soft tissue sarcomas remain controversial. In 86 cases of surgically resected soft tissue sarcoma, intrinsic mRNA levels of MDR1, MRP1, MRP2 and MRP3 were assessed using a quantitative reverse transcriptase-PCR (RT-PCR) method. Moreover, immunohistochemical protein expressions of P-glycoprotein (P-gp), MRP1, MRP2, MRP3 and p53 protein were evaluated in concordant paraffin-embedded material. The mRNA expression and immunohistochemical expression of ABC superfamily transporters were compared to clinicopathologic parameters and proliferative activities as evaluated by the MIB-1-labeling index (LI). Among the various histologic types, malignant peripheral nerve sheath tumor (MPNST) showed significantly high levels of MDR1 (p=0.017) and MRP3 (p=0.0384) mRNA expression, compared to the other tumor types. When the immunohistochemical method was compared to the RT-PCR technique to assess ABC transported expression at the protein and mRNA levels, a significantly close relationship was found between the 2 methods (p<0.05). P-gp expression was significantly correlated with large tumor size (> or =5 cm, p=0.041) and high AJCC stage (stages III and IV) (p=0.0365). Furthermore, cases with nuclear expression of p53 revealed significantly higher levels of MDR1 mRNA expression, compared to those with negative immunoreaction for p53 (p=0.0328). Our results suggest that MDR1/P-gp expression may have an important role to play in tumor progression in the cases of soft tissue sarcoma, and p53 may be one of the active regulators of the MDR1 transcript. In addition, the high levels of both MDR1 and MRP3 mRNA expression in MPNST may help to explain the poor response of this tumor to anticancer-drugs. PMID:15609299

Oda, Yoshinao; Saito, Tsuyoshi; Tateishi, Naomi; Ohishi, Yoshihiro; Tamiya, Sadafumi; Yamamoto, Hidetaka; Yokoyama, Ryohei; Uchiumi, Takeshi; Iwamoto, Yukihide; Kuwano, Michihiko; Tsuneyoshi, Masazumi

2005-05-10

38

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

PubMed

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

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

2014-01-01

39

The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae  

PubMed Central

The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP- binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3- oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins). PMID:8647887

1996-01-01

40

Nucleotide sequence of the Streptococcus gordonii PK488 coaggregation adhesin gene, scaA, and ATP-binding cassette.  

PubMed Central

Human oral viridans group streptococci that coaggregate with Actinomyces naeslundii PK606 express surface proteins related to ScaA, the coaggregation-mediating adhesin of Streptococcus gordonii PK488 (R. N. Andersen, N. Ganeshkumar, and P. E. Kolenbrander, Infect. Immun. 61:981-987, 1993). The nucleotide sequence of the 6,125-bp EcoRI insert of pRA1, containing scaA, the gene encoding ScaA, was determined. Six open reading frames (ORFs) were identified. The orientation of four ORFs, two upstream (ORF 1 and ORF 2) and one downstream (ORF 4) of scaA (ORF 3), indicated transcription in one direction, whereas ORF 5 and ORF 6 were transcribed divergently. Computer analysis of the deduced amino acid sequences identified a consensus binding site for ATP (GxxGxGKS) in the putative 28,054-Da protein encoded by ORF 1. ORF 2 potentially encoded a hydrophobic protein of 29,705 Da with six potential membrane-spanning regions. ScaA was 310 amino acids, 34,787 Da, and contained the lipoprotein consensus sequence LxxC, also reported for the ScaA-related proteins SsaB, FimA, and PsaA from Streptococcus sanguis 12, Streptococcus parasanguis FW213, and Streptococcus pneumoniae R36A, respectively. ORF 4 potentially encoded a 163-amino-acid protein of 17,912 Da, which was nearly identical to the downstream adjacent gene products of ssaB, fimA, and psaA. No significant homology with other proteins was found with the putative ORF 5 gene product, a 229-amino-acid protein of 25,107 Da. ORF 6 was incomplete and encoded a protein larger than 564 amino acids. This putative protein had a consensus Zn2+ binding motif, HExxH, found among bacterial thermolysins and mammalian neutral endopeptidases and was 40% identical to a homologous 210-amino-acid region of human enkephalinase. The genetic organization of ORFs 1, 2, and 3 was similar to those of the bacterial periplasmic-binding protein-dependent transport systems of gram-negative bacteria and binding-lipoprotein-dependent transport systems of gram-positive bacteria, and these genes appeared to encode ABC (ATP-binding cassette) proteins. This report describes a cell-to-cell adherence function associated with an ATP-binding cassette. Images PMID:7927711

Kolenbrander, P E; Andersen, R N; Ganeshkumar, N

1994-01-01

41

ATP-binding cassette transporters and HDL suppress hematopoietic stem cell proliferation.  

PubMed

Elevated leukocyte cell numbers (leukocytosis), and monocytes in particular, promote atherosclerosis; however, how they become increased is poorly understood. Mice deficient in the adenosine triphosphate-binding cassette (ABC) transporters ABCA1 and ABCG1, which promote cholesterol efflux from macrophages and suppress atherosclerosis in hypercholesterolemic mice, displayed leukocytosis, a transplantable myeloproliferative disorder, and a dramatic expansion of the stem and progenitor cell population containing Lin(-)Sca-1(+)Kit+ (LSK) in the bone marrow. Transplantation of Abca1(-/-) Abcg1(-/-) bone marrow into apolipoprotein A-1 transgenic mice with elevated levels of high-density lipoprotein (HDL) suppressed the LSK population, reduced leukocytosis, reversed the myeloproliferative disorder, and accelerated atherosclerosis. The findings indicate that ABCA1, ABCG1, and HDL inhibit the proliferation of hematopoietic stem and multipotential progenitor cells and connect expansion of these populations with leukocytosis and accelerated atherosclerosis. PMID:20488992

Yvan-Charvet, Laurent; Pagler, Tamara; Gautier, Emmanuel L; Avagyan, Serine; Siry, Read L; Han, Seongah; Welch, Carrie L; Wang, Nan; Randolph, Gwendalyn J; Snoeck, Hans W; Tall, Alan R

2010-06-25

42

ATP-Binding Cassette Transporters and HDL Suppress Hematopoietic Stem Cell Proliferation  

PubMed Central

Elevated leukocyte cell numbers (leukocytosis), and monocytes in particular, promote atherosclerosis; however, how they become increased is poorly understood. Mice deficient in the adenosine triphosphate–binding cassette (ABC) transporters ABCA1 and ABCG1, which promote cholesterol efflux from macrophages and suppress atherosclerosis in hypercholesterolemic mice, displayed leukocytosis, a transplantable myeloproliferative disorder, and a dramatic expansion of the stem and progenitor cell population containing Lin? Sca-1+Kit+ (LSK) in the bone marrow. Transplantation of Abca1?/? Abcg1?/? bone marrow into apolipoprotein A-1 transgenic mice with elevated levels of high-density lipoprotein (HDL) suppressed the LSK population, reduced leukocytosis, reversed the myeloproliferative disorder, and accelerated atherosclerosis. The findings indicate that ABCA1, ABCG1, and HDL inhibit the proliferation of hematopoietic stem and multipotential progenitor cells and connect expansion of these populations with leukocytosis and accelerated atherosclerosis. PMID:20488992

Yvan-Charvet, Laurent; Pagler, Tamara; Gautier, Emmanuel L.; Avagyan, Serine; Siry, Read L.; Han, Seongah; Welch, Carrie L.; Wang, Nan; Randolph, Gwendalyn J.; Snoeck, Hans W.; Tall, Alan R.

2011-01-01

43

An ATP binding cassette transporter is required for cuticular wax deposition and desiccation tolerance in the moss Physcomitrella patens.  

PubMed

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

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

2013-10-01

44

PGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in Arabidopsis thaliana RootsW?  

PubMed Central

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid–reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells. PMID:16243904

Terasaka, Kazuyoshi; Blakeslee, Joshua J.; Titapiwatanakun, Boosaree; Peer, Wendy A.; Bandyopadhyay, Anindita; Makam, Srinivas N.; Lee, Ok Ran; Richards, Elizabeth L.; Murphy, Angus S.; Sato, Fumihiko; Yazaki, Kazufumi

2005-01-01

45

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

PubMed Central

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

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

1992-01-01

46

Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA*  

PubMed Central

ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP. PMID:23766512

Doshi, Rupak; van Veen, Hendrik W.

2013-01-01

47

Physicochemical Factors Controlling the Activity and Energy Coupling of an Ionic Strength-gated ATP-binding Cassette (ABC) Transporter*  

PubMed Central

Cells control their volume through the accumulation of compatible solutes. The bacterial ATP-binding cassette transporter OpuA couples compatible solute uptake to ATP hydrolysis. Here, we study the gating mechanism and energy coupling of OpuA reconstituted in lipid nanodiscs. We show that anionic lipids are essential both for the gating and the energy coupling. The tight coupling between substrate binding on extracellular domains and ATP hydrolysis by cytoplasmic nucleotide-binding domains allows the study of transmembrane signaling in nanodiscs. From the tight coupling between processes at opposite sides of the membrane, we infer that the ATPase activity of OpuA in nanodiscs reflects solute translocation. Intriguingly, the substrate-dependent, ionic strength-gated ATPase activity of OpuA in nanodiscs is at least an order of magnitude higher than in lipid vesicles (i.e. with identical membrane lipid composition, ionic strength, and nucleotide and substrate concentrations). Even with the chemical components the same, the lateral pressure (profile) of the nanodiscs will differ from that of the vesicles. We thus propose that membrane tension limits translocation in vesicular systems. Increased macromolecular crowding does not activate OpuA but acts synergistically with ionic strength, presumably by favoring gating interactions of like-charged surfaces via excluded volume effects. PMID:23979139

Karasawa, Akira; Swier, Lotteke J. Y. M.; Stuart, Marc C. A.; Brouwers, Jos; Helms, Bernd; Poolman, Bert

2013-01-01

48

Efficient Purification and Reconstitution of ATP Binding Cassette Transporter B6 (ABCB6) for Functional and Structural Studies*  

PubMed Central

The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 ?m) and an ATPase activity with a Km of 0.99 mm. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6. PMID:23792964

Chavan, Hemantkumar; Taimur Khan, Mohiuddin Md.; Tegos, George; Krishnamurthy, Partha

2013-01-01

49

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states  

PubMed Central

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters. PMID:23716676

Shintre, Chitra A.; Pike, Ashley C. W.; Kim, Jung-In; Barr, Alastair J.; Goubin, Solenne; Shrestha, Leela; Yang, Jing; Berridge, Georgina; Ross, Jonathan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Edwards, Aled M.; Bountra, Chas; Marsden, Brian D.; von Delft, Frank; Bullock, Alex N.; Gileadi, Opher; Burgess-Brown, Nicola A.; Carpenter, Elisabeth P.

2013-01-01

50

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

PubMed Central

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

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

2013-01-01

51

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

PubMed

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

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

2013-08-01

52

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

PubMed

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

Saurin, W; Hofnung, M; Dassa, E

1999-01-01

53

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

PubMed

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

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

2009-08-01

54

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

PubMed Central

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

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

55

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

PubMed Central

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

Cak?r, Birsen; K?l?ckaya, Ozan

2013-01-01

56

Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis*  

PubMed Central

In ATP-binding cassette proteins, the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The intermonomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mm MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant contact models, for the mode of operation of ATP-binding cassette proteins. PMID:23723071

Cooper, Rebecca S.; Altenberg, Guillermo A.

2013-01-01

57

Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida glabrata  

Microsoft Academic Search

Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753-2765, 1999). Deletion of

DOMINIQUE SANGLARD; FRANCOISE ISCHER; JACQUES BILLE

2001-01-01

58

Expression of ATP-binding cassette multidrug transporters in the giant liver fluke Fasciola gigantica and their possible involvement in the transport of bile salts and anthelmintics  

Microsoft Academic Search

ATP-binding cassette (ABC) transporters belong to one of the largest protein families that either import or export a wide\\u000a spectrum of different substrates. Certain members of this superfamily have been implicated in multidrug resistance in various\\u000a types of cancer as well as in pathogenic microorganisms. The role of ABC proteins in parasitic multidrug resistance becomes\\u000a increasingly evident. However, studies on

Supeecha Kumkate; Supatra Chunchob; Tavan Janvilisri

2008-01-01

59

Expression of reverse cholesterol transport proteins ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI) in the retina and retinal pigment epithelium  

Microsoft Academic Search

Aims:Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol transport (RCT) activity to move lipid into BrM, mediated through ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI).Methods:ABCA1 expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) and

K G Duncan; K Hosseini; K R Bailey; H Yang; R J Lowe; M T Matthes; J P Kane; M M LaVail; D M Schwartz; J L Duncan

2009-01-01

60

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

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

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

2013-01-01

61

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

PubMed Central

BioMNY proteins are considered to constitute tripartite biotin transporters in prokaryotes. Recent comparative genomic and experimental analyses pointed to the similarity of BioMN to homologous modules of prokaryotic transporters mediating uptake of metals, amino acids, and vitamins. These systems resemble ATP-binding cassette-containing transporters and include typical ATPases (e.g., BioM). Absence of extracytoplasmic solute-binding proteins among the members of this group, however, is a distinctive feature. Genome context analyses uncovered that only one-third of the widespread bioY genes are linked to bioMN. Many bioY genes are located at loci encoding biotin biosynthesis, and others are unlinked to biotin metabolic or transport genes. Heterologous expression of the bioMNY operon and of the single bioY of the ?-proteobacterium Rhodobacter capsulatus conferred biotin-transport activity on recombinant Escherichia coli cells. Kinetic analyses identified BioY as a high-capacity transporter that was converted into a high-affinity system in the presence of BioMN. BioMNY-mediated biotin uptake was severely impaired by replacement of the Walker A lysine residue in BioM, demonstrating dependency of high-affinity transport on a functional ATPase. Biochemical assays revealed that BioM, BioN, and BioY proteins form stable complexes in membranes of the heterologous host. Expression of truncated bio transport operons, each with one gene deleted, resulted in stable BioMN complexes but revealed only low amounts of BioMY and BioNY aggregates in the absence of the respective third partner. The results substantiate our earlier suggestion of a mechanistically novel group of membrane transporters. PMID:17301237

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

2007-01-01

62

ATP Binding Cassette Transporter G1 Deletion Induces IL-17-dependent Dysregulation of Pulmonary Adaptive Immunity1  

PubMed Central

Mice with genetic deletion of the cholesterol transporter ATP Binding Cassette (ABC)G1 have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1+/+ and Abcg1?/? mice were sensitized to ovalbumin via the airway using low-dose lipopolysaccharide as an adjuvant, and then challenged with ovalbumin aerosol. Naive Abcg1?/? mice displayed increased B cells, CD4+ T cells, CD8+ T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b+ DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1?/? airway, compared to Abcg1+/+, displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard intraperitoneal ovalbumin sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1?/? mice produced reduced IL-5 upon ex vivo ovalbumin challenge. Abcg1?/? CD4+ T cells displayed normal ex vivo differentiation, whereas Abcg1?/? DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17+ ??T cells, and IL-17+ neutrophils were all increased in Abcg1?/? lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1?/? airway. We conclude that Abcg1?/? mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs. PMID:22539789

Draper, David W.; Gowdy, Kymberly M.; Madenspacher, Jennifer H.; Wilson, Rhonda H.; Whitehead, Gregory S.; Nakano, Hideki; Pandiri, Arun R.; Foley, Julie F.; Remaley, Alan T.; Cook, Donald N.; Fessler, Michael B.

2012-01-01

63

Gene expression profiles of ATP-binding cassette transporter A and C subfamilies in mouse retinal vascular endothelial cells.  

PubMed

The purpose of this study was to quantify gene expression levels of the ATP-binding cassette (ABC) transporter A and C subfamilies ABCA1-A9, and ABCC1-6/Mrp1-6, C10/Mrp7 in mouse retinal vascular endothelial cells (RVEC) using a combination of a magnetic isolation method for mouse RVEC and real-time quantitative PCR analysis. The transcript level of endothelial cell markers, such as CD31, Tie-2, claudin-5, occludin, ABCB1a/mdr1a, and ABCG2, were more than 20-fold higher than those in the non-RVEC fraction, suggesting that RVEC in the RVEC fraction are concentrated at least 20-fold compared with those of the non-RVEC fraction. In the ABCA1 to A9 families, the transcript level of ABCA3 and A9 in the RVEC fraction was 1.2- and 32-fold higher than that in the non-RVEC fraction. Although ABCA3 was expressed in both the RVEC and non-RVEC fractions, A9 is predominantly expressed in the RVEC fraction. In the ABCC1 to C6 and C10 families, the transcript level of ABCC3, C4, and C6 in the RVEC fraction was 27-, 251-, and 242-fold higher, respectively, than that in the non-RVEC fraction, suggesting that ABCC3, C4, and C6 are predominantly expressed in the RVEC. In conclusion, ABCA3, ABCA9, ABCC3, ABCC4, and ABCC6 mRNAs are predominantly expressed at the inner blood-retina barrier (inner BRB) and appear to play a major role in the efflux transport of their substrates at the inner BRB. PMID:17574281

Tachikawa, Masanori; Toki, Hidetoh; Tomi, Masatoshi; Hosoya, Ken-ichi

2008-01-01

64

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

PubMed

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

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

2014-10-17

65

Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region.  

PubMed Central

A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly. PMID:9169612

Bakos, E; Klein, I; Welker, E; Szabo, K; Muller, M; Sarkadi, B; Varadi, A

1997-01-01

66

Characterization of the human multidrug resistance protein containing mutations in the ATP-binding cassette signature region.  

PubMed

A number of mutants with single amino acid replacements were generated in the highly conserved ATP-binding cassette (ABC)-signature region (amino acids 531-543) of the N-terminal half of the human multidrug resistance (MDR1) protein. The cDNA variants were inserted into recombinant baculoviruses and the MDR1 proteins were expressed in Spodoptera frugiperda (Sf9) insect cells. The level of expression and membrane insertion of the MDR1 variants was examined by immunostaining, and MDR1 function was followed by measuring drug-stimulated ATPase activity. We found that two mutations, L531R and G534V, practically eliminated MDR1 expression; thus these amino acid replacements seem to inhibit the formation of a stable MDR1 protein structure. The MDR1 variants G534D and I541R were expressed at normal levels with normal membrane insertion, but showed a complete loss of drug-stimulated ATPase activity, while mutant R538M yielded full protein expression but with greatly decreased ATPase activity. Increasing the ATP concentration did not restore MDR1 ATPase activity in these variants. Some amino acid replacements in the ABC-signature region (K536I, K536R, I541T and R543S) affected neither the expression and membrane insertion nor the ATPase function of MDR1. We found no alteration in the drug-sensitivity of ATP cleavage in any of the MDR1 variants that had measurable ATPase activity. These observations suggest that the ABC-signature region is essential for MDR1 protein stability and function, but alterations in this region do not seem to modulate MDR1-drug interactions directly. PMID:9169612

Bakos, E; Klein, I; Welker, E; Szabó, K; Müller, M; Sarkadi, B; Váradi, A

1997-05-01

67

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

PubMed Central

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

Aberuyi, N; Rahgozar, S; Moafi, A

2014-01-01

68

Up-Regulation of the ATP-Binding Cassette Transporter A1 Inhibits Hepatitis C Virus Infection  

PubMed Central

Hepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus–cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy. PMID:24646941

Gondeau, Claire; Douam, Florian; Lebreton, Stephanie; Lagaye, Sylvie; Pol, Stanislas; Helle, Francois; Plengpanich, Wanee; Guerin, Maryse; Bourgine, Maryline; Michel, Marie Louise; Lavillette, Dimitri; Roingeard, Philippe; le Goff, Wilfried; Budkowska, Agata

2014-01-01

69

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

PubMed

Hepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus-cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy. PMID:24646941

Bocchetta, Simone; Maillard, Patrick; Yamamoto, Mami; Gondeau, Claire; Douam, Florian; Lebreton, Stéphanie; Lagaye, Sylvie; Pol, Stanislas; Helle, François; Plengpanich, Wanee; Guérin, Maryse; Bourgine, Maryline; Michel, Marie Louise; Lavillette, Dimitri; Roingeard, Philippe; le Goff, Wilfried; Budkowska, Agata

2014-01-01

70

Drug Resistance Is Conferred on the Model Yeast Saccharomyces cerevisiae by Expression of Full-Length Melanoma-Associated Human ATP-Binding Cassette Transporter ABCB5.  

PubMed

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-? mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance. PMID:25115303

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

2014-10-01

71

NpPDR1, a Pleiotropic Drug Resistance-Type ATP-Binding Cassette Transporter from Nicotiana plumbaginifolia, Plays a Major Role in Plant Pathogen Defense1  

PubMed Central

Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family. PMID:16126865

Stukkens, Yvan; Bultreys, Alain; Grec, Sebastien; Trombik, Tomasz; Vanham, Delphine; Boutry, Marc

2005-01-01

72

Genetic Separation of FK506 Susceptibility and Drug Transport in the Yeast Pdr5 ATP-binding Cassette Multidrug Resistance Transporter  

PubMed Central

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility. PMID:9450972

Egner, Ralf; Rosenthal, Friederike E.; Kralli, Anastasia; Sanglard, Dominique; Kuchler, Karl

1998-01-01

73

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

PubMed

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis. PMID:22635270

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

2012-06-19

74

Lapatinib (Tykerb, GW572016) Reverses Multidrug Resistance in Cancer Cells by Inhibiting the Activity of ATP-Binding Cassette Subfamily B Member 1 and G Member 2  

PubMed Central

Lapatinib is active at the ATP-binding site of tyrosine kinases that are associated with the human epidermal growth factor receptor (EGFR, Her-1, or ErbB1) and Her-2. It is conceivable that lapatinib may inhibit the function of ATP-binding cassette (ABC) transporters by binding to their ATP-binding sites. The aim of this study was to investigate the ability of lapatinib to reverse tumor multidrug resistance (MDR) due to overexpression of ABCB1 and ABCG2 transporters. Our results showed that lapatinib significantly enhanced the sensitivity to ABCB1 or ABCG2 substrates in cells expressing these transporters although a small synergetic effect was observed in combining lapatinib and conventional chemotherapeutic agents in parental sensitive MCF-7 or S1 cells. Lapatinib alone, however, did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in sensitive and resistant cells. Additionally, lapatinib significantly increased the accumulation of doxorubicin or mitoxantrone in ABCB1 or ABCG2 overexpressing cells and inhibited the transport of methotrexate and E217?G by ABCG2. Furthermore, lapatinib stimulated the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2 with [125I]Iodoarylazidoprazosin in a concentration-dependent manner. However, lapatinib did not affect the expression of these transporters at mRNA or protein levels. Importantly, lapatinib also strongly enhanced the effect of paclitaxel on the inhibition of growth of the ABCB1-overexpressing KBv200 cell xenografts in nude mice. Overall, we conclude that lapatinib reverses ABCB1- and ABCG2-mediated MDR by directly inhibiting their transport function. These findings may be useful for cancer combinational therapy with lapatinib in the clinic. PMID:18829547

Dai, Chun-ling; Tiwari, Amit K.; Wu, Chung-Pu; Su, Xiao-dong; Wang, Si-Rong; Liu, Dong-geng; Ashby, Charles R.; Huang, Yan; Robey, Robert W.; Liang, Yong-ju; Chen, Li-ming; Shi, Cheng-Jun; Ambudkar, Suresh V.; Chen, Zhe-Sheng; Fu, Li-wu

2009-01-01

75

Down-regulation of the ATP-binding Cassette Transporter 2 (Abca2) Reduces Amyloid-? Production by Altering Nicastrin Maturation and Intracellular Localization*  

PubMed Central

Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-? (A?) generation. In vitro studies revealed altered ?-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered ?-secretase processing of APP, and thus A? generation, without affecting Notch cleavage. PMID:22086926

Michaki, Vasiliki; Guix, Francesc X.; Vennekens, Krist'l; Munck, Sebastian; Dingwall, Colin; Davis, John B.; Townsend, Danyelle M.; Tew, Kenneth D.; Feiguin, Fabian; De Strooper, Bart; Dotti, Carlos G.; Wahle, Tina

2012-01-01

76

An ATP-binding cassette multidrug-resistance transporter is necessary for tolerance of Gibberella pulicaris to phytoalexins and virulence on potato tubers.  

PubMed

The necrotrophic pathogen Gibberella pulicaris infects potato tubers through wounds that contain fungitoxic secondary metabolites such as the phytoalexins rishitin and lubimin. In order to colonize tuber tissue, the fungus must possess a mechanism to tolerate potato defense compounds. In this paper, we show that a gene, Gpabc1, that codes an ATP-binding cassette (ABC) transporter is required for tolerance to these phytoalexins and for virulence on potato. The Gpabc1 gene, isolated in the course of a differential cDNA screen, shares high sequence homology with the ABC1 gene of Magnaporthe grisea. G. pulicaris mutants deficient in Gpabc1 were still able to metabolize rishitin but lost their tolerance to this phytoalexin as well as their virulence on potato. These results strongly suggest that the Gpabc1-encoded ABC transporter is necessary for tolerance of G. pulicaris to rishitin and that this tolerance is required for virulence on potato. PMID:11876422

Fleissner, André; Sopalla, Claudia; Weltring, Klaus-Michael

2002-02-01

77

Identification and characterization of a Brucella abortus ATP-binding cassette transporter homolog to Rhizobium meliloti ExsA and its role in virulence and protection in mice.  

PubMed

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not fully understood yet. Furthermore, genes that allow Brucella to reach the intracellular niche and to interact with host cells need to be identified. Using the genomic survey sequence (GSS) approach, we identified the gene encoding an ATP-binding cassette (ABC) transporter of B. abortus strain S2308. The deduced amino acid sequence encoded by this gene exhibited 69 and 67% identity with the sequences of the ABC transporters encoded by the exsA genes of Rhizobium meliloti and Mesorhizobium loti, respectively. Additionally, B. abortus ExsA, like R. meliloti and M. loti ExsA, possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Furthermore, ortholog group analysis placed B. abortus ExsA in ortholog group 6 of ABC transporters more likely to be involved in bacterial pathogenesis. In R. meliloti, ExsA is an exopolysaccharide transporter essential for alfalfa root nodule invasion and establishment of infection. To test the role of ExsA in Brucella pathogenesis, an exsA deletion mutant was constructed. Replacement of the wild-type exsA by recombination was demonstrated by Southern blot analysis of Brucella genomic DNA. Decreased survival in mice of the Brucella DeltaexsA mutant compared to the survival of parental strain S2308 demonstrated that ExsA is critical for full bacterial virulence. Additionally, the B. abortus exsA deletion mutant was used as a live vaccine. Challenge experiments revealed that the exsA mutant strain induced superior protective immunity in BALB/c mice compared to the protective immunity induced by strain S19 or RB51. PMID:12183550

Rosinha, G M S; Freitas, Daniela A; Miyoshi, Anderson; Azevedo, Vasco; Campos, Eleonora; Cravero, Silvio L; Rossetti, Osvaldo; Splitter, Gary; Oliveira, S C

2002-09-01

78

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

PubMed Central

The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd2+ transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1pK669M) was unable to complement the fusion defect of ycf1? vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1? vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion. PMID:23658021

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

2013-01-01

79

An ATP-binding cassette pleiotropic drug transporter protein is required for xenobiotic tolerance and antagonism in the fungal biocontrol agent Clonostachys rosea.  

PubMed

ATP-binding cassette (ABC) transporters mediate active efflux of natural and synthetic toxicants and are considered to be important for drug tolerance in microorganisms. In biological control agents (BCA), ABC transporters can play important roles in antagonism by providing protection against toxins derived from the fungal prey and by mediating the secretion of endogenous toxins. In the present study, we generated deletion and complementation strains of the ABC transporter abcG5 in the fungal BCA Clonostachys rosea to study its role in xenobiotic tolerance and antagonism. Gene expression analysis shows induced expression of abcG5 in the presence of the Fusarium mycotoxin zearalenone (ZEA), secreted metabolites of F. graminearum, and different classes of fungicides. Phenotypic analysis of abcG5 deletion and complementation strains showed that the deletion strains were more sensitive towards F. graminearum culture filtrates, ZEA, and iprodione- and mefenoxam-based fungicides, thus suggesting the involvement of abcG5 in cell protection. The ?abcG5 strains displayed reduced antagonism towards F. graminearum in a plate confrontation assay. Furthermore, the ?abcG5 strains failed to protect barley seedlings from F. graminearium foot rot disease. These data show that the abcG5 ABC transporter is important for xenobiotic tolerance and biocontrol traits in C. rosea. PMID:24654977

Dubey, Mukesh K; Jensen, Dan Funck; Karlsson, Magnus

2014-07-01

80

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

PubMed Central

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

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

2003-01-01

81

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

PubMed Central

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

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

2009-01-01

82

Involvement of a Soybean ATP-Binding Cassette-Type Transporter in the Secretion of Genistein, a Signal Flavonoid in Legume-Rhizobium Symbiosis1  

PubMed Central

Legume plants have an ability to fix atmospheric nitrogen into nutrients via symbiosis with soil microbes. As the initial event of the symbiosis, legume plants secrete flavonoids into the rhizosphere to attract rhizobia. Secretion of flavonoids is indispensable for the establishment of symbiotic nitrogen fixation, but almost nothing is known about the membrane transport mechanism of flavonoid secretion from legume root cells. In this study, we performed biochemical analyses to characterize the transport mechanism of flavonoid secretion using soybean (Glycine max) in which genistein is a signal flavonoid. Plasma membrane vesicles prepared from soybean roots showed clear transport activity of genistein in an ATP-dependent manner. This transport activity was inhibited by sodium orthovanadate, a typical inhibitor of ATP-binding cassette (ABC) transporters, but was hardly affected by various ionophores, such as gramicidin D, nigericin, or valinomycin, suggesting involvement of an ABC transporter in the secretion of flavonoids from soybean roots. The Km and Vmax values of this transport were calculated to be 158 ?m and 322 pmol mg protein?1 min?1, respectively. Competition experiments using various flavonoids of both aglycone and glucoside varieties suggested that this ABC-type transporter recognizes genistein and daidzein, another signaling compound in soybean root exudates, as well as other isoflavonoid aglycones as its substrates. Transport activity was constitutive regardless of the availability of nitrogen nutrition. This is, to our knowledge, the first biochemical characterization of the membrane transport of flavonoid secretion from roots. PMID:17556512

Sugiyama, Akifumi; Shitan, Nobukazu; Yazaki, Kazufumi

2007-01-01

83

An atomic detail model for the human ATP binding cassette transporter P-glycoprotein derived from disulfide cross-linking and homology modeling.  

PubMed

The multidrug resistance P-glycoprotein mediates the extrusion of chemotherapeutic drugs from cancer cells. Characterization of the drug binding and ATPase activities of the protein have made it the paradigm ATP binding cassette (ABC) transporter. P-glycoprotein has been imaged at low resolution by electron cryo-microscopy and extensively analyzed by disulphide cross-linking, but a high resolution structure solved ab initio remains elusive. Homology models of P-glycoprotein were generated using the structure of a related prokaryotic ABC transporter, the lipid A transporter MsbA, as a template together with structural data describing the dimer interface of the nucleotide binding domains (NBDs). The first model, which maintained the NBD:transmembrane domain (TMD) interface of MsbA, did not satisfy previously published cross-linking data. This suggests that either P-glycoprotein has a very different structure from MsbA or that the published E. coli MsbA structure does not reflect a physiological state. To distinguish these alternatives, we mapped the interface between the two TMDs of P-glycoprotein experimentally by chemical cross-linking of introduced triple-cysteine residues. Based on these data, a plausible atomic model of P-glycoprotein could be generated using the MsbA template, if the TMDs of MsbA are reoriented with respect to the NBDs. This model will be important for understanding the mechanism of P-glycoprotein and other ABC transporters. PMID:14563687

Stenham, Daniella R; Campbell, Jeff D; Sansom, Mark S P; Higgins, Christopher F; Kerr, Ian D; Linton, Kenneth J

2003-12-01

84

Lipid Absorption Defects in Intestine-specific Microsomal Triglyceride Transfer Protein and ATP-binding Cassette Transporter A1-deficient Mice*  

PubMed Central

We have previously described apolipoprotein B (apoB)-dependent and -independent cholesterol absorption pathways and the role of microsomal triglyceride transfer protein (MTP) and ATP-binding cassette transporter A1 (ABCA1) in these pathways. To assess the contribution of these pathways to cholesterol absorption and to determine whether there are other pathways, we generated mice that lack MTP and ABCA1, individually and in combination, in the intestine. Intestinal deletions of Mttp and Abca1 decreased plasma cholesterol concentrations by 45 and 24%, respectively, whereas their combined deletion reduced it by 59%. Acute cholesterol absorption was reduced by 28% in the absence of ABCA1, and it was reduced by 92–95% when MTP was deleted in the intestine alone or together with ABCA1. MTP deficiency significantly reduced triglyceride absorption, although ABCA1 deficiency had no effect. ABCA1 deficiency did not affect cellular lipids, but Mttp deficiency significantly increased intestinal levels of triglycerides and free fatty acids. Accumulation of intestinal free fatty acids, but not triglycerides, in Mttp-deficient intestines was prevented when mice were also deficient in intestinal ABCA1. Combined deficiency of these genes increased intestinal fatty acid oxidation as a consequence of increased expression of peroxisome proliferator-activated receptor-? (PPAR?) and carnitine palmitoyltransferase 1? (CPT1?). These studies show that intestinal MTP and ABCA1 are critical for lipid absorption and are the main determinants of plasma and intestinal lipid levels. Reducing their activities might lower plasma lipid concentrations. PMID:24019513

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

2013-01-01

85

Structural and functional characterization of an orphan ATP-binding cassette ATPase involved in manganese utilization and tolerance in Leptospira spp.  

PubMed

Pathogenic Leptospira species are the etiological agents of the widespread zoonotic disease leptospirosis. Most organisms, including Leptospira, require divalent cations for proper growth, but because of their high reactivity, these metals are toxic at high concentrations. Therefore, bacteria have acquired strategies to maintain metal homeostasis, such as metal import and efflux. By screening Leptospira biflexa transposon mutants for their ability to use Mn(2+), we have identified a gene encoding a putative orphan ATP-binding cassette (ABC) ATPase of unknown function. Inactivation of this gene in both L. biflexa and L. interrogans strains led to mutants unable to grow in medium in which iron was replaced by Mn(2+), suggesting an involvement of this ABC ATPase in divalent cation uptake. A mutation in this ATPase-coding gene increased susceptibility to Mn(2+) toxicity. Recombinant ABC ATPase of the pathogen L. interrogans exhibited Mg(2+)-dependent ATPase activity involving a P-loop motif. The structure of this ATPase was solved from a crystal containing two monomers in the asymmetric unit. Each monomer adopted a canonical two-subdomain organization of the ABC ATPase fold with an ?/? subdomain containing the Walker motifs and an ? subdomain containing the ABC signature motif (LSSGE). The two monomers were arranged in a head-to-tail orientation, forming a V-shaped particle with all the conserved ABC motifs at the dimer interface, similar to functional ABC ATPases. These results provide the first structural and functional characterization of a leptospiral ABC ATPase. PMID:24123817

Benaroudj, Nadia; Saul, Frederick; Bellalou, Jacques; Miras, Isabelle; Weber, Patrick; Bondet, Vincent; Murray, Gerald L; Adler, Ben; Ristow, Paula; Louvel, Hélène; Haouz, Ahmed; Picardeau, Mathieu

2013-12-01

86

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

PubMed Central

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 ?-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

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

2013-01-01

87

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

PubMed Central

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

Roohparvar, Ramin; Huser, Aurelie; Zwiers, Lute-Harm; De Waard, Maarten A.

2007-01-01

88

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4*  

PubMed Central

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport. PMID:23144455

Biswas-Fiss, Esther E.; Affet, Stephanie; Ha, Malissa; Biswas, Subhasis B.

2012-01-01

89

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

PubMed Central

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

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

90

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

PubMed Central

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) ? 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 PPAR? response elements (PPRE). Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated specific binding of PPAR? 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 PPAR? 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

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

2014-01-01

91

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p  

Microsoft Academic Search

Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we

Irena Ivnitski-Steele; Ann R. Holmes; Erwin Lamping; Brian C. Monk; Richard D. Cannon; Larry A. Sklar

2009-01-01

92

High expression of ATP-binding cassette transporter ABCC11 in breast tumors is associated with aggressive subtypes and low disease-free survival  

PubMed Central

ATP-binding cassette (ABC) transporters are membrane proteins that efflux various compounds from cells, including chemotherapeutic agents, and are known to affect multidrug resistance. Recent reports disagree on whether ABCC11 is a risk factor for breast tumorigenesis, but its expression in breast cancer is poorly investigated. We hypothesized that both frequency and expression levels of ABC transporters in breast tumors would vary by cancer subtype, and be associated with prognosis. Here, we constructed a tissue microarray breast tumor samples from 281 patients, and analyzed expressions of ABCB1, ABCC1, ABCC11, and ABCG2 immunohistochemically. Breast cancer subtypes were determined by immunohistochemistry of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2). Protein expression was correlated to clinicopathological characteristics, clinical follow-up, and pathological complete response to neoadjuvant chemotherapy. The tissue microarray comprised 191 luminal A (68.0%), 17 luminal B (6.0%), 27 HER2 (9.0%), and 46 triple-negative (16.4%) samples. ABCC1 and ABCC11 expressions were associated with significantly shorter disease-free survival (P = 0.027 and P = 0.003, respectively). ABCC1, ABCC11, and ABCG2, but not ABCB1, were expressed significantly more, and more frequently, in aggressive subtypes. Patients with HER2+ and triple-negative tumor subtypes that expressed high levels of ABCC11 had significantly worse disease-free survival (P = 0.017 and P < 0.001, respectively). We have shown, for the first time, that ABCC1, ABCC11 and ABCG2 are highly expressed in aggressive breast cancer subtypes, and that tumor ABCC11 expression is associated with poor prognosis. PMID:23288347

Yamada, Akimitsu; Ishikawa, Takashi; Ota, Ikuko; Kimura, Mariko; Shimizu, Daisuke; Tanabe, Mikiko; Chishima, Takashi; Sasaki, Takeshi; Ichikawa, Yasushi; Morita, Satoshi; Yoshiura, Koh-ichiro; Takabe, Kazuaki; Endo, Itaru

2013-01-01

93

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

PubMed

Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C28 and C30 fatty acids or ?-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier. PMID:25041515

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

2014-10-01

94

Functional and Structural Characterization of Polysaccharide Co-polymerase Proteins Required for Polymer Export in ATP-binding Cassette Transporter-dependent Capsule Biosynthesis Pathways*  

PubMed Central

Neisseria meningitidis serogroup B and Escherichia coli K1 bacteria produce a capsular polysaccharide (CPS) that is composed of ?2,8-linked polysialic acid (PSA). Biosynthesis of PSA in these bacteria occurs via an ABC (ATP-binding cassette) transporter-dependent pathway. In N. meningitidis, export of PSA to the surface of the bacterium requires two proteins that form an ABC transporter (CtrC and CtrD) and two additional proteins, CtrA and CtrB, that are proposed to form a cell envelope-spanning export complex. CtrA is a member of the outer membrane polysaccharide export (OPX) family of proteins, which are proposed to form a pore to mediate export of CPSs across the outer membrane. CtrB is an inner membrane protein belonging to the polysaccharide co-polymerase (PCP) family. PCP proteins involved in other bacterial polysaccharide assembly systems form structures that extend into the periplasm from the inner membrane. There is currently no structural information available for PCP or OPX proteins involved in an ABC transporter-dependent CPS biosynthesis pathway to support their proposed roles in polysaccharide export. Here, we report cryo-EM images of purified CtrB reconstituted into lipid bilayers. These images contained molecular top and side views of CtrB and showed that it formed a conical oligomer that extended ?125 Å from the membrane. This structure is consistent with CtrB functioning as a component of an envelope-spanning complex. Cross-complementation of CtrA and CtrB in E. coli mutants with defects in genes encoding the corresponding PCP and OPX proteins show that PCP-OPX pairs require interactions with their cognate partners to export polysaccharide. These experiments add further support for the model of an ABC transporter-PCP-OPX multiprotein complex that functions to export CPS across the cell envelope. PMID:21454677

Larue, Kane; Ford, Robert C.; Willis, Lisa M.; Whitfield, Chris

2011-01-01

95

Disruption of N-linked glycosylation enhances ubiquitin-mediated proteasomal degradation of the human ATP-binding cassette transporter ABCG2.  

PubMed

The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP/MXR/ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and an N-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. In the present study, we aimed to analyze quantitatively the impact of the N-linked glycan on the protein stability of ABCG2. For this purpose, we incorporated one single copy of ABCG2 cDNA into a designated site of genomic DNA in Flp-In-293 cells to stably express ABCG2 or its variant proteins. When ABCG2 wild type-expressing cells were incubated with various N-linked glycosylation inhibitors, tunicamycin profoundly suppressed the protein expression level of ABCG2 and, accordingly, reduced the ABCG2-mediated cellular resistance to the cancer chemotherapeutic SN-38. When Asn596 was converted to Gln596, the resulting variant protein was not glycosylated, and its protein level was about one-third of the wild type level in Flp-In-293 cells. Treatment with MG132, a proteasome inhibitor, increased the level of the variant protein. Immunoblotting with anti-ubiquitin IgG1k after immunoprecipitation of ABCG2 revealed that the N596Q protein was ubiquitinated at levels that were significantly enhanced by treatment with MG132. Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. In conclusion, we propose that the N-linked glycan at Asn596 is important for stabilizing de novo-synthesized ABCG2 and that disruption of this linkage results in protein destabilization and enhanced ubiquitin-mediated proteasomal degradation. PMID:19909340

Nakagawa, Hiroshi; Wakabayashi-Nakao, Kanako; Tamura, Ai; Toyoda, Yu; Koshiba, Shoko; Ishikawa, Toshihisa

2009-12-01

96

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

PubMed Central

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

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

2013-01-01

97

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

PubMed Central

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

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

1999-01-01

98

ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal  

PubMed Central

The visual cycle is a series of enzyme-catalyzed reactions which converts all-trans-retinal to 11-cis-retinal for the regeneration of visual pigments in rod and cone photoreceptor cells. Although essential for vision, 11-cis-retinal like all-trans-retinal is highly toxic due to its highly reactive aldehyde group and has to be detoxified by either reduction to retinol or sequestration within retinal-binding proteins. Previous studies have focused on the role of the ATP-binding cassette transporter ABCA4 associated with Stargardt macular degeneration and retinol dehydrogenases (RDH) in the clearance of all-trans-retinal from photoreceptors following photoexcitation. How rod and cone cells prevent the accumulation of 11-cis-retinal in photoreceptor disk membranes in excess of what is required for visual pigment regeneration is not known. Here we show that ABCA4 can transport N-11-cis-retinylidene-phosphatidylethanolamine (PE), the Schiff-base conjugate of 11-cis-retinal and PE, from the lumen to the cytoplasmic leaflet of disk membranes. This transport function together with chemical isomerization to its all-trans isomer and reduction to all-trans-retinol by RDH can prevent the accumulation of excess 11-cis-retinal and its Schiff-base conjugate and the formation of toxic bisretinoid compounds as found in ABCA4-deficient mice and individuals with Stargardt macular degeneration. This segment of the visual cycle in which excess 11-cis-retinal is converted to all-trans-retinol provides a rationale for the unusually high content of PE and its long-chain unsaturated docosahexaenoyl group in photoreceptor membranes and adds insight into the molecular mechanisms responsible for Stargardt macular degeneration. PMID:24707049

Quazi, Faraz; Molday, Robert S.

2014-01-01

99

Effect of triptolide on the regulation of ATP?binding cassette transporter A1 expression in lipopolysaccharide?induced acute lung injury of rats.  

PubMed

The aim of this study was to investigate the effect of triptolide on ATP?binding cassette transporter A1 (ABCA1) expression in lipopolysaccharide (LPS)?induced acute lung injury (ALI) in rats. Thirty male Sprague Dawley rats weighing 200?250 g were randomly divided into six groups: Normal (N, n=5), Control (C, n=5), LPS (L, n=5), Triptolide 25 µg (TP1, n=5), Triptolide 50 µg (TP2, n=5) and Triptolide 100 µg (TP3, n=5). The N group was not administered anything; the C group was administered 5 ml/kg normal saline intravenously and 7.5 ml/kg 1% dimethylsulfoxide (DMSO) intraperitoneally; the L group was administered 5 mg/kg 0.1% LPS and 1% DMSO; and the TP1, TP2 and TP3 groups were separately injected with 0.1% LPS and 25, 50 or 100 µg/kg triptolide, respectively. All groups had the same liquid?injection volume. Arterial blood gases, tumor necrosis factor?? (TNF??) and ABCA1 expression and general pathology were examined following the treatments. It was found that increasing the triptolide dose in the TP1?3 groups resulted in an increase in the expression of ABCA1 mRNA and protein. As compared with the L group, the ABCA1 expression showed a significant increase in TP2 and TP3 groups (P<0.05). In addition, the expression level of TNF?? was significantly increased in the L and TP1 groups, as compared with that in the N or C groups (P<0.05). Conversely, a marked decrease in TNF?? expression was detected in the TP2 and TP3 groups, as compared with the L or TP1 groups (P<0.05). In conclusion, this study found that triptolide could promote the expression of ABCA1 mRNA and protein and inhibit other inflammatory factors during LPS?induced ALI in rats. Regulating the expression of ABCA1 may be one of the protective mechanisms of triptolide. Furthermore, triptolide?induced increases in ABCA1 expression occurred in a dose?dependent manner between 25 and 100 µg/kg. PMID:25323823

Chen, Jun; Gao, Jianlin; Yang, Jianping; Zhang, Yukun; Wang, Lina

2014-12-01

100

Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters.  

PubMed Central

Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters. One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF. The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones. PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E. coli cells carrying the E. chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP). The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E. coli. For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S. marcescens 8000 and their secretion of LipA and PrtA was analyzed. This demonstrated that a unique exporter, the Lip apparatus, in S. marcescens secretes both LipA and PrtA. Hybrid exporters were tested for secretion of HasA and LipA. The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity. LipA, HasA, and E. chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters. Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems. These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters. PMID:9244262

Akatsuka, H; Binet, R; Kawai, E; Wandersman, C; Omori, K

1997-01-01

101

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

SciTech Connect

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.

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

2011-11-04

102

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

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

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

2014-09-19

103

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

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 7?-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

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

104

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

PubMed Central

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

2013-01-01

105

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

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 < 0.05 for all). Serum lipid parameters were also correlated with sex, age, body mass index, alcohol consumption, and blood pressure in both ethnic groups (P < 0.05-0.001). Conclusion The present study suggests that the V825I polymorphism in the ABCA1 gene is associated with male serum HDL-C and ApoAI levels in the Han, and serum TC levels in the Bai Ku Yao populations. The difference in the association of V825I polymorphism and serum lipid levels between the two ethnic groups might partly result from different ABCA1 gene-enviromental interactions. PMID:21247457

2011-01-01

106

GCN20, a novel ATP binding cassette protein, and GCN1 reside in a complex that mediates activation of the eIF-2 alpha kinase GCN2 in amino acid-starved cells.  

PubMed Central

GCN2 is a protein kinase that phosphorylates the alpha-subunit of translation initiation factor 2 (eIF-2) and thereby stimulates translation of GCN4 mRNA in amino acid-starved cells. We isolated a null mutation in a previously unidentified gene, GCN20, that suppresses the growth-inhibitory effect of eIF-2 alpha hyperphosphorylation catalyzed by mutationally activated forms of GCN2. The deletion of GCN20 in otherwise wild-type strains impairs derepression of GCN4 translation and reduces the level of eIF-2 alpha phosphorylation in vivo, showing that GCN20 is a positive effector of GCN2 kinase function. In accordance with this conclusion, GCN20 was co-immunoprecipitated from cell extracts with GCN1, another factor required to activate GCN2, and the two proteins interacted in the yeast two-hybrid system. We conclude that GCN1 and GCN20 are components of a protein complex that couples the kinase activity of GCN2 to the availability of amino acids. GCN20 is a member of the ATP binding cassette (ABC) family of proteins and is closely related to ABC proteins identified in Caenorhabditis elegans, rice and humans, suggesting that the function of GCN20 may be conserved among diverse eukaryotic organisms. Images PMID:7621831

Vazquez de Aldana, C R; Marton, M J; Hinnebusch, A G

1995-01-01

107

Pathogen-Responsive Expression of a Putative ATP-Binding Cassette Transporter Gene Conferring Resistance to the Diterpenoid Sclareol Is Regulated by Multiple Defense Signaling Pathways in Arabidopsis1  

PubMed Central

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory. PMID:14526118

Campbell, Emma J.; Schenk, Peer M.; Kazan, Kemal; Penninckx, Iris A.M.A.; Anderson, Jonathan P.; Maclean, Don J.; Cammue, Bruno P.A.; Ebert, Paul R.; Manners, John M.

2003-01-01

108

An Aeromonas salmonicida gene which influences a-protein expression in Escherichia coli encodes a protein containing an ATP-binding cassette and maps beside the surface array protein gene.  

PubMed Central

A conserved Aeromonas salmonicida gene (abcA) affecting expression of the surface array protein gene (vapA) in Escherichia coli was identified. The 924-bp gene starts 205 bp after vapA and codes for a protein with a deduced molecular weight (M(r)) of 34,015 containing an N-terminal P-loop and significant homology to the ATP-binding cassette transport protein superfamily. AbcA was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by using T7 polymerase expression and DNA-directed translation and was copurified with the sarkosyl-soluble cytoplasmic membrane fraction. The protein displayed aberrant migration during SDS-PAGE. A lacZ fusion containing 128 bp of upstream sequence and 387 bases in the 5' end of abcA was constructed, and the beta-galactosidase activity of the abcA-lacZ fusion gene was shown to be similar in E. coli and A. salmonicida. The 130,000-M(r) AbcA-LacZ fusion protein was purified, and by using an ATP affinity column, the 129 AbcA N-terminal P-loop-containing residues were shown to bind ATP. Images PMID:8491726

Chu, S; Trust, T J

1993-01-01

109

Normal Formation of a Subset of Intestinal Granules in Caenorhabditis elegans Requires ATP-binding Cassette Transporters HAF-4 and HAF-9, Which Are Highly Homologous to Human Lysosomal Peptide Transporter TAP-Like  

PubMed Central

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects. PMID:19403699

Kawai, Hiromi; Tanji, Takahiro; Shiraishi, Hirohisa; Yamada, Mitsuo; Iijima, Ryoko; Inoue, Takao; Kezuka, Yasuko; Ohashi, Kazuaki; Yoshida, Yasuo; Tohyama, Koujiro; Gengyo-Ando, Keiko; Mitani, Shohei; Arai, Hiroyuki; Maeda, Masatomo

2009-01-01

110

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

PubMed Central

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

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

2013-01-01

111

Cholesteryl ester transfer protein and ATP-binding cassette transporter A1 genotype alter the atorvastatin and simvastatin efficacy: time for genotype-guided therapy?  

PubMed

We compared the efficacy of atorvastatin with simvastatin according to cholesteryl ester transfer protein (CETP) and adenosine triphosphate-binding cassette transporter A1 (ABCA1) genes. Patients treated with atorvastatin (n = 254) or simvastatin (n = 332) were genotyped for CETP (TaqIB and I405V) and ABCA1 (R219K) genetic variants. For genotype B1B2, atorvastatin compared with simvastatin treatment resulted in a greater decrease in total cholesterol (35.4% vs 31.6%, P = .035) and a lower increase in high-density lipoprotein cholesterol (2% vs 8%, P = .05). For genotype B2B2, atorvastatin compared with simvastatin treatment resulted in a lower decrease in low-density lipoprotein cholesterol (31.85 vs 42%, P = .029). For genotypes RR and KK, atorvastatin compared with simvastatin treatment resulted in a greater decrease of triglycerides (27% vs 17% and 35% vs 15%, respectively; P = .02 for all comparisons). The TaqIB and R219K (opposite to I405V) gene polymorphisms seem to modify the response to lipid-lowering therapy with simvastatin or atorvastatin treatment. PMID:22584245

Kolovou, Genovefa; Kolovou, Vana; Mihas, Constantinos; Giannakopoulou, Vasiliki; Vasiliadis, Iannis; Boussoula, Elena; Kollia, Aikaterini; Boutsikou, Maria; Katsiki, Niki; Mavrogeni, Sophie

2013-05-01

112

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

PubMed

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

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

2013-03-01

113

The Arabidopsis ATP-binding Cassette Protein AtMRP5/AtABCC5 Is a High Affinity Inositol Hexakisphosphate Transporter Involved in Guard Cell Signaling and Phytate Storage*  

PubMed Central

Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule. PMID:19797057

Nagy, Reka; Grob, Hanne; Weder, Barbara; Green, Porntip; Klein, Markus; Frelet-Barrand, Annie; Schjoerring, Jan K.; Brearley, Charles; Martinoia, Enrico

2009-01-01

114

ATP-binding Cassette Transporter A1 Mediates the Beneficial Effects of the Liver X Receptor Agonist GW3965 on Object Recognition Memory and Amyloid Burden in Amyloid Precursor Protein/Presenilin 1 Mice*  

PubMed Central

The cholesterol transpoter ATP-binding cassette transporter A1 (ABCA1) moves lipids onto apolipoproteins including apolipoprotein E (apoE), which is the major cholesterol carrier in the brain and an established genetic risk factor for late-onset Alzheimer disease (AD). In amyloid mouse models of AD, ABCA1 deficiency exacerbates amyloidogenesis, whereas ABCA1 overexpression ameliorates amyloid load, suggesting a role for ABCA1 in A? metabolism. Agonists of liver X receptors (LXR), including GW3965, induce transcription of several genes including ABCA1 and apoE, and reduce A? levels and improve cognition in AD mice. However, the specific role of ABCA1 in mediating beneficial responses to LXR agonists in AD mice is unknown. We evaluated behavioral and neuropathogical outcomes in GW3965-treated female APP/PS1 mice with and without ABCA1. Treatment of APP/PS1 mice with GW3965 increased ABCA1 and apoE protein levels. ABCA1 was required to observe significantly elevated apoE levels in brain tissue and cerebrospinal fluid upon therapeutic (33 mg/kg/day) GW3965 treatment. At 33 mg/kg/day, GW3965 was also associated with a trend toward redistribution of A? to the carbonate-soluble pool independent of ABCA1. APP/PS1 mice treated with either 2.5 or 33 mg/kg/day of GW3965 showed a clear trend toward reduced amyloid burden in hippocampus and whole brain, whereas APP/PS1-treated mice lacking ABCA1 failed to display reduced amyloid load in the whole brain and showed trends toward increased hippocampal amyloid. Treatment of APP/PS1 mice with either dose of GW3965 completely restored novel object recognition memory to wild-type levels, which required ABCA1. These results suggest that ABCA1 contributes to several beneficial effects of the LXR agonist GW3965 in APP/PS1 mice. PMID:20739291

Donkin, James J.; Stukas, Sophie; Hirsch-Reinshagen, Veronica; Namjoshi, Dhananjay; Wilkinson, Anna; May, Sharon; Chan, Jeniffer; Fan, Jianjia; Collins, Jon; Wellington, Cheryl L.

2010-01-01

115

Association of Three Common Single Nucleotide Polymorphisms of ATP Binding Cassette G8 Gene with Gallstone Disease: A Meta-Analysis  

PubMed Central

Background In this study, we evaluated the association between these polymorphisms and gallstone disease using meta-analysis and compared the hepatic ABCG5/G8 mRNA expression and biliary lipids composition in patients with different genotypes of T400K and Y54C. Methods Data were analyzed using the Stata/SE 11.0 software and a random- effects model was applied irrespective of between-study heterogeneity. Hepatic mRNA expression of ABCG5/G8 genes in 182 patients with gallstone disease and 35 gallstone-free patients who underwent cholecystectomy were determined using real-time PCR. Genotypes of Y54C and T400K in the ABCG8 gene were determined by allelic discrimination using either genomic DNA or hepatic cDNA as template by Taqman assays. Biliary compostion in gallbladder bile was assayed in these patients as well. Results Ten papers including 13 cohorts were included for the final analysis. In the genotype model, the overall association between genotype with gallstone was significant for D19H (OR?=?2.43, 95%CI: 2.23–2.64, P<0.001), and for Y54C (OR?=?1.36, 95%CI: 1.01–1.83, P?=?0.044), or T400K (OR?=?1.17, 95%CI: 0.96–1.43. P?=?0.110). In allele model, minor alleles of D19H polymorphism (allele D: OR?=?2.25, 95%CI: 2.10–2.42, P<0.001) and of T400K polymorphism (allele K: OR?=?1.18, 95%CI: 1.06–1.31, P<0.001) were related with an increased risk of gallstone disease. However, minor allele of Y54C polymorphism (allele Y, OR?=?1.08, 95%CI: 0.96–1.21, P?=?0.146) was not related with gallstone disease. I2 statistics indicated no significant between-study heterogeneity for all genetic models for any of the three polymorphisms. Funnel plot and Egger’s test suggested the absence of publication bias as well. However, no association of T400K and Y54C polymorphism with hepatic ABCG8/G5 mRNA expression or biliary lipids composition was found. Conclusions Our study showed strong association of D19H polymorphism with gallstone disease. T400K and Y54C polymorphism, though to a less extent, may also relate with gallstone disease. PMID:24498041

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

2014-01-01

116

Phytosterols reduce cholesterol absorption by inhibition of 27-hydroxycholesterol generation, liver X receptor ? activation, and expression of the basolateral sterol exporter ATP-binding cassette A1 in Caco-2 enterocytes.  

PubMed

Phytosterol-enriched foods are increasingly marketed to lower cholesterol levels and atherosclerosis in the general population. Phytosterols reduce cholesterol absorption, but the molecular mechanism is controversial. We therefore investigated the phytosterol effects on cholesterol metabolism in human enterocyte, hepatocyte, and macrophage models relevant for sterol absorption, reverse transport, and excretion. Isomolar sitosterol (50 ?mol/L) was less effectively taken up by enterocytes than cholesterol but suppressed apical cholesterol uptake by 50% (P < 0.01) and basolateral secretion by two-thirds (P < 0.01) whether added in micelles or ethanol or complexed to cyclodextrin. In contrast, enterocytes handled nanomolar (3)H-sitosterol similarly to cholesterol. Enterocytes selectively oxidized all sterols to 27-hydroxy- and 27-carboxy-sterols. Conversion rates were much lower for sitosterol (0.05 ± 0.02 nmol/mg protein) and campesterol (0.48 ± 0.10) compared with cholesterol (3.73 ± 0.60) (P < 0.001). 27-Hydroxycholesterol (27OH-C) activated liver-X-receptor alpha (LXR?) (P < 0.01) and stimulated ATP-binding cassette transporter (ABC) A1 expression (P < 0.001) and basolateral systemic cholesterol secretion from enterocytes (P < 0.05). In co-incubations, phytosterols inhibited 27OH-C generation by sterol 27-hydroxylase (P < 0.001) and reduced LXR?-mediated ABCA1 expression (P < 0.01) and basolateral systemic cholesterol secretion. In contrast, ABCG8 transcription and apical sterol resecretion was unchanged by LXR? activation in human enterocytes. Exogenous LXR? agonists reverted sterol selectivity and phytosterol cholesterol interaction. Due to constitutive apical expression of ABCG5/G8 and LXR?-enhanced basolateral expression of ABCA1 in enterocytes, interference of phytosterols with the generation of the dominating LXR?-agonist 27OH-C blocks the self-priming component of cholesterol absorption. This local LXR? antagonism of dietary phytosterols contributes to sterol selectivity and reduces fractional cholesterol absorption and preloading of nascent HDL with dietary cholesterol. PMID:22535758

Brauner, Reinhard; Johannes, Christian; Ploessl, Florian; Bracher, Franz; Lorenz, Reinhard L

2012-06-01

117

Catalog of 605 single-nucleotide polymorphisms (SNPs) among 13 genes encoding human ATP-binding cassette transporters: ABCA4, ABCA7, ABCA8, ABCD1, ABCD3, ABCD4, ABCE1, ABCF1, ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8.  

PubMed

Single-nucleotide polymorphisms (SNPs) at some gene loci are useful as markers of individual risk for adverse drug reactions or susceptibility to complex diseases. We have been focusing on identifying SNPs in and around genes encoding drug-metabolizing enzymes and transporters, and have constructed several high-density SNP maps of such regions. Here we report SNPs at additional loci, specifically 13 genes belonging to the superfamily of ATP-binding cassette transporters ( ABCA4, ABCA7, ABCA8, ABCD1, ABCD3, ABCD4, ABCE1, ABCF1, ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8). Sequencing a total of 416 kb of genomic DNA from 48 Japanese volunteers identified 605 SNPs among these 13 loci: 14 in 5' flanking regions, 5 in 5' untranslated regions, 37 within coding elements, 529 in introns, 8 in 3' untranslated regions, and 12 in 3' flanking regions. By comparing our data with SNPs deposited in the dbSNP database of the National Center for Biotechnology Information (US) and with published reports, we determined that 491 (81%) of the SNPs reported here were novel. We also detected 107 genetic variations of other types among the loci examined (insertion-deletions or mono- di-, or trinucleotide polymorphisms). The high-density SNP maps we constructed on the basis of these data should provide useful information for investigating associations between genetic variations and common diseases or responsiveness to drug therapy. PMID:12111378

Iida, Aritoshi; Saito, Susumu; Sekine, Akihiro; Mishima, Chihiro; Kitamura, Yuri; Kondo, Kimei; Harigae, Satoko; Osawa, Saori; Nakamura, Yusuke

2002-01-01

118

Functional hot spots in human ATP-binding cassette transporter  

E-print Network

intrahepatic cholestasis type 2; CBAVD, congenital bilateral absence of the vas deferens; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane regulator; CMD1O, cardiomyopathy dilated type 1O; CORD3, cone; Grant sponsor: Cystic Fibrosis Foundation (CFTR2). Libusha Kelly and Hisayo Fukushima contributed

Sali, Andrej

119

PATTERNS & PHENOTYPES ATP-Binding Cassette (ABC) Transporter  

E-print Network

are membrane proteins that regulate intracellular concentrations of myriad compounds and ions. There are >100 was re- stricted to secondary mesenchyme cells and their precursors. Fluorescent protein fusions showed homeo- stasis, potassium channel regulation, pigmentation, and xenobiotic efflux. Detailed

120

Structural model of ATP-binding proteing associated with cystic fibrosis, multidrug resistance and bacterial transport  

Microsoft Academic Search

THE ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization (reviewed in refs 1-3). This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export

Stephen C. Hyde; Paul Emsley; Michael J. Hartshorn; Michael M. Mimmack; Uzi Gileadi; Stephen R. Pearce; Maurice P. Gallagher; Deborah R. Gill; Roderick E. Hubbard; Christopher F. Higgins

1990-01-01

121

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

PubMed Central

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

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

2008-01-01

122

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

PubMed

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

Yu, Fang; De Luca, Vincenzo

2013-09-24

123

ATP-binding cassette transporters are required for efficient RNA interference in Caenorhabditis elegans  

E-print Network

in RNAi have been identified using C. elegans, the list is far from complete. C. elegans remains an important genetics tool for the elu- cidation of RNAi mechanisms in multicellular animals. Ge- netic screening is facilitated by a number of protocols...) targets the TCF/LEF1 transcription factor and produces sterility in young animals reared on this food. unc-22 food (Timmons and Fire, 1998) targets the muscle-specific unc-22 transcript and induces a twitching phenotype. Other feeding strains were obtained...

Timmons, Lisa; Hull, Dawn; Han, Wang; Echalier, B.; Sundaram, P.

2006-08-01

124

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2*  

PubMed Central

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2?,5?-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2?,5?-dihydroxychalcone (2?,5?-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 ?m), indicating competitive inhibition. In contrast, 2?,5?-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter. PMID:20332504

Brechbuhl, Heather M.; Gould, Neal; Kachadourian, Remy; Riekhof, Wayne R.; Voelker, Dennis R.; Day, Brian J.

2010-01-01

125

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

PubMed

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

Aszalos, Adorjan

2008-12-01

126

Putative ATP-binding cassette transporter is essential for Brucella ovis pathogenesis in mice.  

PubMed

Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis-induced genital disease. Two mutant strains were generated in this study (the ?abcAB and ?hmg strains). The B. ovis ?abcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain (P < 0.001). Conversely, the ?hmg strain infected mice at the same level as WT B. ovis, suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the ?abcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the ?abcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo. PMID:21300772

Silva, Teane M A; Paixão, Tatiane A; Costa, Erica A; Xavier, Mariana N; Sá, Joicy Cortez; Moustacas, Valéria S; den Hartigh, Andreas B; Carvalho Neta, Alcina V; Oliveira, Sérgio C; Tsolis, Renée; Santos, Renato L

2011-04-01

127

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein  

E-print Network

Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein Sheref S. Mansy1 op- timization of a non-biological protein derived from a library of random amino acid sequences sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily

Heller, Eric

128

Mechanical Modulation of ATP-binding Affinity of V1-ATPase*  

PubMed Central

V1-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F1-ATPase (the closest relative of V1-ATPase evolutionarily), the role of ATP binding for V1-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V1-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V1-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V1-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (kon) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (koff) was exponentially reduced. The angle dependence of the koff of V1-ATPase was significantly smaller than that of F1-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V1-ATPase. When V1-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, kon was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V1-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions. PMID:23155048

Tirtom, Naciye Esma; Okuno, Daichi; Nakano, Masahiro; Yokoyama, Ken; Noji, Hiroyuki

2013-01-01

129

Gene Expression Profiling of Transporters in the Solute Carrier and ATP-Binding Cassette Superfamilies in Human Eye Substructures  

PubMed Central

The barrier epithelia of the cornea and retina control drug and nutrient access to various compartments of the human eye. While ocular transporters are likely to play a critical role in homeostasis and drug delivery, little is known about their expression, localization and function. In this study, the mRNA expression levels of 445 transporters, metabolic enzymes, transcription factors and nuclear receptors were profiled in five regions of the human eye: cornea, iris, ciliary body, choroid and retina. Through RNA expression profiling and immunohistochemistry, several transporters were identified as putative targets for drug transport in ocular tissues. Our analysis identified SLC22A7 (OAT2), a carrier for the anti-viral drug, acyclovir, in the corneal epithelium, in addition to ABCG2 (BCRP), an important xenobiotic efflux pump, in retinal nerve fibers and the retinal pigment epithelium. Collectively, our results provide an understanding of the transporters that serve to maintain ocular homeostasis and which may be potential targets for drug delivery to deep compartments of the eye. PMID:23268600

Dahlin, Amber; Geier, Ethan; Stocker, Sophie L.; Cropp, Cheryl D.; Grigorenko, Elena; Bloomer, Michele; Siegenthaler, Julie; Xu, Lu; Basile, Anthony S.; Tang-Liu, Diane D-S.; Giacomini, Kathy

2014-01-01

130

Brucella ovis lacking a species-specific putative ATP-binding cassette transporter is attenuated but immunogenic in rams.  

PubMed

Ovine brucellosis caused by Brucella ovis is considered one of the most important reproductive diseases of rams worldwide. This study aimed to characterize the kinetics of infection of a ?abcAB B. ovis mutant strain in rams. Twelve 1-year-old crossbred rams were used. Six rams were challenged with 2 mL of a suspension containing 1.2×10(9) CFU/mL of B. ovis strain ATCC25840 (wild type) by intraprepucial inoculation and additional 50 ?L in each conjunctival sac of a suspension containing 1.2×10(10) CFU/mL of the same strain. The other six rams were challenged with an equivalent number of CFU of the mutant strain ?abcAB B. ovis through the same routes. Serum samples for serology and semen and urine samples for bacteriologic culture and PCR were collected weekly during 24 weeks. At 24 weeks post infection, tissue samples were collected for bacteriologic culture and PCR. All rams inoculated with wild type or the ?abcAB strain seroconverted at the fourth week post infection, remaining positive up to the 16th week post infection. PCR and bacteriology demonstrated that only rams inoculated with the wild type strain shed the organism in semen and urine. Lymphocytes from rams inoculated with wild type or ?abcAB B. ovis had significantly higher proliferation in response to B. ovis antigens when compared with unstimulated controls. Tissue bacteriology and PCR detected B. ovis in all rams challenged with the wild type strain, whereas only one ?abcAB-infected ram had a positive iliac lymph node sample by PCR. PMID:24075357

Silva, Ana Patrícia C; Macêdo, Auricélio A; Costa, Luciana F; Turchetti, Andréia P; Bull, Valquíria; Pessoa, Moisés S; Araújo, Márcio S S; Nascimento, Ernane F; Martins-Filho, Olindo A; Paixão, Tatiane A; Santos, Renato L

2013-12-27

131

XENOBIOTIC REGULATION OF THE ATP BINDING CASSETTE TRANSPORTER ABCB6 AND ITS SIGNIFICANCE TO HEPATIC HEME HOMEOSTASIS  

E-print Network

Porphobilinogen dehydratase PBR Peripheral-type benzodiazepine receptor PCN 5-pregnen-3?-ol-20-one-16?-carbonitrile POR Cytochrome P450 reductase PPIX Protoporphyrin IX PPO Protoporphyrin oxidase PXR Pregnane X receptor TCDD 2... 4.1.3. TCDD induces Abcb6 expression in both mice and humans 85 Figure 4.1.4. A functional AhR pathway is required for B[a]P-mediated up-regulation of Abcb6 87 Figure 4.1.5. Active AhR response element in human and mouse 5?-flanking...

Chavan, Hemantkumar Dilip

2013-12-31

132

Up-Regulation of the ATP-Binding Cassette Transporter A1 Inhibits Hepatitis C Virus Infection  

E-print Network

, France, 6 Unite� Trafic Membranaire et Pathogene`se, Institut Pasteur, Paris, France, 7 Unite� d. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent

Paris-Sud XI, Université de

133

Apatinib (YN968D1) reverses multidrug resistance by inhibiting the efflux function of multiple ATP-binding cassette transporters  

PubMed Central

Apatinib, a small-molecule multi-targeted tyrosine kinase inhibitor, is in phase III clinical trial for treatment of patients with non-small cell lung cancer and gastric cancer in China. In this study, we determined the effect of apatinib on the interaction of specific antineoplastic compounds with P-glycoprotein (P-gp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2). Our results showed that apatinib significantly enhanced the cytotoxicity of ABCB1 or ABCG2 substrate drugs in KBv200, MCF-7/adr and HEK293/ABCB1 cells overexpressing ABCB1 and S1-M1-80, MCF-7/FLV1000 and HEK293/ABCG2-R2 cells overexpressing ABCG2 (wild-type). In contrast, apatinib did not alter the cytotoxicity of specific substrates in the parental cells and cells overexpressing ABCC1. Apatinib significantly increased the intracellular accumulation of rhodamine 123 and doxorubicin in the multidrug resistance (MDR) cells. Furthermore, apatinib significantly inhibited the photolabeling of both ABCB1 and ABCG2 with [125I]-iodoarylazidoprazosin in a concentration-dependent fashion. The ATPase activity of both ABCB1 and ABCG2 was significantly increased by apatinib. However, apatinib, at a concentration the produced a reversal of MDRl, did not significantly alter the expression of the ABCB1 or ABCG2 protein or mRNA levels or the phosphorylation of AKT and ERK1/2. Importantly, apatinib significantly enhanced the effect of paclitaxel against the ABCB1 resistant KBv200 cancer cell xenografts in nude mice. In conclusion, apatinib reverses ABCB1- and ABCG2-mediated MDR by inhibiting their transport function, but not by blocking AKT or ERK1/2 pathway or downregulating ABCB1 or ABCG2 expression. Apatinib may be useful in circumventing MDR to other conventional antineoplastic drugs. PMID:20876799

Mi, Yan-jun; Liang, Yong-ju; Huang, Hong-bing; Zhao, Hong-yun; Wu, Chung-Pu; Wang, Fang; Tao, Li-yang; Zhang, Chuan-zhao; Dai, Chun-Ling; Tiwari, Amit K.; Ma, Xiao-xu; Wah To, Kenneth Kin; Ambudkar, Suresh V.; Chen, Zhe-Sheng; Fu, Li-wu

2010-01-01

134

Differential Sensitivities of the Human ATP-Binding Cassette Transporters ABCG2 and P-Glycoprotein to Cyclosporin A  

E-print Network

such as human immuno- deficiency virus protease inhibitors (Kim et al., 1998; Lee et al., 1998). Therefore-glycoprotein (P-gp) are two ABC transporters that, when overexpressed, are capable of extruding a variety A is neither a substrate nor an inhibitor of the human ABCG2 transporter, under the conditions

Hrycyna, Christine A.

135

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase  

Microsoft Academic Search

A modified ‘cold chase’ technique was used to study tight [14C]ADP and [14C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF0F1). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF0F1 or CF1 reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time

Alexander N. Malyan

2006-01-01

136

Critical role of ?-phosphate in structural transition of Na,K-ATPase upon ATP binding  

NASA Astrophysics Data System (ADS)

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that ?-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while ?-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the ``E1-open'' to ``E1-closed'' conformation ready for phosphorylation.

Petrushanko, Irina Yu.; Mitkevich, Vladimir A.; Anashkina, Anastasia A.; Klimanova, Elizaveta A.; Dergousova, Elena A.; Lopina, Olga D.; Makarov, Alexander A.

2014-06-01

137

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase.  

PubMed

Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P; Diffley, John F X

2014-09-01

138

Critical role of ?-phosphate in structural transition of Na,K-ATPase upon ATP binding.  

PubMed

Active transport of sodium and potassium ions by Na,K-ATPase is accompanied by the enzyme conformational transition between E1 and E2 states. ATP and ADP bind to Na,K-ATPase in the E1 conformation with similar affinity but the properties of enzyme in complexes with these nucleotides are different. We have studied thermodynamics of Na,K-ATPase binding with adenine nucleotides at different temperatures using isothermal titration calorimetry. Our data indicate that ?-phosphate is involved in complex formation by increasing the affinity of adenine nucleotides to Na,K-ATPase by an order of magnitude, while ?-phosphate does not affect it. ATP binding to Na,K-ATPase in contrast to ADP binding generates a structural transition in the enzyme, which is consistent with the movement of a significant portion of the surface area to a solvent-protected state. We propose that ATP binding leads to convergence of the nucleotide-binding and phosphorylation domains transferring the enzyme from the "E1-open" to "E1-closed" conformation ready for phosphorylation. PMID:24893715

Petrushanko, Irina Yu; Mitkevich, Vladimir A; Anashkina, Anastasia A; Klimanova, Elizaveta A; Dergousova, Elena A; Lopina, Olga D; Makarov, Alexander A

2014-01-01

139

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

PubMed

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

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

2010-09-01

140

Complexed Structures of Formylglycinamide Ribonucleotide Amidotransferase from Thermotoga maritima Describe a Novel ATP Binding Protein Superfamily  

SciTech Connect

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. SmPurL 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 unliganded form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analogue, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. The 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. Surprising discoveries, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculation about the regulatory role of the auxiliary site in formation of the PurLSQ complex as well as the evolutionary relationship of PurLs from different organisms.

Morar, Mariya; Anand, Ruchi; Hoskins, Aaron A.; Stubbe, JoAnne; Ealick, Steven E. (MIT); (Cornell)

2008-09-11

141

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

PubMed Central

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

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

2013-01-01

142

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

PubMed

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

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

2014-10-17

143

ATP binding to the ? subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities.  

PubMed

ATP binding to the ? subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ? subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ? subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ? subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ? subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ? subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ? subunit was in its extended-state conformation. The present study reveals a novel role of the ? subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1. PMID:21510843

Kadoya, Fumitaka; Kato, Shigeyuki; Watanabe, Kei; Kato-Yamada, Yasuyuki

2011-07-01

144

Identification of Mutations in Regions Corresponding to the Two Putative Nucleotide (ATP)Binding Folds of the Cystic Fibrosis Gene  

Microsoft Academic Search

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

Bat-Sheva Kerem; Julian Zielenski; Danuta Markiewicz; Dominique Bozon; Ephraim Gazit; Jacob Yahav; Dara Kennedy; John R. Riordan; Francis S. Collins; Johanna M. Rommens; Lap-Chee Tsui

1990-01-01

145

Inhibition of cholesterol absorption associated with a PPAR -dependent increase in ABC binding cassette transporter A1 in mice  

Microsoft Academic Search

Dietary supplementation with the peroxisome proliferator-activated receptor ? (PPAR ? ) ligand WY 14,643 gave rise to a 4- to 5-fold increase in the expression of mRNA for the ATP binding cassette transporter A1 (ABCA1) in the intestine of normal mice. There was no effect in the intes- tine of PPAR ? -null mice. Consumption of a high-choles- terol diet

Brian L. Knight; Dilip D. Patel; Sandy M. Humphreys; David Wiggins; Geoffrey F. Gibbons

2003-01-01

146

Structure-guided development of specific pyruvate dehydrogenase kinase inhibitors targeting the ATP-binding pocket.  

PubMed

Pyruvate dehydrogenase kinase isoforms (PDKs 1-4) negatively regulate activity of the mitochondrial pyruvate dehydrogenase complex by reversible phosphorylation. PDK isoforms are up-regulated in obesity, diabetes, heart failure, and cancer and are potential therapeutic targets for these important human diseases. Here, we employed a structure-guided design to convert a known Hsp90 inhibitor to a series of highly specific PDK inhibitors, based on structural conservation in the ATP-binding pocket. The key step involved the substitution of a carbonyl group in the parent compound with a sulfonyl in the PDK inhibitors. The final compound of this series, 2-[(2,4-dihydroxyphenyl)sulfonyl]isoindoline-4,6-diol, designated PS10, inhibits all four PDK isoforms with IC50 = 0.8 ?M for PDK2. The administration of PS10 (70 mg/kg) to diet-induced obese mice significantly augments pyruvate dehydrogenase complex activity with reduced phosphorylation in different tissues. Prolonged PS10 treatments result in improved glucose tolerance and notably lessened hepatic steatosis in the mouse model. The results support the pharmacological approach of targeting PDK to control both glucose and fat levels in obesity and type 2 diabetes. PMID:24356970

Tso, Shih-Chia; Qi, Xiangbing; Gui, Wen-Jun; Wu, Cheng-Yang; Chuang, Jacinta L; Wernstedt-Asterholm, Ingrid; Morlock, Lorraine K; Owens, Kyle R; Scherer, Philipp E; Williams, Noelle S; Tambar, Uttam K; Wynn, R Max; Chuang, David T

2014-02-14

147

Three-Dimensional Structures Reveal Multiple ADP/ATP Binding Modes  

SciTech Connect

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.

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

2011-12-31

148

ATP binding to human serine racemase is cooperative and modulated by glycine.  

PubMed

The N-methyl D-aspartate (NMDA) receptors play a key role in excitatory neurotransmission, and control learning, memory and synaptic plasticity. Their activity is modulated by the agonist glutamate and by the co-agonists d-serine and glycine. In the human brain, d-serine is synthesized from l-serine by the dimeric pyridoxal 5'-phosphate-dependent enzyme serine racemase, which also degrades l- and d-serine to pyruvate and ammonia. The dependence of l- and d-serine ?-elimination and l-serine racemization activities on ATP concentration was characterized, and was found to be strongly cooperative, with Hill coefficients close to 2 and apparent ATP dissociation constants ranging from 0.22 to 0.41 mm. ATP binding to the holo-enzyme, monitored by the fluorescence changes of the coenzyme, was also determined to be cooperative, with an apparent dissociation constant of 0.24 mm. Glycine, an active-site ligand, increased the serine racemase affinity for ATP by ~ 22-fold, abolishing cooperativity. Conversely, ATP increased the non-cooperative glycine binding 15-fold. These results indicate cross-talk between allosteric and active sites, leading to the stabilization of two alternative protein conformations with ATP affinities of ~ 10 ?M and 1.8 mm, as evaluated within the Monod, Wyman and Changeux model. Therefore, intracellular ATP and glycine control d-serine homeostasis, and, indirectly, NMDA receptor activity. Because hyper- and hypo-activation of NMDA receptors are associated with neuropathologies, the development of allosteric drugs modulating serine racemase activity is a promising therapeutic strategy. PMID:23992455

Marchetti, Marialaura; Bruno, Stefano; Campanini, Barbara; Peracchi, Alessio; Mai, Nicole; Mozzarelli, Andrea

2013-11-01

149

ACID: annotation of cassette and integron data  

E-print Network

Background: Although integrons and their associated gene cassettes are present in ~10% of bacteria and can represent up to 3% of the genome in which they are found, very few have been properly identified and annotated in ...

Boucher, Yan

150

Involvement of ectodomain Leu 214 in ATP binding and channel desensitization of the P2X4 receptor.  

PubMed

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains. PMID:24762105

Zhang, Longmei; Xu, Huijuan; Jie, Yanling; Gao, Chao; Chen, Wanjuan; Yin, Shikui; Samways, Damien S K; Li, Zhiyuan

2014-05-13

151

The ATP Binding Cassette Transporter Gene CgCDR1 from Candida glabrata Is Involved in the Resistance of Clinical Isolates to Azole Antifungal Agents  

Microsoft Academic Search

The resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrata clinical isolates recovered from two separate AIDS patients. The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itracon- azole and ketoconazole. Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was

DOMINIQUE SANGLARD; FRANCOISE ISCHER; DAVID CALABRESE; PAUL A. MAJCHERCZYK; JACQUES BILLE

1999-01-01

152

Ethanolic Extract of Propolis Promotes Reverse Cholesterol Transport and the Expression of ATP-Binding Cassette Transporter A1 and G1 in Mice  

Microsoft Academic Search

The ethanolic extract of propolis (EEP) is beneficial in increasing high density lipoprotein (HDL) cholesterol (HDL-C) and\\u000a diminishing risks of atherosclerosis. In this study, we examined the effects of EEP on reverse cholesterol transport in mice.\\u000a 3H -cholesterol laden macrophage was injected intraperitoneally into mice fed by gastric gavage with EEP. Plasma lipid level\\u000a was determined and 3H-cholesterol was traced

Yang Yu; Yanhong Si; Guohua Song; Tian Luo; Jiafu Wang; Shucun Qin

153

Chalcogenopyrylium compounds as modulators of the ATP-binding cassette transporters P-glycoprotein (P-gp/ABCB1) and multidrug resistance protein 1 (MRP1/ABCC1).  

PubMed

Twenty-seven chalcogenopyrylium derivatives varying in the heteroatom of the pyrylium core and substituents at the 2-, 4-, and 6-positions were examined for their effect on human MRP1-mediated uptake of tritiated estradiol glucuronide into inside-out membrane vesicles, their affinity for and ability to stimulate the ATPase activity of purified human P-glycoprotein (P-gp)-His(10), and their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant cells. Differences in their effects on MRP1 and P-gp activity were noted, and a second set of thiopyrylium compounds with systematic substituent changes was examined to refine these differences further. Derivatives with tert-butyl substituents in the 2- and 6-positions had the lowest inhibitory activity toward both transporters. Derivatives with thioamide functionality in the 4-position were more active against MRP1 than derivatives with amide functionality. Conversely, derivatives with amide functionality in the 4-position were more active in P-gp than derivatives with thioamide functionality. PMID:22533905

Ebert, Sean P; Wetzel, Bryan; Myette, Robert L; Conseil, Gwenaëlle; Cole, Susan P C; Sawada, Geri A; Loo, Tip W; Bartlett, M Claire; Clarke, David M; Detty, Michael R

2012-05-24

154

Stimulation of CD36 and the key effector of reverse cholesterol transport ATP-binding cassette A1 in monocytoid cells by niacin  

Microsoft Academic Search

Niacin, the first lipid lowering drug shown to improve survival after myocardial infarction, decreases LDL and increases HDL cholesterol levels. These effects cannot fully be explained by its suspected mechanism of action, inhibition of lipolysis and hepatic VLDL synthesis. Niacin has also been shown to interfere with the cyclic AMP (cAMP)\\/protein kinase A (PKA) pathway and massively stimulate prostaglandin D2

Tina Rubic; Matthias Trottmann; Reinhard L Lorenz

2004-01-01

155

The ATP binding cassette transporter A1 (ABCA1) modulates the development of aortic atherosclerosis in C57BL\\/6 and apoE-knockout mice  

Microsoft Academic Search

Identification of mutations in the ABCA1 transporter (ABCA1) as the genetic defect in Tangier disease has generated interest in modulating atherogenic risk by enhancing ABCA1 gene expression. To investigate the role of ABCA1 in atherogenesis, we analyzed diet-induced atherosclerosis in transgenic mice overexpressing human ABCA1 (hABCA1-Tg) and spontaneous lesion formation in hABCA1-Tg x apoE-knockout (KO) mice. Overexpression of hABCA1 in

C. W. Joyce; M J Amar; G Lambert; B L Vaisman; B Paigen; Fruchart J Najib; R F Hoyt; E D Neufeld; A T Remaley; D S Fredrickson; H B Brewer; Fojo S Santamarina

2002-01-01

156

Cloning and expression analysis of the ATP-binding cassette transporter gene MFABC1 and the alternative oxidase gene MfAOX1 from Monilinia fructicola.  

PubMed

Brown rot, caused by Moniliniafructicola (G Wint) Honey, is a serious disease of peach in all commercial peach production areas in the USA, including South Carolina where it has been primarily controlled by pre-harvest application of 14-alpha demethylation (DMI) fungicides for more than 15 years. Recently, the Qo fungicide azoxystrobin was registered for brown rot control and is currently being investigated for its potential as a DMI fungicide rotation partner because of its different mode of action. In an effort to investigate molecular mechanisms of DMI and Qo fungicide resistance in M fructicola, the ABC transporter gene MfABC1 and the alternative oxidase gene MfAOX1 were cloned to study their potential role in conferring fungicide resistance. The MfABC1 gene was 4380 bp in length and contained one intron of 71 bp. The gene revealed high amino acid homologies with atrB from Aspergillus nidulans (Eidam) Winter, an ABC transporter conferring resistance to many fungicides, including DMI fungicides. MfABC1 gene expression was induced after myclobutanil and propiconazole treatment in isolates with low sensitivity to the same fungicides, and in an isolate with high sensitivity to propiconazole. The results suggest that the MfABC1 gene may be a DMI fungicide resistance determinant in M fructicola. The alternative oxidase gene MfAOX1 from M fructicola was cloned and gene expression was analyzed. The MfAOX1 gene was 1077 bp in length and contained two introns of 54 and 67 bp. The amino acid sequence was 63.8, 63.8 and 57.7% identical to alternative oxidases from Venturia inaequalis (Cooke) Winter, Aspergillus niger van Teighem and A nidulans, respectively. MfAOX1 expression in some but not all M fructicola isolates was induced in mycelia treated with azoxystrobin. Azoxystrobin at 2 microg ml(-1) significantly induced MfAOX1 expression in isolates with low MfAOX1 constitutive expression levels. PMID:14561072

Schnabel, Guido; Dait, Qun; Paradkar, Manjiri R

2003-10-01

157

Heat shock protein hsp70 protects cells from thermal stress even after deletion of its ATP-binding domain.  

PubMed Central

Retroviral-mediated gene transfer experiments show that rodent cells become heat resistant when stably and constitutively expressing a cloned human gene encoding an intact human 70-kDa heat shock protein (hsp70). Cells expressing higher levels of the hsp70 protein generally tolerate thermal stress better, whereas cells expressing either of two mutated hsp70-encoding genes, one with a 4-base pair out-of-frame deletion and one with an in-frame deletion of codons 438-618, are heat sensitive. These results provide strong evidence that expression of hsp70 leads directly to thermal tolerance. Surprisingly, cells expressing a mutant hsp70 of a human gene missing codons 120-428 are, nevertheless, heat resistant. Because the deleted region of this mutant contains the ATP-binding domain of human hsp70, this domain appears dispensable in the hsp70-mediated protection of cells from thermal stress. Images PMID:1549562

Li, G C; Li, L; Liu, R Y; Rehman, M; Lee, W M

1992-01-01

158

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

SciTech Connect

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.

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

2009-01-01

159

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

SciTech Connect

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.

Rhee, Dong Keun [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Cho, Bon A [School of Life Sciences and Biotechnology, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Hyong Bai [Department of Bioinformatics, Korea University, Yeongigun, Chungnam 339-700 (Korea, Republic of)]. E-mail: hbkim5212@hotmail.com

2005-06-03

160

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

SciTech Connect

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.

Nakai, A.; Hirayama, C.; Ohtsuka, K.; Hirayoshi, K.; Nagata, K. (Kyoto Univ. (Japan))

1990-06-01

161

Oxidation-induced intramolecular disulfide bond inactivates mitogen-activated protein kinase kinase 6 by inhibiting ATP binding  

PubMed Central

Mitogen-activated protein kinase kinase 6 (MKK6) is a member of the mitogen-activated protein kinase (MAPK) kinase (MAP2K) subfamily that specifically phosphorylates and activates the p38 MAPKs. Based on both biochemical and cellular assays, we found that MKK6 was extremely sensitive to oxidation: It was inactivated by oxidation and its kinase activity was fully restored upon treatment with a reducing agent. Detailed mechanistic studies showed that cysteines 109 and 196, two of the six cysteines in MKK6, formed an intramolecular disulfide bond upon oxidation that inactivated MKK6 by inhibiting its ATP binding. This mechanism is distinct from that seen in other redox-sensitive kinases. The two cysteines involved in intramolecular disulfide formation are conserved in all seven members of the MAP2K family. Consistently, we confirmed that other MAP2Ks were also sensitive to oxidation. Our work reveals that MKK6 and other MAP2Ks are a distinct class of cellular redox sensors. PMID:21078955

Diao, Yarui; Liu, Wei; Wong, Catherine C. L.; Wang, Xi; Lee, Kaman; Cheung, Po-yan; Pan, Lifeng; Xu, Tao; Han, Jiahuai; Yates, John R.; Zhang, Mingjie; Wu, Zhenguo

2010-01-01

162

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

SciTech Connect

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.

Kerem, B.; Zielenski, J.; Markiewicz, D.; Bozon, D.; Kennedy, D.; Rommens, J.M. (Hospital for Sick Children, Toronto, Ontario (Canada)); Gazit, E. (Tel Aviv Univ. (Israel)); Yahav, J. (Chaim Sheba Medical Center, Tel Hashomer (Israel)); Riordan, J.R. (Univ. of Toronto, Ontario (Canada)); Collins, F.S. (Univ. of Michigan, Ann Arbor (United States)); Tsui, Lapchee (Hospital for Sick Children, Toronto, Ontario (Canada) Univ. of Toronto, Ontario (Canada))

1990-11-01

163

ATP Binding to Hemoglobin Response Gene 1 Protein Is Necessary for Regulation of the Mating Type Locus in Candida albicans*  

PubMed Central

HBR1 (hemoglobin response gene 1) is an essential gene in Candida albicans that positively regulates mating type locus MTL? gene expression and thereby regulates cell type-specific developmental genes. Hbr1p contains a phosphate-binding loop (P-loop), a highly conserved motif characteristic of ATP- and GTP-binding proteins. Recombinant Hbr1p was isolated in an oligomeric state that specifically bound ATP with Kd ?2 ?m. ATP but not ADP, AMP, GTP, or dATP specifically protected Hbr1p from proteolysis by trypsin. Site-directed mutagenesis of the highly conserved P-loop lysine (K22Q) and the less conserved glycine (G19S) decreased the binding affinity for soluble ATP and ATP immobilized through its ?-phosphate. ATP bound somewhat more avidly than ATP?S to wild type and mutant Hbr1p. Although Hbr1p exhibits sequence motifs characteristic of adenylate kinases, and adenylate kinase and ATPase activities have been reported for the apparent human ortholog of Hbr1p, assays for adenylate kinase activity, autophosphorylation, and ATPase activity proved negative. Overexpression of wild type but not the mutant forms of Hbr1p restored MTl?2 expression in an HBR1/hbr1 mutant, indicating that ATP binding to the P-loop is necessary for this function of Hbr1p. PMID:21372131

Peterson, Alexander W.; Pendrak, Michael L.; Roberts, David D.

2011-01-01

164

Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio  

Microsoft Academic Search

BACKGROUND: Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such

Yan Boucher; Camilla L Nesbø; Michael J Joss; Andrew Robinson; Bridget C Mabbutt; Michael R Gillings; W Ford Doolittle; HW Stokes

2006-01-01

165

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

PubMed

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na+,K+)-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na+ and K+ of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na+,K+)-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na+ and K+. However, in contrast to Na+, a threshold K+ concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations.Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na+,K+)-ATPase compared to the vertebrate enzyme. PMID:18796291

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

2008-11-15

166

ATP sequestration by a synthetic ATP-binding protein leads to novel phenotypic changes in Escherichia coli.  

PubMed

Artificial proteins that bind key metabolites with high affinity and specificity hold great promise as new tools in synthetic biology, but little has been done to create such molecules and examine their effects on living cells. Experiments of this kind have the potential to expand our understanding of cellular systems, as certain phenotypes may be physically realistic but not yet observed in nature. Here, we examine the physiology and morphology of a population of Escherichia coli as they respond to a genetically encoded, non-biological ATP-binding protein. Unlike natural ATP-dependent proteins, which transiently bind ATP during metabolic transformations, the synthetic protein DX depletes the concentration of intracellular ATP and ADP by a mechanism of protein-mediated ligand sequestration. The resulting ATP/ADP imbalance leads to an adaptive response in which a large population of bacilli cells transition to a filamentous state with dense lipid structures that segregate the cells into compartmentalized units. A wide range of biochemical and microscopy techniques extensively characterized these novel lipid structures, which we have termed endoliposomes. We show that endoliposomes adopt well-defined box-like structures that span the full width of the cell but exclude the synthetic protein DX. We further show that prolonged DX exposure causes a large fraction of the population to enter a viable-but-non-culturable state that is not easily reversed. Both phenotypes correlate with strong intracellular changes in ATP and ADP concentration. We suggest that artificial proteins, such as DX, could be used to control and regulate specific targets in metabolic pathways. PMID:23181457

Korch, Shaleen B; Stomel, Joshua M; León, Megan A; Hamada, Matt A; Stevenson, Christine R; Simpson, Brent W; Gujulla, Sunil K; Chaput, John C

2013-02-15

167

The hypertrophic cardiomyopathy myosin mutation R453C alters ATP binding and hydrolysis of human cardiac ?-myosin.  

PubMed

The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long ?-helix, helix O, and to Switch-2 via the fifth strand of the central ?-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecular activities. We report here the first detailed biochemical kinetic analysis of the motor domain of the human ?-cardiac myosin carrying the R453C mutation. A recent report of the same mutation (Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., and Spudich, J. A. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 12607-12612) found reduced ATPase and in vitro motility but increased force production using an optical trap. Surprisingly, our results show that the mutation alters few biochemical kinetic parameters significantly. The exceptions are the rate constants for ATP binding to the motor domain (reduced by 35%) and the ATP hydrolysis step/recovery stroke (slowed 3-fold), which could be the rate-limiting step for the ATPase cycle. Effects of the mutation on the recovery stroke are consistent with a perturbation of Switch-2 closure, which is required for the recovery stroke and the subsequent ATP hydrolysis. PMID:24344137

Bloemink, Marieke; Deacon, John; Langer, Stephen; Vera, Carlos; Combs, Ariana; Leinwand, Leslie; Geeves, Michael A

2014-02-21

168

The Hypertrophic Cardiomyopathy Myosin Mutation R453C Alters ATP Binding and Hydrolysis of Human Cardiac ?-Myosin*  

PubMed Central

The human hypertrophic cardiomyopathy mutation R453C results in one of the more severe forms of the myopathy. Arg-453 is found in a conserved surface loop of the upper 50-kDa domain of the myosin motor domain and lies between the nucleotide binding pocket and the actin binding site. It connects to the cardiomyopathy loop via a long ?-helix, helix O, and to Switch-2 via the fifth strand of the central ?-sheet. The mutation is, therefore, in a position to perturb a wide range of myosin molecular activities. We report here the first detailed biochemical kinetic analysis of the motor domain of the human ?-cardiac myosin carrying the R453C mutation. A recent report of the same mutation (Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., and Spudich, J. A. (2013) Proc. Natl. Acad. Sci. U.S.A. 110, 12607–12612) found reduced ATPase and in vitro motility but increased force production using an optical trap. Surprisingly, our results show that the mutation alters few biochemical kinetic parameters significantly. The exceptions are the rate constants for ATP binding to the motor domain (reduced by 35%) and the ATP hydrolysis step/recovery stroke (slowed 3-fold), which could be the rate-limiting step for the ATPase cycle. Effects of the mutation on the recovery stroke are consistent with a perturbation of Switch-2 closure, which is required for the recovery stroke and the subsequent ATP hydrolysis. PMID:24344137

Bloemink, Marieke; Deacon, John; Langer, Stephen; Vera, Carlos; Combs, Ariana; Leinwand, Leslie; Geeves, Michael A.

2014-01-01

169

Zinc and ATP Binding of the Hexameric AAA-ATPase PilF from Thermus thermophilus: ROLE IN COMPLEX STABILITY, PILIATION, ADHESION, TWITCHING MOTILITY, AND NATURAL TRANSFORMATION.  

PubMed

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

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

2014-10-31

170

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

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.

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

1988-06-01

171

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

PubMed Central

CaMKII (Ca2+/calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The ? isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII ? at Ser26, a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at Ser26, we generated a phosphospecific Ser26 antibody and demonstrated an increase in Ser26 phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of Ser26 affects the kinase activity, we mutated Ser26 to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in Thr287 autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at Ser26 of CaMKII ? inhibits the catalytic activity of the enzyme towards its autophosphorylation site at Thr287 most probably by blocking ATP binding. We propose that Ser26 phosphorylation constitutes an important mechanism for switching off CaMKII activity. PMID:23289753

Yilmaz, Mehtap; Gangopadhyay, Samudra S.; Leavis, Paul; Grabarek, Zenon; Morgan, Kathleen G.

2013-01-01

172

Decipher the Mechanisms of Protein Conformational Changes Induced by Nucleotide Binding through Free-Energy Landscape Analysis: ATP Binding to Hsp70  

PubMed Central

ATP regulates the function of many proteins in the cell by transducing its binding and hydrolysis energies into protein conformational changes by mechanisms which are challenging to identify at the atomic scale. Based on molecular dynamics (MD) simulations, a method is proposed to analyze the structural changes induced by ATP binding to a protein by computing the effective free-energy landscape (FEL) of a subset of its coordinates along its amino-acid sequence. The method is applied to characterize the mechanism by which the binding of ATP to the nucleotide-binding domain (NBD) of Hsp70 propagates a signal to its substrate-binding domain (SBD). Unbiased MD simulations were performed for Hsp70-DnaK chaperone in nucleotide-free, ADP-bound and ATP-bound states. The simulations revealed that the SBD does not interact with the NBD for DnaK in its nucleotide-free and ADP-bound states whereas the docking of the SBD was found in the ATP-bound state. The docked state induced by ATP binding found in MD is an intermediate state between the initial nucleotide-free and final ATP-bound states of Hsp70. The analysis of the FEL projected along the amino-acid sequence permitted to identify a subset of 27 protein internal coordinates corresponding to a network of 91 key residues involved in the conformational change induced by ATP binding. Among the 91 residues, 26 are identified for the first time, whereas the others were shown relevant for the allosteric communication of Hsp70 s in several experiments and bioinformatics analysis. The FEL analysis revealed also the origin of the ATP-induced structural modifications of the SBD recently measured by Electron Paramagnetic Resonance. The pathway between the nucleotide-free and the intermediate state of DnaK was extracted by applying principal component analysis to the subset of internal coordinates describing the transition. The methodology proposed is general and could be applied to analyze allosteric communication in other proteins. PMID:24348227

Nicolai, Adrien; Delarue, Patrice; Senet, Patrick

2013-01-01

173

RAC: Repository of Antibiotic resistance Cassettes  

PubMed Central

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 ?-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 and improve the quality of annotations of gene cassettes in integrons associated with antibiotic resistance. Database URL: http://www2.chi.unsw.edu.au/rac. PMID:22140215

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

2011-01-01

174

Chemistry & Biology 13, 139147, February 2006 2006 Elsevier Ltd All rights reserved DOI 10.1016/j.chembiol.2005.10.015 Directed Evolution of ATP Binding Proteins  

E-print Network

.chembiol.2005.10.015 Directed Evolution of ATP Binding Proteins from a Zinc Finger Domain by Using mRNA Display molecular targets, but alterna- tive protein scaffolds are increasingly being used for the directed evolution of proteins with novel molecular recognition properties. We have designed a combina- torial

Heller, Eric

175

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

PubMed Central

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

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

2013-01-01

176

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

PubMed Central

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.

Karlin, Justin; Fischhaber, Paula L.

2013-01-01

177

Automatic loading of composite tape using cassettes  

NASA Astrophysics Data System (ADS)

This patent application relates generally to laminating and tape-laying systems, and more specifically to a system of automatically loading composite tape onto tape-laying machine using cassettes. Automated composite laminating work centers reduce manufacturing costs and provide for high rates of production. A system for the automatic and continuous loading of composite tape into a tape-laying machine is disclosed. The system provides composite tape from a plurality of cassettes which are held in the magazine of a tape loading system. The tape loading system has a tape drive which feeds tape from a cassette in an active position to the tape-laying system in controlled amounts. The tape loading system also has a cassette transfer system which pushes a full cassette from a standby position into the active position when the cassette in the active position exhausts its supply of tape.

Hailey, S. I.

1986-02-01

178

MgATP binding to the nucleotide-binding domains of the eukaryotic cytoplasmic chaperonin induces conformational changes in the putative substrate-binding domains.  

PubMed Central

The eukaryotic cytosolic chaperonins are large heterooligomeric complexes with a cylindrical shape, resembling that of the homooligomeric bacterial counterpart, GroEL. In analogy to GroEL, changes in shape of the cytosolic chaperonin have been detected in the presence of MgATP using electron microscopy but, in contrast to the nucleotide-induced conformational changes in GroEL, no details are available about the specific nature of these changes. The present study identifies the structural regions of the cytosolic chaperonin that undergo conformational changes when MgATP binds to the nucleotide binding domains. It is shown that limited proteolysis with trypsin in the absence of MgATP cleaves each of the eight subunits approximately in half, generating two fragments of approximately 30 kDa. Using mass spectrometry (MS) and N-terminal sequence analysis, the cleavage is found to occur in a narrow span of the amino acid sequence, corresponding to the peptide binding regions of GroEL and to the helical protrusion, recently identified in the structure of the substrate binding domain of the archeal group II chaperonin. This proteolytic cleavage is prevented by MgATP but not by ATP in the absence of magnesium, ATP analogs (MgATPyS and MgAMP-PNP) or MgADP. These results suggest that, in analogy to GroEL, binding of MgATP to the nucleotide binding domains of the cytosolic chaperonin induces long range conformational changes in the polypeptide binding domains. It is postulated that despite their different subunit composition and substrate specificity, group I and group II chaperonins may share similar, functionally-important, conformational changes. Additional conformational changes are likely to involve a flexible helix-loop-helix motif, which is characteristic for all group II chaperonins. PMID:9684884

Szpikowska, B. K.; Swiderek, K. M.; Sherman, M. A.; Mas, M. T.

1998-01-01

179

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

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.

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

180

amdSYM, a new dominant recyclable marker cassette for Saccharomyces cerevisiae  

PubMed Central

Despite the large collection of selectable marker genes available for Saccharomyces cerevisiae, marker availability can still present a hurdle when dozens of genetic manipulations are required. Recyclable markers, counterselectable cassettes that can be removed from the targeted genome after use, are therefore valuable assets in ambitious metabolic engineering programs. In the present work, the new recyclable dominant marker cassette amdSYM, formed by the Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene (Aspergillus nidulans amdS), is presented. The amdSYM cassette confers S. cerevisiae the ability to use acetamide as sole nitrogen source. Direct repeats flanking the amdS gene allow for its efficient recombinative excision. As previously demonstrated in filamentous fungi, loss of the amdS marker cassette from S. cerevisiae can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in S. cerevisiae. Furthermore, using astute cassette design, amdSYM excision can be performed without leaving a scar or heterologous sequences in the targeted genome. The present work therefore demonstrates that amdSYM is a useful addition to the genetic engineering toolbox for Saccharomyces laboratory, wild, and industrial strains. PMID:23253382

Solis-Escalante, Daniel; Kuijpers, Niels GA; Bongaerts, Nadine; Bolat, Irina; Bosman, Lizanne; Pronk, Jack T; Daran, Jean-Marc; Daran-Lapujade, Pascale

2013-01-01

181

Cassette-based digital mammography.  

PubMed

Over the past several years, digital mammography systems have been installed clinically across North America in small but growing numbers. A photostimulable phosphor-based full-field digital mammography image was evaluated in this investigation. Commonly known as computed radiography (CR), its use closely mimics the screen-film mammography paradigm. System performance using modulation transfer function (MTF) and detective quantum efficiency (DQE) metrics show MTF(2.5 mm(-1)) = 0.5, DQE(2.5 mm(-1)) = 0.3, and MTF(5.0 mm(-1)) = 0.2, DQE(5.0 mm(-1)) = 0.05, for a 26 kVp beam, 0.03 mm molybdenum tube filtration, 4.5 cm tissue attenuation, and 15 mR incident exposure to the detector. Slightly higher DQE values were measured at 32 kVp with 0.025 mm rhodium tube filtration. CR mammography advantages include the ability to use existing mammography machines, where multiple rooms can be converted to "digital" operation, which allows overall cost savings compared to integrated digital mammography systems. Chief disadvantages include the labor-intensive handling of the cassettes prior to and after the imaging exam, lack of a direct interface to the x-ray system for recording technique parameters, and relatively slow processing time. Clinical experience in an IRB-approved research trial has suggested that digital mammography with photostimulable storage phosphors and a dedicated CR reader is a viable alternative to conventional screen-film mammography. PMID:15453806

Seibert, J A; Boone, J M; Cooper, V N; Lindfors, K K

2004-10-01

182

Cassette for handling banknotes or the like  

DOEpatents

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.

Lundblad, Leif (Haradsvagen 102, S-141 41 Huddinge, SE)

1981-08-11

183

A disposable microfluidic cassette for DNA amplification and detection.  

PubMed

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

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

2006-01-01

184

Acceptance Inspection for Audio Cassette Recorders.  

ERIC Educational Resources Information Center

A series of inspections for cassette recorders that can be performed to assure that the devices are acceptable is described. The inspections can be completed in 20 minutes and can be performed by instructional personnel. The series of inspection procedures includes tests of the intelligibility of audio, physical condition, tape speed, impulse…

Smith, Edgar A.

185

Antibiotic-resistance cassettes for Bacillus subtilis  

Microsoft Academic Search

The genes encoding resistance to four different antibiotics (erythromycin, kanamycin, tetracycline and spectinomycin) were cloned in the polylinker of various Escherichia coli plasmid vectors. These cassettes can be inserted into cloned Bacillus subtilis (Bs) genes and used to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.

Anne-Marie Guérout-Fleury; Kamran Shazand; Niels Frandsen; Patrick Stragier

1995-01-01

186

An improved counterselection cassette for use in Haemophilus influenzae.  

PubMed

Counterselectable cassettes are extremely useful in molecular biology and allow for the creation of unmarked deletion mutants or the introduction of point mutations. I have constructed an inducible sacB cassette, using the tetracycline repressor. When used in tandem with a kanamycin-resistance marker, the cassette was successful in creating unmarked mutants in Haemophilus influenzae. The inducible nature of the cassette avoids some of the common problems associated with the utilization of sacB in counterselection. PMID:22037605

Johnston, Jason W

2012-01-15

187

Mutation of the ATP-binding pocket of SSA1 indicates that a functional interaction between Ssa1p and Ydj1p is required for post-translational translocation into the yeast endoplasmic reticulum.  

PubMed Central

The translocation of proteins across the yeast ER membrane requires ATP hydrolysis and the action of DnaK (hsp70) and DnaJ homologues. In Saccharomyces cerevisiae the cytosolic hsp70s that promote post-translational translocation are the products of the Ssa gene family. Ssa1p maintains secretory precursors in a translocation-competent state and interacts with Ydj1p, a DnaJ homologue. Although it has been proposed that Ydj1p stimulates the ATPase activity of Ssa1p to release preproteins and engineer translocation, support for this model is incomplete. To this end, mutations in the ATP-binding pocket of SSA1 were constructed and examined both in vivo and in vitro. Expression of the mutant Ssa1p's slows wild-type cell growth, is insufficient to support life in the absence of functional Ssa1p, and results in a dominant effect on post-translational translocation. The ATPase activity of the purified mutant proteins was not enhanced by Ydj1p and the mutant proteins could not bind an unfolded polypeptide substrate. Our data suggest that a productive interaction between Ssa1p and Ydj1p is required to promote protein translocation. PMID:11014801

McClellan, A J; Brodsky, J L

2000-01-01

188

A Cassette Based System for Hydrogen Storage and Delivery  

SciTech Connect

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.

Britton Wayne E.

2006-11-29

189

High-throughput pharmacokinetics: cassette dosing.  

PubMed

The profound technological advances that are now occurring early in the drug discovery process have enabled lead identification groups to deliver very large numbers of promising compounds to the project teams responsible for lead optimization and candidate selection. This success has applied significant pressure to the 'traditional' selection processes performed during the preclinical optimization stages of a new medicine, where compounds with the optimal balance of potency, selectivity, safety and pharmacokinetics, are identified for progression using an iterative synthesis and testing process. Thus, the need exists for higher-throughput methods of determining pharmacokinetic parameters to enable rational decisions to be made on large numbers of compounds. Protocols detailing the administration of mixtures of compounds, cassette dosing, to single animals have been used successfully to increase throughput, and, at the same time address ethical considerations by reducing animal usage. Typically, cassettes of up to ten compounds have been administered in one dose via the intravenous or oral routes. The samples produced have then been analyzed by mass spectrometry. PMID:19649913

Bayliss, M K; Frick, L W

1999-01-01

190

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

DOEpatents

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.

Lindenmeyer, C.W.

1993-01-26

191

21 CFR 892.1860 - Radiographic film/cassette changer.  

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2014-04-01

192

21 CFR 892.1850 - Radiographic film cassette.  

... 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. (a) Identification. A...

2014-04-01

193

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2013 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2013-04-01

194

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2011 CFR

... 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. (a) Identification. A...

2011-04-01

195

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2012 CFR

... 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. (a) Identification. A...

2012-04-01

196

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2012 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2012-04-01

197

21 CFR 892.1860 - Radiographic film/cassette changer.  

Code of Federal Regulations, 2011 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1860 Radiographic film/cassette changer. (a) Identification. A...

2011-04-01

198

21 CFR 892.1850 - Radiographic film cassette.  

Code of Federal Regulations, 2013 CFR

... 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. (a) Identification. A...

2013-04-01

199

Gene activation regresses atherosclerosis, promotes health, and enhances longevity  

Microsoft Academic Search

BACKGROUND: Lifestyle factors and pharmacological compounds activate genetic mechanisms that influence the development of atherosclerotic and other diseases. This article reviews studies on natural and pharmacological gene activation that promotes health and enhances longevity. RESULTS: Living habits including healthy diet and regular physical activity, and pharmacotherapy, upregulate genes encoding enzymes and apolipoprotein and ATP-binding cassette transporters, acting in metabolic processes

Pauli V Luoma

2010-01-01

200

A Portable Analyzer for Pouch-Actuated, Immunoassay Cassettes  

PubMed Central

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

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

2011-01-01

201

Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae  

PubMed Central

Background Manipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time. Results To overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 ?m ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection. Conclusions These vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided. PMID:24161108

2013-01-01

202

2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate and its Cr(H2O)4 and Co(NH3)4 complex derivatives are new fluorescent tools for labelling ATP binding sites of Na+/K+-ATPase.  

PubMed

2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K+-ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na+/K+-ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, Kd=200 microM) and the high (E1, Kd=22 microM) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Ki=(KdE1*KdE2)1/2, the experimentally measured inhibition constant of 66 microM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium(III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na+/K+-ATPase in a specific way. PMID:9728479

Linnertz, H; Lastres Becker, I; Krumscheid, R; Amler, E; Thoenges, D; Schoner, W

1997-01-01

203

Directory of Spoken-Voice Audio-Cassettes, 1972.  

ERIC Educational Resources Information Center

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…

McKee, Gerald, Ed.

204

Cassette Commentary: An Approach to the Teaching of Expository Writing.  

ERIC Educational Resources Information Center

One convenient and time saving method of evaluating student compositions employs a tape recorder and cassettes. In addition to once or twice weekly classroom sessions spent on traditional composition techniques, the instructor regularly schedules individual tutoring sessions with each student. One to two days before the individual session, the…

Medlicott, Alexander, Jr.

205

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

NASA Technical Reports Server (NTRS)

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.

Hughes, J. M., II

1975-01-01

206

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

ERIC Educational Resources Information Center

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…

Adhikarya, Ronny; Colle, Royal D.

207

Pouring and running a protein gel by reusing commercial cassettes.  

PubMed

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

Hwang, Alexander C; Grey, Paris H; Cuddy, Katrina; Oppenheimer, David G

2012-01-01

208

Cassettes for solid-oxide fuel cell stacks and methods of making the same  

DOEpatents

Solid-oxide fuel cell (SOFC) stack assembly designs are consistently investigated to develop an assembly that provides optimal performance, and durability, within desired cost parameters. A new design includes a repeat unit having a SOFC cassette and being characterized by a three-component construct. The three components include an oxidation-resistant, metal window frame hermetically joined to an electrolyte layer of a multi-layer, anode-supported ceramic cell and a pre-cassette including a separator plate having a plurality of vias that provide electrical contact between an anode-side collector within the pre-cassette and a cathode-side current collector of an adjacent cell. The third component is a cathode-side seal, which includes a standoff that supports a cathode channel spacing between each of the cassettes in a stack. Cassettes are formed by joining the pre-cassette and the window frame.

Weil, K. Scott; Meinhardt, Kerry D; Sprenkle, Vincent L

2012-10-23

209

A New Class of Genetic Element, Staphylococcus Cassette Chromosome mec, Encodes Methicillin Resistance in Staphylococcus aureus  

Microsoft Academic Search

We have previously shown that the methicillin-resistance gene mecA of Staphylococcus aureus strain N315 is localized within a large (52-kb) DNA cassette (designated the staphylococcal cassette chromosome mec (SCCmec)) inserted in the chromosome. By sequence determination of the entire DNA, we identified two novel genes (designated cassette chromosome recombinase genes (ccrA and ccrB)) encoding polypeptides having a partial homology to

Y. Katayama; T. Ito; K. Hiramatsu

2000-01-01

210

Is this charred material from a VHS video cassette?  

NASA Astrophysics Data System (ADS)

At his residence, a victim in a double homicide had installed a home-built video surveillance system. The suspects either knew of or discovered this system and removed it. In a backyard at a location associated with the suspects was a barrel used for burning trash. Could charred debris recovered from a metal bowl found among the contents of the barrel be the remains of a VHS video cassette? A positive answer to the question was obtained through a combination of optical microscopy, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and Energy Dispersive Spectroscopy (EDS).

Fruchtenicht, Tara; Blackledge, Robert D.; Williams, Teresa R.

2010-06-01

211

PharmGKB Submission Update: IV. PMT Submissions of Genetic Variations in ATP-  

E-print Network

published on December 1, 2005 Category: genotype Project: Pharmacogenetics of Membrane Transporters Table 1 IDs. Pharmacogenetic Significance: Genetic variation in the ATP-binding cassette (ABC) family

Sali, Andrej

212

An AC electrokinetics facilitated biosensor cassette for rapid pathogen identification.  

PubMed

To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieving robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency through electrokinetics induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetics for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future. PMID:23626988

Ouyang, Mengxing; Mohan, Ruchika; Lu, Yi; Liu, Tingting; Mach, Kathleen E; Sin, Mandy L Y; McComb, Mason; Joshi, Janhvi; Gau, Vincent; Wong, Pak Kin; Liao, Joseph C

2013-07-01

213

AC Electrokinetics Facilitated Biosensor Cassette for Rapid Pathogen Identification  

PubMed Central

To develop a portable point-of-care system based on biosensors for common infectious diseases such as urinary tract infection, the sensing process needs to be implemented within an enclosed fluidic system. On chip sample preparation of clinical samples remains a significant obstacle to achieve robust sensor performance. Herein AC electrokinetics is applied in an electrochemical biosensor cassette to enhance molecular convection and hybridization efficiency though electrokinetic induced fluid motion and Joule heating induced temperature elevation. Using E. coli as an exemplary pathogen, we determined the optimal electrokinetic parameters for detecting bacterial 16S rRNA in the biosensor cassette based on the current output, signal-to-noise ratio, and limit of detection. In addition, a panel of six probe sets targeting common uropathogenic bacteria was demonstrated. The optimized parameters were also validated using patient-derived clinical urine samples. The effectiveness of electrokinetic for on chip sample preparation will facilitate the implementation of point-of-care diagnosis of urinary tract infection in the future. PMID:23626988

Ouyang, Mengxing; Mohan, Ruchika; Lu, Yi; Liu, Tingting; Mach, Kathleen E.; Sin, Mandy L. Y.; McComb, Mason; Joshi, Janhvi; Gau, Vincent

2013-01-01

214

Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species  

Microsoft Academic Search

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky–and blunt–end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used

Mulalo B. Nthangeni; Faranani Ramagoma; Matsobane G. Tlou; Derek Litthauer

2005-01-01

215

Transcriptional regulation of the ABCC6 gene and the background of impaired function of missense disease-causing mutations  

PubMed Central

The human ATP-binding cassette family C member 6 (ABCC6) gene encodes an ABC transporter protein expressed primarily in the liver and to a lesser extent in the kidneys and the intestines. We review here the mechanisms of this restricted tissue-specific expression and the role of hepatocyte nuclear factor 4? which is responsible for the expression pattern. Detailed analyses uncovered further regulators of the expression of the gene pointing to an intronic primate-specific regulator region, an activator of the expression of the gene by binding CCAAT/enhancer-binding protein beta, which interacts with other proteins acting in the proximal promoter. This regulatory network is affected by various environmental stimuli including oxidative stress and the extracellular signal-regulated protein kinases 1 and 2 pathway. We also review here the structural and functional consequences of disease-causing missense mutations of ABCC6. A significant clustering of the missense disease-causing mutations was found at the domain–domain interfaces. This clustering means that the domain contacts are much less permissive to amino acid replacements than the rest of the protein. We summarize the experimental methods resulting in the identification of mutants with preserved transport activity but failure in intracellular targeting. These mutants are candidates for functional rescue by chemical chaperons. The results of such research can provide the basis of future allele-specific therapy of ABCC6-mediated disorders like pseudoxanthoma elasticum or the generalized arterial calcification in infancy. PMID:23483032

Arányi, Tamás; Bacquet, Caroline; de Boussac, Hugues; Ratajewski, Marcin; Pomozi, Viola; Fülöp, Krisztina; Brampton, Christopher N.; Pulaski, Lukasz; Saux, Olivier Le; Váradi, András

2013-01-01

216

A new generic column switching system for quantitation in cassette dosing using LC\\/MS\\/MS  

Microsoft Academic Search

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

T. Ohkawa; Y. Ishida; E. Kanaoka; K. Takahashi; H. Okabe; T. Matsumoto; S. Nakamoto; J. Tamada; M. Koike; T. Yoshikawa

2003-01-01

217

Expression of Antibiotic Resistance Genes in the Integrated Cassettes of Integrons  

Microsoft Academic Search

Plasmids containing cloned integron fragments which differ only with respect to either the sequence of the promoter(s) or the number and order of inserted cassettes were used to examine the expression of resistance genes encoded in integron-associated gene cassettes. All transcripts detected commenced at the common promoter Pant, and alterations in the sequence of Pant affected the level of resistance

CHRISTINA M. COLLIS; ANDRUTH M. HALL

1995-01-01

218

Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons  

Microsoft Academic Search

Resistance of gram-negative organisms to antibiotics such as ?-lactams, aminoglycosides, trimethoprim and chloramphenicol is caused by many different acquired genes, and a substantial proportion of these are part of small mobile elements known as gene cassettes. A gene cassette consists of the gene and a downstream sequence, known as a 59-base element (59-be), that acts as a specific recombination site.

Ruth M. Hall; Christina M. Collis

1998-01-01

219

Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species.  

PubMed

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing "universal" degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus. PMID:15722149

Nthangeni, Mulalo B; Ramagoma, Faranani; Tlou, Matsobane G; Litthauer, Derek

2005-05-01

220

Quantitative assessment and reduction of long-term autoradiographic background  

SciTech Connect

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.

Traub, R.K.; Famous, L.; Krishnan, R.; Olson, K.R.

1990-01-01

221

Combination air sampling cassette and nutrient media dish  

US Patent & Trademark Office Database

A combination air sampling cassette and nutrient media dish having base, orifice plate, and nutrient media dish assembly for the collection of airborne particles. The orifice plate includes a plurality of holes that brings the nutrient media dish into fluid communication with the ambient air. A pump is connected to the air outlet in the base to pull air into the orifice plate through the holes, over the culture media, and out through the air outlet. As the air passes through the holes in the orifice plate it is accelerated and results in the selected impaction of particles in the culture media. A cover fits over the assembly to protect the culture media prior to and after sampling.

2002-10-29

222

Effect of fenofibrate therapy and ABCA1 polymorphisms on high-density lipoprotein subclasses in the Genetics of Lipid Lowering Drugs and Diet Network  

Microsoft Academic Search

Background: Previous studies have shown that ATP-binding cassette transporter 1 (ABCA1) polymorphisms associated with increased ABCA1 expression result in increased small HDL (high-density lipoprotein) subclass particle concentration. This study examines the effect of treatment with fenofibrate, a drug known to bind peroxisome proliferator-activated receptor alpha (PPAR?) which increases the expression of ABCA1 gene, on lipoprotein subclass profiles of individuals stratified

Michael Y. Tsai; Jose M. Ordovas; Na Li; Robert J. Straka; Naomi Q. Hanson; Valerie L. Arends; Donna Arnett

2010-01-01

223

The spectrum of retinal phenotypes caused by mutations in the ABCA4 gene  

Microsoft Academic Search

Background The majority of studies on the retina-specific ATP-binding cassette transporter ( ABCA4) gene have focussed on molecular genetic analysis; comparatively few studies have described the clinical aspects of ABCA4-associated retinal disorders. In this study, we demonstrate the spectrum of retinal dystrophies associated with ABCA4 gene mutations. Methods Nine well-documented patients representing distinct phenotypes in the continuum of ABCA4-related disorders

B. Jeroen Klevering; Alessandra Maugeri; Frans P. M. Cremers; Carel B. Hoyng

2005-01-01

224

Common Genetic Variation in ABCA1 Is Associated With Altered Lipoprotein Levels and a Modified Risk for Coronary Artery Disease  

Microsoft Academic Search

Background—Low plasma HDL cholesterol (HDL-C) is associated with an increased risk of coronary artery disease (CAD). We recently identified the ATP-binding cassette transporter 1 (ABCA1) as the major gene underlying the HDL deficiency associated with reduced cholesterol efflux. Mutations within the ABCA1 gene are associated with decreased HDL-C, increased triglycerides, and an increased risk of CAD. However, the extent to

Susanne M. Clee; Aeilko H. Zwinderman; James C. Engert; Karin Y. Zwarts; Henri O. F. Molhuizen; Kirsten Roomp; J. Wouter Jukema; Michel van Wijland; Marjel van Dam; Thomas J. Hudson; Angela Brooks-Wilson; John J. P. Kastelein; Michael R. Hayden

2010-01-01

225

Statistical Mechanics Analysis of ATP Binding to a Multisubunit Enzyme  

NASA Astrophysics Data System (ADS)

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.

Zhang, Yun-Xin

2014-10-01

226

Supplemental Data Directed Evolution of ATP Binding Proteins  

E-print Network

GA 3') and gc132 (5' ACC TAG TCT CCC CTT TCT CAC GGT GCG CTT GAA GAA GCC TTT ACA GCC CTC ACA GGA 765 765 765 765 765 765 765 765 765 765 765 765 ATC TCC ACA GAT GGC GCA G 3'; 5 is a mixture of bases127 (5'GCT AAT ACG ACT CAC TAT AGG GAC AAT TAC TAT TTA CAA TTA CAA TGG ACT ACA AGG ACG ACG A 3

Heller, Eric

227

Delineating a Conserved Genetic Cassette Promoting Outgrowth of Body Appendages  

PubMed Central

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 ?-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

Lin, Congxing; Yin, Yan; Bell, Sheila M.; Veith, G. Michael; Chen, Hong; Huh, Sung-Ho; Ornitz, David M.; Ma, Liang

2013-01-01

228

The GoldenBricks assembly: A standardized one-shot cloning technique for complete cassette assembly  

E-print Network

BBF RFC 92 proposes a new standard assembly method for the Parts Registry. The method makes one-shot cloning of a complete eukaryotic or prokaryotic cassette possible in one day while keeping compatibility with the BBF RFC ...

Pauthenier, Cyrille

2012-11-05

229

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2014-04-01

230

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2010 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2010-04-01

231

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2013 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2013-04-01

232

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2012 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2012-04-01

233

21 CFR 892.1880 - Wall-mounted radiographic cassette holder.  

Code of Federal Regulations, 2011 CFR

... Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES RADIOLOGY DEVICES Diagnostic Devices § 892.1880 Wall-mounted radiographic cassette holder. (a)...

2011-04-01

234

How To Choose Audio and Video Tape and Cassettes That Best Fit Your Needs  

ERIC Educational Resources Information Center

An audiovisual consultant presents a guide to selecting suitable and cost effective tape and cassettes. He describes and illustrates physical properties of tapes and includes a list of manufacturers and suppliers. (MF)

Smith, Judson

1978-01-01

235

Recordings for Children. A Selected List of Records and Cassettes. Fourth Edition.  

National Technical Information Service (NTIS)

This booklet, compiled by three experts in children's recordings, provides a selected list of records and cassettes which can accommodate a broad range of informational and recreational requirements of children. Some of the subjects covered are children's...

E. E. Thomas

1980-01-01

236

Integron Gene Cassettes: A Repository of Novel Protein Folds with Distinct Interaction Sites  

PubMed Central

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-?, ?+? and ?/? 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

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

237

Cassette EEG sleep recordings in Gilles de la Tourette syndrome.  

PubMed

Tourette syndrome (TS) patients often complain of sleep problems, and questionnaire studies indicate that sleep disturbance is frequent. Decreased slow wave sleep and increased awakenings have been reported in laboratory polysomnography in TS patients, and a serotoninergic disorder of arousal has been postulated. We recorded outpatient sleep in 20 patients newly diagnosed with TS utilizing a 4-channel cassette EEG system. The newly-diagnosed patients were predominantly male, and ranged in age from 10 to 36 years. Some had taken psychotropic medications in the past, but none had been treated systematically for TS. Seven patients had chronic tics only, 8 had tics and attention deficit-hyperactivity, and 5 had tics plus obsessions and compulsions. None had other medical, neurologic, or psychiatric disorders. All were nocturnal sleepers, and were recorded in their usual sleeping environments and routines. TS patients had reduced sleep, decreased sleep efficiency, increased awakenings, and decreased slow wave sleep. Tic patients had increased nocturnal awakenings and movements, particularly those who had tics during sleep. Sleep fragmentation and loss of slow wave sleep was most marked in TS patients with attention deficit-hyperactivity. Sleep latency was increased, REM sleep reduced, and REM sleep latency decreased in TS patients with obsessions and compulsions. These findings accord with previous reports of sleep disturbance in TS, and suggest that these disturbances may vary with TS symptoms. Chronic tics may persist in sleep and cause awakenings, TS with attention deficit may be associated with a disorder of arousal and alertness, and obsessions and compulsions may be manifestations of a biochemical disturbance involving paradoxical sleep. PMID:1628407

Drake, M E; Hietter, S A; Bogner, J E; Andrews, J M

1992-07-01

238

Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids  

PubMed Central

Background Terpenoids constitute a large family of natural products, attracting commercial interest for a variety of uses as flavours, fragrances, drugs and alternative fuels. Saccharomyces cerevisiae offers a versatile cell factory, as the precursors of terpenoid biosynthesis are naturally synthesized by the sterol biosynthetic pathway. Results S. cerevisiae wild type yeast cells, selected for their capacity to produce high sterol levels were targeted for improvement aiming to increase production. Recyclable integration cassettes were developed which enable the unlimited sequential integration of desirable genetic elements (promoters, genes, termination sequence) at any desired locus in the yeast genome. The approach was applied on the yeast sterol biosynthetic pathway genes HMG2, ERG20 and IDI1 resulting in several-fold increase in plant monoterpene and sesquiterpene production. The improved strains were robust and could sustain high terpenoid production levels for an extended period. Simultaneous plasmid-driven co-expression of IDI1 and the HMG2 (K6R) variant, in the improved strain background, maximized monoterpene production levels. Expression of two terpene synthase enzymes from the sage species Salvia fruticosa and S. pomifera (SfCinS1, SpP330) in the modified yeast cells identified a range of terpenoids which are also present in the plant essential oils. Co-expression of the putative interacting protein HSP90 with cineole synthase 1 (SfCinS1) also improved production levels, pointing to an additional means to improve production. Conclusions Using the developed molecular tools, new yeast strains were generated with increased capacity to produce plant terpenoids. The approach taken and the durability of the strains allow successive rounds of improvement to maximize yields. PMID:21276210

2011-01-01

239

Use of a ura5+-lys7+ cassette to construct unmarked gene knock-ins in Schizosaccharomyces pombe.  

PubMed

While the counterselectable Schizosaccharomyces pombe ura4(+) gene can be used to prepare a site in the S. pombe genome to receive an unmarked mutant allele (loss of ura4(+) confers 5FOA-resistant (5FOA(R)) growth), the desired unmarked knock-in strains are generally outnumbered by spontaneously arising 5FOA(R) mutants. Relative to the same approach using the homologous URA3(+) gene in Saccharomyces cerevisiae, knock-ins in S. pombe are harder to identify due to a lower efficiency of homologous recombination and a relatively high background of spontaneous 5FOA(R) colonies. To develop an improved method for identifying cells receiving unmarked mutant alleles, we first determined that 5FOA(R) strains carry mutations in either of two genes; ura4(+) and ura5(+). We then cloned the S. pombe ura5(+) orotate phosphoribosyltransferase gene and constructed a 2.1 kb cassette containing ura5(+) together with the S. pombe lys7(+) gene. Using this doubly marked cassette to disrupt the sck1(+) kinase gene, we can distinguish between strains created by homologous knock-in of unmarked wild-type or kinase-dead alleles and spontaneously arising ura4(-) and ura5(-) mutants by screening 5FOA(R) colonies for the loss of the lys7(+) marker. The utility of this system, especially when the phenotype for the strain carrying the knock-in allele is indistinguishable from that of the disruption strain, is borne out by the fact that ~95% of 5FOA(R) colonies in our studies arose from background ura4(-) and ura5(-) mutations. PMID:22198627

Mudge, Dayna K; Hoffman, Catherine A; Lubinski, Tristan J; Hoffman, Charles S

2012-02-01

240

Erythromycin Esterase Gene ere(A) Is Located in a Functional Gene Cassette in an Unusual Class 2 Integron  

PubMed Central

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

Biskri, Latefa; Mazel, Didier

2003-01-01

241

Erythromycin esterase gene ere(A) is located in a functional gene cassette in an unusual class 2 integron.  

PubMed

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

Biskri, Latefa; Mazel, Didier

2003-10-01

242

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

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 ?M, P = 0.017; 10 ?M, P = 0.001; 25 ?M, P = 0.008; and 50 ?M, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics. PMID:21821808

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

243

A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms  

NASA Technical Reports Server (NTRS)

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.

Hedenstierna, K. O.; Lee, Y. H.; Yang, Y.; Fox, G. E.

1993-01-01

244

Diverse Gene Cassettes in Class 1 Integrons of Facultative Oligotrophic Bacteria of River Mahananda, West Bengal, India  

PubMed Central

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 (?2) 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, Moraxellaceae, Pseudomonadaceae, Aeromonadaceae and Enterobacteriaceae. Conclusions Oligotrophic bacteria are good sources of novel genes as well as potential reservoirs of antibiotic resistance gene casettes. PMID:23951238

Chakraborty, Ranadhir; Kumar, Arvind; Bhowal, Suparna Saha; Mandal, Amit Kumar; Tiwary, Bipransh Kumar; Mukherjee, Shriparna

2013-01-01

245

The Evolution of the Bacterial Luciferase Gene Cassette (lux) as a Real-Time Bioreporter  

PubMed Central

The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted. PMID:22368493

Close, Dan; Xu, Tingting; Smartt, Abby; Rogers, Alexandra; Crossley, Robert; Price, Sarah; Ripp, Steven; Sayler, Gary

2012-01-01

246

Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens.  

PubMed

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

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

247

Mass spectrometry imaging of cassette-dosed drugs for higher throughput pharmacokinetic and biodistribution analysis.  

PubMed

Cassette dosing of compounds for preclinical drug plasma pharmacokinetic analysis has been shown to be a powerful strategy within the pharmaceutical industry for increasing throughput while decreasing the number of animals used. Presented here for the first time is data on the application of a cassette dosing strategy for label-free tissue distribution studies. The aim of the study was to image the spatial distribution of eight nonproprietary drugs (haloperidol, bufuralol, midazolam, clozapine, terfenadine, erlotinib, olanzapine, and moxifloxacin) in multiple tissues after oral and intravenous cassette dosing (four compounds per dose route). An array of mass spectrometry imaging technologies, including matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI), liquid extraction surface analysis tandem mass spectrometry (LESA-MS/MS), and desorption electrospray ionization mass spectrometry (DESI-MS) was used. Tissue analysis following intravenous and oral administration of discretely and cassette-dosed compounds demonstrated similar relative abundances across a range of tissues indicating that a cassette dosing approach was applicable. MALDI MSI was unsuccessful in detecting all of the target compounds; therefore, DESI MSI, a complementary mass spectrometry imaging technique, was used to detect additional target compounds. In addition, by adapting technology used for tissue profiling (LESA-MS/MS) low spatial resolution mass spectrometry imaging (?1 mm) was possible for all targets across all tissues. This study exemplifies the power of multiplatform MSI analysis within a pharmaceutical research and development (R&D) environment. Furthermore, we have illustrated that the cassette dosing approach can be readily applied to provide combined, label-free pharmacokinetic and drug distribution data at an early stage of the drug discovery/development process while minimizing animal usage. PMID:25084360

Swales, John G; Tucker, James W; Strittmatter, Nicole; Nilsson, Anna; Cobice, Diego; Clench, Malcolm R; Mackay, C Logan; Andren, Per E; Takáts, Zoltán; Webborn, Peter J H; Goodwin, Richard J A

2014-08-19

248

Identification, susceptibility, and detection of integron-gene cassettes of Arcanobacterium pyogenes in bovine endometritis.  

PubMed

The present study aimed to identify, determine the susceptibility, and detect gene cassettes of Arcanobacterium (Actinomyces) pyogenes isolates from cows with endometritis. Arcanobacterium pyogenes isolates were identified first by using the API Coryne Vit system test, and further through PCR. Minimum inhibitory concentrations of 23 antimicrobial agents against A. pyogenes were tested using standard broth microdilution assays according to the protocols of the Clinical and Laboratory Standards Institute. The genes of integrons I and II were amplified by PCR using specific primers. Thirty-two A. pyogenes isolates were isolated from 136 endometritic cows in the Hohhot region. Antibiotic susceptibility tests revealed that all isolates were highly sensitive to fluoroquinolones (100%), macrolides (approximately 81.2 to 100%) and florfenicol (90.6%), aminoglycosides (approximately 15.6 to 81.2%), and tetracyclines (approximately 43.7 to 68.7%). However, 53.1% were resistant to clindamycin, approximately 50 to 65.6% were resistant to penicillins, and approximately 37.5 to 71.9% were resistant to cephalosporins. One hundred percent were resistant to sulfonamides and bacitracin zinc. The integrons were further confirmed by sequencing. No class II integrons were detected, whereas 50% (n = 16) of the A. pyogenes isolates were positive for the presence of the intI I gene, but only 13 contained gene cassettes. Sequence analysis of gene cassettes revealed 6 gene cassettes, 4 of which encode resistant determinants of aminoglycosides (aadA1, aadA5, aadA24, and aadB) and 1 of which encodes the resistance gene of chloramphenicol (cmlA6). The function of the sixth identified cassette, designated ORF1, is unknown. The gene cassette arrays aadA24-ORF1, aadA5, and aadA1-addB-cmlA6 were found in 46.13% (6/13), 38.46% (5/13), and 38.46% (5/13) of the isolates, respectively. These cassettes segregated according to a consistent pattern, with aadA5 always alone, ORF1 always with aadA24, and aadA1-aadB and cmlA6 always together. Most of the positive integrons existed in the multiresistant isolates (n = approximately 3 to 7), indicating that the integrons played an important role in the dissemination and spread of antimicrobial resistance. This is the first report of A. pyogenes infections in dairy cows in China and of detection of gene cassettes and integrons in A. pyogenes. PMID:19620647

Liu, M-C; Wu, C-M; Liu, Y-C; Zhao, J-C; Yang, Y-L; Shen, J-Z

2009-08-01

249

Nanoparticle-delivered VEGF-silencing cassette and suicide gene expression cassettes inhibit colon carcinoma growth in vitro and in vivo  

Microsoft Academic Search

The strategies for tumor-specific expression of suicide genes and target tumor angiogenesis have been tested in tumors. However,\\u000a the anti-tumor efficacy of the combination of these two strategies, particularly, delivering suicide gene and anti-angiogenesis\\u000a agent by nanoparticles, has not yet been evaluated in colon carcinoma. We constructed a cassette to silence VEGF-A expression\\u000a and express a fused yCDglyTK gene driven

Aimin Leng; Jing Yang; Ting Liu; Jianfang Cui; Xiu-hua Li; Yanan Zhu; Ting Xiong; Yuxiang Chen

250

A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids.  

PubMed

An inexpensive, hand-held, point-of-care, disposable, self-contained immunoassay cassette comprised of air pouches for pumping, a metering chamber, reagents storage chambers, a mixer, and a lateral flow strip was designed, constructed, and tested. The assay was carried out in a consecutive flow format. The detection was facilitated with up-converting phosphor (UCP) reporter particles. The automated, timely pumping of the various reagents was driven by a spring-loaded timer. The utility of the cassette was demonstrated by detecting antibodies to HIV in saliva samples and further evaluated with a non-contagious, haptenized DNA assay. The cassette has several advantages over dip sticks such as sample preprocessing, integrated storage of reagents, and automated operation that reduces operator errors and training. The cassette and actuator described herein can readily be extended to detect biomarkers of other diseases in body fluids and other fluids at the point of care. The system is particularly suitable for resource-poor countries, where funds and trained personnel are in short supply. PMID:19255658

Liu, Changchun; Qiu, Xianbo; Ongagna, Serge; Chen, Dafeng; Chen, Zongyuan; Abrams, William R; Malamud, Daniel; Corstjens, Paul L A M; Bau, Haim H

2009-03-21

251

Analytica Chimica Acta 559 (2006) 3744 High-throughput pharmacokinetic method: Cassette dosing in mice  

E-print Network

of whole blood from serial bleeds (up to 10 per animal), by LC/MS/MS. An accuracy (88­100%) and precision dramatically increases speed of data collection while dramatically reducing cost and animal usage. The results. © 2005 Elsevier B.V. All rights reserved. Keywords: Pharmacokinetics; Serial bleeding; Cassette dosing

Hammock, Bruce D.

252

A Novel Cassette Method for Probe Evaluation in the Designed Biochips  

PubMed Central

A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip. PMID:24897111

Zinkevich, Vitaly; Sapojnikova, Nelly; Mitchell, Julian; Kartvelishvili, Tamar; Asatiani, Nino; Alkhalil, Samia; Bogdarina, Irina; Al-Humam, Abdulmohsen A.

2014-01-01

253

Integrase-Mediated Recombination of the veb1 Gene Cassette Encoding an Extended-Spectrum ?-Lactamase  

PubMed Central

The veb1 gene cassette encodes the extended spectrum ?-lactamase, VEB-1 that is increasingly isolated from worldwide Gram-negative rods. Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively. PMID:23251590

Aubert, Daniel; Naas, Thierry; Nordmann, Patrice

2012-01-01

254

Synthesis of Novel Tyrosinyl FRET Cassettes, Terminators, and Their Potential Use in DNA Sequencing  

Microsoft Academic Search

Fluorescence resonance energy transfer (FRET) dye labeled cassettes and terminators with one or more donor dyes (fluorescein) and acceptor dye (rhodamine dyes) with benzofuran or tyrosine linker moieties were synthesized. These terminators were evaluated for their energy transfer and DNA sequencing potential using thermostable DNA polymerase.

T. Sudhakar Rao; Weihong Zhang; Haiguang Xiao; Parke Flick; Shiv Kumar; Satyam Nampalli

2003-01-01

255

STS-37 crewmembers watch Pilot Cameron juggle cassettes on OV-104's middeck  

NASA Technical Reports Server (NTRS)

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.

1991-01-01

256

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids.  

PubMed

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

Chen, Dafeng; Mauk, Michael; Qiu, Xianbo; Liu, Changchun; Kim, Jitae; Ramprasad, Sudhir; Ongagna, Serge; Abrams, William R; Malamud, Daniel; Corstjens, Paul L A M; Bau, Haim H

2010-08-01

257

An integrated, self-contained microfluidic cassette for isolation, amplification, and detection of nucleic acids  

PubMed Central

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

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

258

A new integron carrying VIM2 metallo-?-lactamase gene cassette in a Serratia marcescens isolate  

Microsoft Academic Search

Serratia marcescens is an important nosocomial pathogen which is often resistant to multiple antimicrobial agents. An imipenem-resistant S. marcescens isolate from a urine specimen was found to carry a blaVIM-2 gene cassette on a class 1 integron. This finding indicates that blaVIM-2 is presently spreading even to Serratia spp. in Korea, which could compromise the usefulness of carbapenem in the

Jong Hwa Yum; Dongeun Yong; Kyungwon Lee; Hyon-Suk Kim; Yunsop Chong

2002-01-01

259

Development of a low resource RNA extraction cassette based on surface tension valves  

PubMed Central

Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid-air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents by using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low resource setting. PMID:21604768

Bordelon, Hali; Adams, Nicholas M.; Klemm, Amy S.; Russ, Patricia K.; Williams, John V.; Talbot, H. Keipp; Wright, David W.; Haselton, Frederick R.

2011-01-01

260

A Field Comparison of the IOM Inhalable Aerosol Sampler and a Modified 37-mm Cassette  

Microsoft Academic Search

This research focused on comparing a modified 37-mm (Mod37) sampling cassette with an IOM inhalable dust sampler. Paired IOM and Mod37 breathing-zone air samples were collected for workers engaged in corrosion control maintenance operations on several types of aircraft at several U.S. Air Force bases in the United States. Sampled operations included hand and power sanding, blow-down and wipe-down to

R. E. Clinkenbeard; E. C. England; D. L. Johnson; N. A. Esmen; T. A. Hall

2002-01-01

261

An rpsL Cassette, Janus, for Gene Replacement through Negative Selection in Streptococcus pneumoniae  

Microsoft Academic Search

Natural genetic transformation offers a direct route by which synthetic gene constructs can be placed into the single circular chromosome of Streptococcus pneumoniae. However, the lack of a general negative-selection marker has hampered the introduction of constructs that do not confer a selectable phenotype. A 1.3-kb cassette was constructed comprising a kanamycin (Kn) resistance marker (kan) and a counterselectable rpsL

C. K. Sung; H. Li; J. P. Claverys; D. A. Morrison

2001-01-01

262

Development of a low-resource RNA extraction cassette based on surface tension valves.  

PubMed

Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low-resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low-resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid-air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low-resource setting. PMID:21604768

Bordelon, Hali; Adams, Nicholas M; Klemm, Amy S; Russ, Patricia K; Williams, John V; Talbot, H Keipp; Wright, David W; Haselton, Frederick R

2011-06-01

263

Cassette vectors directing expression of T cell receptor genes in transgenic mice  

Microsoft Academic Search

We describe a pair of cassette vectors that can be used to express rearranged T cell receptor genes in transgenic mice. Short DNA fragments containing rearranged V? and V? segments are readily amplified from T cells and introduced between artificial cloning sites. Transgene-derived mRNAs are transcribed under the control of the natural TCR? and -? promoter\\/enhancer elements. Using this vector,

Valérie Kouskoff; Kathy Signorelli; Christophe Benoist; Diane Mathis

1995-01-01

264

Expression of the Domain Cassette 8 Plasmodium falciparum Erythrocyte Membrane Protein 1 Is Associated with Cerebral Malaria in Benin  

PubMed Central

Background Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi-conserved types defined by tandem runs of specific domains (“domain cassettes” (DC)). The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection. Methods Parasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM). We assessed the transcript level of var genes by RT-qPCR and the expression of PfEMP-1 proteins by LC-MS/MS. Results Var genes encoding DC8 and Group A PfEMP-1 were transcribed more often and at higher levels in cerebral malaria vs. uncomplicated malaria patients. LC-MS/MS identified peptides from group A, DC8 PfEMP-1 more frequently in cerebral malaria than in uncomplicated malaria and pregnancy-associated malaria samples. Conclusion This is the first study to show association between PfEMP-1 subtype and disease outcome by direct analysis of parasites proteome. The results corroborate that group A and specifically the PfEMP-1 types DC8 are universally associated with cerebral malaria. This is a crucial observation for promoting studies on malaria pathogenesis. PMID:23922654

Bertin, Gwladys I.; Lavstsen, Thomas; Guillonneau, Francois; Doritchamou, Justin; Wang, Christian W.; Jespersen, Jakob S.; Ezimegnon, Sem; Fievet, Nadine; Alao, Maroufou J.; Lalya, Francis; Massougbodji, Achille; Ndam, Nicaise Tuikue; Theander, Thor G.; Deloron, Philippe

2013-01-01

265

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates.  

PubMed

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA(+) and VLRC(+) lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the ?? and ?? T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes. PMID:25228758

Das, Sabyasachi; Li, Jianxu; Holland, Stephen J; Iyer, Lakshminarayan M; Hirano, Masayuki; Schorpp, Michael; Aravind, L; Cooper, Max D; Boehm, Thomas

2014-10-14

266

Characterization of the cassette containing genes for type 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae.  

PubMed

The capsular polysaccharide is the major virulence factor of Streptococcus pneumoniae. Previously, we identified and cloned a region from the S. pneumoniae chromosome specific for the production of type 3 capsular polysaccharide. Now, by sequencing the region and characterizing mutations genetically and in an in vitro capsule synthesis assay, we have assigned putative functions to the products of the type-specific genes. Using DNA from the right end of the region in mapping studies, we have obtained further evidence indicating that the capsule genes of each serotype are contained in a gene cassette located adjacent to this region. We have cloned the region flanking the left end of the cassette from the type 3 chromosome and have found that it is repeated in the S. pneumoniae chromosome. The DNA sequence and hybridization data suggest a model for recombination of the capsule gene cassettes that not only describes the replacement of capsule genes, but also suggests an explanation for binary capsule type formation, and the creation of novel capsule types. PMID:7869055

Dillard, J P; Vandersea, M W; Yother, J

1995-03-01

267

Genomic donor cassette sharing during VLRA and VLRC assembly in jawless vertebrates  

PubMed Central

Lampreys possess two T-like lymphocyte lineages that express either variable lymphocyte receptor (VLR) A or VLRC antigen receptors. VLRA+ and VLRC+ lymphocytes share many similarities with the two principal T-cell lineages of jawed vertebrates expressing the ?? and ?? T-cell receptors (TCRs). During the assembly of VLR genes, several types of genomic cassettes are inserted, in step-wise fashion, into incomplete germ-line genes to generate the mature forms of antigen receptor genes. Unexpectedly, the structurally variable components of VLRA and VLRC receptors often possess partially identical sequences; this phenomenon of module sharing between these two VLR isotypes occurs in both lampreys and hagfishes. By contrast, VLRA and VLRC molecules typically do not share their building blocks with the structurally analogous VLRB receptors that are expressed by B-like lymphocytes. Our studies reveal that VLRA and VLRC germ-line genes are situated in close proximity to each other in the lamprey genome and indicate the interspersed arrangement of isotype-specific and shared genomic donor cassettes; these features may facilitate the shared cassette use. The genomic structure of the VLRA/VLRC locus in lampreys is reminiscent of the interspersed nature of the TCRA/TCRD locus in jawed vertebrates that also allows the sharing of some variable gene segments during the recombinatorial assembly of TCR genes. PMID:25228758

Das, Sabyasachi; Li, Jianxu; Holland, Stephen J.; Iyer, Lakshminarayan M.; Hirano, Masayuki; Schorpp, Michael; Aravind, L.; Cooper, Max D.; Boehm, Thomas

2014-01-01

268

Conceptual Design Studies of the KSTAR Bay-Nm Cassette and Thomson Scattering Optics  

SciTech Connect

A Multi-Channel Thomson Scattering System viewing the edge and core of the KSTAR plasma will be installed at the mid-plane port Bay-N. An engineering design study was undertaken at PPPL in collaboration with the Korea Basic Science Institute (KBSI) to determine the optimal optics and cassette design. Design criteria included environmental, mechanical and optical factors. All of the optical design options have common design features; the Thomson Scattering laser, an in-vacuum shutter, a quartz heat shield and primary vacuum window, a set of optical elements and a fiber optic bundle. Neutron radiation damage was a major factor in the choice of competing lens-based and mirror-based optical designs. Both the mirror based design and the lens design are constrained by physical limits of the Bay-N cassette and interference with the Bay-N micro-wave launcher. The cassette will contain the optics and a rail system for maintenance of the optics.

Feder R., Ellis R., Johnson D., Park H., Lee H.G.

2005-09-26

269

Evidence for dynamic exchange of qac gene cassettes between class 1 integrons and other integrons in freshwater biofilms.  

PubMed

Class 1 integrons carried by pathogens have acquired over 100 different gene cassettes encoding resistance to antimicrobial compounds, helping to generate a crisis in the management of infectious disease. It is presumed that these cassettes originated from environmental bacteria, but exchange of gene cassettes has surprisingly never been demonstrated outside laboratory or clinical contexts. We aimed to identify a natural environment where such exchanges might occur, and determine the phylogenetic range of participating integrons. Here we examine freshwater biofilms and show that families of cassettes conferring resistance to quaternary ammonium compounds (qac) are found on class 1 integrons identical to those from clinical contexts, on sequence variants of class 1 integrons only known from natural environments, and on other diverse classes of integrons only known from the chromosomes of soil and freshwater Proteobacteria. We conclude that gene cassettes might be readily shared between different integron classes found in environmental, commensal and pathogenic bacteria. This suggests that class 1 integrons in pathogens have access to a vast pool of gene cassettes, any of which could confer a phenotype of clinical relevance. Exploration of this resource might allow identification of resistance or virulence genes before they become part of multi-drug-resistant human pathogens. PMID:19459951

Gillings, Michael R; Holley, Marita P; Stokes, H W

2009-06-01

270

Characterization of integrons and their cassettes in Escherichia coli and Salmonella isolates from poultry in Korea.  

PubMed

Ninety-nine Escherichia coli and 33 Salmonella isolates were assessed for antimicrobial susceptibility (disc diffusion test). Sulfonamide and tetracycline resistance genes were identified through PCR, and class 1 and class 2 integrons with resistance gene cassettes were identified with PCR followed by sequencing. Salmonella (63.6%) and E. coli (85.8%) isolates were multidrug resistant (resistance to 3 or more antimicrobials), and the highest incidences of resistance were observed for tetracycline, nalidixic acid, and sulfamethoxazole. The sul1, sul2, tetA, and tetB resistance determinant genes were predominant in E. coli, whereas only sul2 and tetA were identified in Salmonella isolates. In the E. coli isolates, 54 (54.5%) class 1 integrons, 6 (6.1%) class 2 integrons, and 5 (5.1%) class 1 and class 2 integrons together were detected, whereas only 3 (9.1%) integrons were found in the Salmonella serovars. Around 87% of the integrons in E. coli harbored resistance gene cassettes conferring resistance to streptomycin/spectinomycin (aadA, aminoglycoside resistance gene), trimethoprim (dfrA, dihydrofolate reductase gene), streptothricin [sat1 and sat2 (streptothricin acetyltransferase), and estX (putative esterases)]. The most common gene cassettes were aadA1+dfrA1 and dfrA1+sat2+aadA1 in class 1 and class 2 integrons, respectively. Other cassettes including aadA5+dfrA7, dfrA12+aadA2, aadA2+aadA1+dfrA12, and aadA5+aadA2/dfrA7 were also identified. Among the Salmonella serovars, Salmonella Malmoe harbored aadA1+dfrA1 and dfrA12+sat2+aadA1 genes. The aadA1, aadA2, sat2, and dfrA1 had wide variation in similarity among themselves and from previously reported genes worldwide. The diverse gene cassettes could be responsible for the prominent resistance profiles observed and a potential source for dissemination of antimicrobial resistance determinants to other bacteria. PMID:24135609

Dessie, Hirut Kidie; Bae, Dong Hwa; Lee, Young Ju

2013-11-01

271

Inter-domain Communication Mechanisms in an ABC Importer: A Molecular Dynamics Study of the MalFGK2E Complex  

Microsoft Academic Search

ATP-Binding Cassette transporters are ubiquitous membrane proteins that convert the energy from ATP-binding and hydrolysis into conformational changes of the transmembrane region to allow the translocation of substrates against their concentration gradient. Despite the large amount of structural and biochemical data available for this family, it is still not clear how the energy obtained from ATP hydrolysis in the ATPase

A. Sofia F. Oliveira; António M. Baptista; Cláudio M. Soares

2011-01-01

272

Ambient Influenza and Avian Influenza Virus during Dust Storm Days and Background Days  

PubMed Central

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

Chen, Pei-Shih; Tsai, Feng Ta; Lin, Chien Kun; Yang, Chun-Yuh; Chan, Chang-Chuan; Young, Chea-Yuan; Lee, Chien-Hung

2010-01-01

273

Diversity of antibiotic resistance genes and staphylococcal cassette chromosome mec elements in faecal isolates of coagulase-negative staphylococci from Nigeria  

PubMed Central

Background Coagulase-negative staphylococci (CoNS) are opportunistic pathogens found as colonisers of the human gut. This study was carried out to examine the genetic resistance mechanisms in faecal isolates of CoNS. The study investigated 53 non-duplicate CoNS isolates obtained from the fresh stool samples of apparently healthy subjects in the community of Ile-Ife, South-Western Nigeria. Antibiotic susceptibility testing was assessed by the disc diffusion test while antibiotic resistance genes were analysed by PCR. mecA positive isolates were analysed by Staphylococcal Chromosome Cassette mec (SCCmec) and cassette chromosome recombinase (ccr) complex typing methods. Results Resistance genes were detected only in isolates that showed resistance by phenotypic screening. The aac(6?)–aph(2?) gene was detected in all the three isolates resistant to gentamicin. Four of the five erythromycin resistant isolates were positive for the ermC gene, the remaining isolate carried the msrA gene. The tetK gene was detected in 6 of the 7 tetracycline resistant isolates while 4 possessed the tetM gene. Three of the isolates (S. haemolyticus, S. xylosus and S. capitis) had both genes. Several SCCmec types were found: SCCmec I- ccrAB?2-?2 (4 isolates: 3 S. epidermidis, 1 S. warneri), SCCmecIVb- ccrAB?2-?3 (1 isolate: S. epidermidis), SCCmecIVd- ccrAB?2-?3 (8 isolates: 3 S. epidermidis, 2 S. xylosus, 1 S. saprophyticus, 1 S. warneri, 1 S. capitis), and untypable (2 isolates: S. epidermidis). Conclusion This genetic background could be a reservoir for interspecies gene transfer among CoNS and S. aureus in the intestinal tract. PMID:24766644

2014-01-01

274

Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species  

PubMed Central

We have investigated to what extent natural transformation acting on free DNA substrates can facilitate transfer of mobile elements including transposons, integrons and/or gene cassettes between bacterial species. Naturally transformable cells of Acinetobacter baylyi were exposed to DNA from integron-carrying strains of the genera Acinetobacter, Citrobacter, Enterobacter, Escherichia, Pseudomonas, and Salmonella to determine the nature and frequency of transfer. Exposure to the various DNA sources resulted in acquisition of antibiotic resistance traits as well as entire integrons and transposons, over a 24 h exposure period. DNA incorporation was not solely dependent on integrase functions or the genetic relatedness between species. DNA sequence analyses revealed that several mechanisms facilitated stable integration in the recipient genome depending on the nature of the donor DNA; homologous or heterologous recombination and various types of transposition (Tn21-like and IS26-like). Both donor strains and transformed isolates were extensively characterized by antimicrobial susceptibility testing, integron- and cassette-specific PCRs, DNA sequencing, pulsed field gel electrophoreses (PFGE), Southern blot hybridizations, and by re-transformation assays. Two transformant strains were also genome-sequenced. Our data demonstrate that natural transformation facilitates interspecies transfer of genetic elements, suggesting that the transient presence of DNA in the cytoplasm may be sufficient for genomic integration to occur. Our study provides a plausible explanation for why sequence-conserved transposons, IS elements and integrons can be found disseminated among bacterial species. Moreover, natural transformation of integron harboring populations of competent bacteria revealed that interspecies exchange of gene cassettes can be highly efficient, and independent on genetic relatedness between donor and recipient. In conclusion, natural transformation provides a much broader capacity for horizontal acquisitions of genetic elements and hence, resistance traits from divergent species than previously assumed. PMID:22876180

Domingues, Sara; Harms, Klaus; Fricke, W. Florian; Johnsen, Pal J.; da Silva, Gabriela J.; Nielsen, Kaare Magne

2012-01-01

275

Marine integrons containing novel integrase genes, attachment sites, attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays  

PubMed Central

In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5?kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like ? propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome. PMID:21248857

Elsaied, Hosam; Stokes, Hatch W; Kitamura, Keiko; Kurusu, Yasurou; Kamagata, Yoichi; Maruyama, Akihiko

2011-01-01

276

REGULATION OF ABCG5 AND ABCG8 STEROL TRANSPORTERS IN BILIARY CHOLESTEROL ELIMINATION, REVERSE CHOLESTEROL TRANSPORT AND DYSLIPIDEMIA.  

E-print Network

??ATP-binding cassette transporters ABCA1 and ABCG1 initiate reverse cholesterol transport generating HDL particles, whereas ABCG5/G8 promote biliary cholesterol secretion thereby facilitating the last step of… (more)

Sabeva, Nadezhda Steliyanova

2011-01-01

277

The metabolic profile of phenylbutyric acid and its antioxidant capacity in vervet monkeys / Wilhelmina Johanna van der Linde.  

E-print Network

??X–linked adrenoleukodystrophy (X–ALD) is the most common peroxisomal enzyme deficiency disorder, characterized by inborn mutations in the ABCD1 gene, an ATP–binding cassette (ABC) half–transporter. The… (more)

Van der Linde, Wilhelmina Johanna

2010-01-01

278

Recombination-Induced Tag Exchange (RITE) Cassette Series to Monitor Protein Dynamics in Saccharomyces cerevisiae  

PubMed Central

Proteins are not static entities. They are highly mobile, and their steady-state levels are achieved by a balance between ongoing synthesis and degradation. The dynamic properties of a protein can have important consequences for its function. For example, when a protein is degraded and replaced by a newly synthesized one, posttranslational modifications are lost and need to be reincorporated in the new molecules. Protein stability and mobility are also relevant for the duplication of macromolecular structures or organelles, which involves coordination of protein inheritance with the synthesis and assembly of newly synthesized proteins. To measure protein dynamics, we recently developed a genetic pulse-chase assay called recombination-induced tag exchange (RITE). RITE has been successfully used in Saccharomyces cerevisiae to measure turnover and inheritance of histone proteins, to study changes in posttranslational modifications on aging proteins, and to visualize the spatiotemporal inheritance of protein complexes and organelles in dividing cells. Here we describe a series of successful RITE cassettes that are designed for biochemical analyses, genomics studies, as well as single cell fluorescence applications. Importantly, the genetic nature and the stability of the tag switch offer the unique possibility to combine RITE with high-throughput screening for protein dynamics mutants and mechanisms. The RITE cassettes are widely applicable, modular by design, and can therefore be easily adapted for use in other cell types or organisms. PMID:23708297

Terweij, Marit; van Welsem, Tibor; van Deventer, Sjoerd; Verzijlbergen, Kitty F.; Menendez-Benito, Victoria; Ontoso, David; San-Segundo, Pedro; Neefjes, Jacques; van Leeuwen, Fred

2013-01-01

279

United States Geological Survey (USGS) FM cassette seismic-refraction recording system  

SciTech Connect

In this two chapter report, instrumentation used to collect seismic data is described. This data acquisition system has two parts: (1) portable anolog seismic recorders and related ``hand-held-testers`` (HHT) and (2) portable digitizing units. During the anolog recording process, ground motion is sensed by a 2-Hz vertical-component seismometer. The voltage output from the seismometer is split without amplification and sent to three parallel amplifier circuit boards. Each circuit board amplifiers the seismic signal in three stages and then frequency modulates the signal. Amplification at the last two stages can be set by the user. An internal precision clock signal is also frequency modulated. The three data carrier frequencies, the clock carrier frequency, and a tape-speed compensation carrier frequency are summed and recorded on a recorded on a cassette tape. During the digitizing process, the cassette tapes are played back and the signals are demultiplexed and demodulated. An anolog-to-digital converter converts the signals to digital data which are stored on 8-inch floppy disks. 7 refs., 19 figs., 6 tabs.

Murphy, J.M.

1988-12-31

280

A comparison of aerosol sampling techniques: "open" versus "closed-face" filter cassettes.  

PubMed

Accepted practice by most professional industrial hygienists in government and industry is to use "closed-face" filter cassette techniques as standard sampling procedures for the majority of aerosols. A two-phase, field study was conducted to determine whether a gravimetric bias exists between "open" and "closed-face" sampling methods. Phase I involved an in-depth analysis of the potential gravimetric viability as it applies to an industrial paint spray mist, and Phase II was a series of pilot studies, of small sample base, to determine if this phenomena exists over a range of aerosol types. Dusts of wood, grain, cellulose, Portland cement and perlite, welding fumes, and chromic acid mist were sampled in Phase II. Paired breathing zone samples, "open" and "closed-face", 37 mm, 3-piece filter cassettes were utilized in both phases of the study. In both phases of the study, "open-face" concentrations were consistently higher than "closed-face" concentrations, with the exception of cellulose dust. Based on the concentration for both sampling techniques, the data suggests that "closed-face" sampling techniques (4.0 mm inlet diameter) might be size selective against large particles. This could lead to an underestimation of a worker's total aerosol exposure. PMID:7435380

Beaulieu, H J; Fidino, A V; Arlington, K L; Buchan, R M

1980-10-01

281

Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.  

PubMed

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

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

282

Fully Automated Data Collection Using PAM and the Development of PAM/SPACE Reversible Cassettes  

NASA Astrophysics Data System (ADS)

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.

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

283

Sewage treatment plant serves as a hot-spot reservoir of integrons and gene cassettes.  

PubMed

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

Ma, Liping; Zhang, Xu-Xiang; Zhao, Fuzheng; Wu, Bing; Cheng, Shupei; Yang, Liuyan

2013-04-01

284

Evaluation of D-1 tape and cassette characteristics: Moisture content of Sony and Ampex D-1 tapes when delivered  

NASA Astrophysics Data System (ADS)

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.

Ashton, Gary

285

Evaluation of D-1 tape and cassette characteristics: Moisture content of Sony and Ampex D-1 tapes when delivered  

NASA Technical Reports Server (NTRS)

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.

Ashton, Gary

1993-01-01

286

Antiproliferative prostaglandins and the MRP\\/GS-X pump role in cancer immunosuppression and insight into new strategies in cancer gene therapy 1 1 Abbreviations: ABC, ATP-binding cassette transporter ATPase; CP-PG, cyclopentenone prostaglandin; GSH, glutathione; GSSG, glutathione disulfide; G S-conjugate, glutathione S-conjugate; MRP\\/GS-X pump, Mg 2+-dependent vanadate-sensitive G S-conjugate export ATPase; hsp, heat shock protein; MDR, multidrug resistance; MRP, multidrug resistance-associated protein; MTL therapy, MRP\\/GS-X pump-transfected lymphocyte therapy; NF-?B, nuclear factor kappa B; PG, prostaglandin; and PPAR-?, peroxisome proliferator-activated receptor-gamma  

Microsoft Academic Search

A dramatic complication in late-stage cancer patients is host immunosuppression. Cyclopentenone prostaglandins (CP-PGs) overproduced in cancer may impair the function of the immune system. These agents, if produced at high concentrations, are powerful cytostatic and cytotoxic compounds that may arrest cell proliferation and immune response in cancer. Lymphoid tissues of tumor-bearing animals accumulate large amounts of CP-PGs, whereas the tumor

Paulo Ivo Homem de Bittencourt; Rui Curi

2001-01-01

287

Cosmic Microwave Background Tutorials  

NSDL National Science Digital Library

Probing whether space is curved or flat, cosmologists have been searching for clues in ripples in the universe's microwave background left from the big bang. These tutorials explain the cosmic microwave background for neophytes, as well as more advanced readers.

Hu, Wayne

2003-10-10

288

Building Background Knowledge  

ERIC Educational Resources Information Center

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.

Neuman, Susan B.; Kaefer, Tanya; Pinkham, Ashley

2014-01-01

289

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

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…

Educational Products Information Exchange Inst., Stony Brook, NY.

290

?C31 integrase mediates efficient cassette exchange in the zebrafish germline  

PubMed Central

Site specific recombinases (SSRs) are powerful tools for genome manipulation, used in diverse organisms including Drosophila melanogaster, mouse, Arabidopsis, zebrafish, and human cultured cells. The integrase from the bacteriophage ?C31 belongs to the large serine family of integrases, and in contrast to other widely used SSRs such as Cre and Flp, recombination is directional and therefore irreversible. We have developed a vector system for recombinase mediated cassette exchange (RMCE) in the zebrafish, allowing swapping of the coding sequence in an integrated transgene. Utilizing codon–optimized ?C31 integrase RNA bearing the 3?UTR from the nanos1 gene, we replaced the egfp coding sequence of an integrated reporter transgene with mCherry coding sequence. Recombination was achieved at high efficiency in both somatic cells and in the germline. We demonstrate an effective approach to RMCE, increasing the repertoire of tools available to manipulate the zebrafish genome. PMID:21805532

Hu, Gui; Goll, Mary G.; Fisher, Shannon

2013-01-01

291

Integron Gene Cassettes and Degradation of Compounds Associated with Industrial Waste: The Case of the Sydney Tar Ponds  

PubMed Central

Integrons are genetic platforms that accelerate lateral gene transfer (LGT) among bacteria. They were first detected on plasmids bearing single and multiple drug resistance determinants in human pathogens, and it is abundantly clear that integrons have played a major role in the evolution of this public health menace. Similar genetic elements can be found in nonpathogenic environmental bacteria and in metagenomic environmental DNA samples, and it is reasonable to suppose that integrons have facilitated microbial adaptation through LGT in niches outside infectious disease wards. Here we show that a heavily impacted estuary, exposed for almost a century to products of coal and steel industries, has developed a rich and unique cassette metagenome, containing genes likely to aid in the catabolism of compounds associated with industrial waste found there. In addition, we report that the most abundant cassette recovered in this study is one that encodes a putative LysR protein. This autoregulatory transcriptional regulator is known to activate transcription of linked target genes or unlinked regulons encoding diverse functions including chlorocatechol and dichlorophenol catabolism. Finally, only class 1 integrase genes were amplified in this study despite using different primer sets, and it may be that the cassettes present in the Tar Ponds will prove to be associated with class 1 integrase genes. Nevertheless, our cassette library provides a snapshot of a complex evolutionary process involving integron-meditated LGT likely to be important in natural bioremediation. PMID:19390587

Koenig, Jeremy E.; Sharp, Christine; Dlutek, Marlena; Curtis, Bruce; Joss, Michael; Boucher, Yan; Doolittle, W. Ford

2009-01-01

292

Popular music as popular expression in North India and the Bhojpuri region, from cassette culture to VCD culture  

Microsoft Academic Search

In the 1980s the Indian popular music scene was revolutionized by the advent of audio cassettes that dramatically decentralized production and precipitated the rise of syncretic folk-pop hybrids aimed at diverse regional audiences. In the years around 2000, the new medium of the VCD, or video compact disc, came to exert a similarly prodigious impact, enabling inexpensive popular music recordings

Peter Manuel

2012-01-01

293

Determination of mean growth parameters of bacterial colonies immobilized in gelatin gel using a laser gel-cassette scanner  

Microsoft Academic Search

The potential of a laser gel-cassette scanner (LGS) for rapid, quantitative measurement of the effect of environmental factors such as pH, temperature and humectants on the lag and doubling times of microorganisms is explored. The quantitative relationships between the laser light scattering intensity and colony radius, mean lag time and doubling time are analysed and a measurement protocol is formulated

K. M. Wright; H. P. Coleman; A. R. Mackie; M. L. Parker; T. F. Brocklehurst; D. R. Wilson; B. P. Hills

2000-01-01

294

Design and construction of an in-plant activation cassette for transgene expression and recombinant protein production in plants.  

PubMed

Virus-based transgene expression systems have become particularly valuable for recombinant protein production in plants. The dual-module in-plant activation (INPACT) expression platform consists of a uniquely designed split-gene cassette incorporating the cis replication elements of Tobacco yellow dwarf geminivirus (TYDV) and an ethanol-inducible activation cassette encoding the TYDV Rep and RepA replication-associated proteins. The INPACT system is essentially tailored for recombinant protein production in stably transformed plants and provides both inducible and high-level transient transgene expression with the potential to be adapted to diverse crop species. The construction of a novel split-gene cassette, the inducible nature of the system and the ability to amplify transgene expression via rolling-circle replication differentiates this system from other DNA- and RNA-based virus vector systems used for stable or transient recombinant protein production in plants. Here we provide a detailed protocol describing the design and construction of a split-gene INPACT cassette, and we highlight factors that may influence optimal activation and amplification of gene expression in transgenic plants. By using Nicotiana tabacum, the protocol takes 6-9 months to complete, and recombinant proteins expressed using INPACT can accumulate to up to 10% of the leaf total soluble protein. PMID:24705598

Dugdale, Benjamin; Mortimer, Cara L; Kato, Maiko; James, Tess A; Harding, Robert M; Dale, James L

2014-05-01

295

Design, Syntheses and Biological Applications of Through-bond Energy Transfer Cassettes and Novel Non-covalently Cell Penetrating Peptides  

E-print Network

A xanthene-BODIPY cassette is used as a ratiometric intracellular pH reporter for imaging protein-dye conjugates in living cells. A model was hypothesized to explain the pH-dependent energy transfer efficiencies from the donor to the acceptor based...

Han, Junyan

2012-02-14

296

Bacterial resistance evolution by recruitment of super-integron gene cassettes.  

PubMed

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

Rowe-Magnus, Dean A; Guerout, Anne-Marie; Mazel, Didier

2002-03-01

297

Localization and properties of a silencing element near the mat3-M mating-type cassette of Schizosaccharomyces pombe.  

PubMed Central

Transcription is repressed in a segment of Schizosaccharomyces pombe chromosome II that encompasses the mat2-P and mat3-M mating-type cassettes. Chromosomal deletion analysis revealed the presence of a repressor element within 500 bp of mat3-M. This element acted in synergy with the trans-acting factors Swi6, Clr1, Clr2, Clr3, and Clr4 and had several properties characteristic of silencers: it did not display promoter specificity, being able to silence not only the M mating-type genes but also the S. pombe ura4 and ade6 genes placed on the centromere-distal side of the mat3-M cassette; it could repress a gene when placed further than 2.6 kb from the promoter and it acted in both orientations, although with different efficiencies, the natural orientation repressing more stringently than the reverse. Following deletion of this element, two semistable states of expression of the mat3-M region were observed and these two states could interconvert. The deletion did not affect gene expression in the vicinity of the mat2-P cassette, 11 kb away from mat3-M. Conversely, deleting 1.5 kb on the centromere-proximal side of the mat2-P cassette, which was previously shown to partially derepress transcription around mat2-P, had no effect on gene expression near mat3-M. A double deletion removing the mat2-P and mat3-M repressor elements had the same effect as the single deletions on their respective cassettes when assayed in cells of the M mating type. These observations allow us to refine a model proposing that redundant pathways silence the mating type region of S. pombe. PMID:10049914

Thon, G; Bjerling, K P; Nielsen, I S

1999-01-01

298

Background Check Presentation  

E-print Network

·July 1, 2012 ­ The UNM Division of Human Resources engaged in an RFP process to identify a NationalBackground Check Presentation Kim Herron-Singleton, Recruitment & UNMTemps Services Manager #12 check website to include BC and FP processes and resources · New background check request e-mail box

New Mexico, University of

299

Background stratospheric aerosol layer  

SciTech Connect

Balloonborne aerosol particle counter measurements are used in studying the stratospheric sulfate layer at Laramie, Wyoming, during 1978 and 1979, a 2-year volcanically quiescent period in which the layer appears to have been in a near equilibrium background state. Subtracting the background aerosol concentration from data obtained during an earlier volcanically active period indicates that the actual decay rate of volcanic aerosol is over 30% faster than one would obtain without this correction. At background, the aerosol size distribution is found to remain remarkably constant between the tropopause and an altitude of approx.25 km, with a sudden transition to a distribution dominated by smaller particles above this altitude. The observations, in some respects, compare favorably with equilibrium one-dimensional stratospheric aerosol models and thus to some extent support the concept of relatively inert tropospheric sulfurous gases, such as carbonyl sulfide and carbon disulfide, as the main background stratospheric aerosol sulfur source. Models which incorporate sulfur chemistry are apparently not able to predict the observed variation of particle size with altitude. The 2-year background period is not long enough in itself to establish long-term trends. The eruption of Mt. St. Helens in May 1980 has considerably disrupted the background stratospheric aerosol which will probably not recover for several years. A comparison of the 1978--79 observations with Junge's original measurements made some 20 years earlier, also during a period void of volcanic perturbations, does not preclude a long-term increase in the background stratospheric aerosol level.

Hofmann, D.J.; Rosen, J.M.

1981-01-01

300

Diffuse Background Radiation  

E-print Network

A new determination of the upper limit to the cosmic diffuse background radiation, at ~110 nm, of 300 photons s-1 cm-2 sr-1 nm-1, is placed in the context of diffuse background measurements across the entire electromagnetic spectrum, including new optical, infrared, visible, and gamma-ray background measurements. The possibility that observed excess diffuse visible radiation is due to redshifted cosmological Lyman alpha recomination radiation is explored. Also, a new standard of units for the display of spectra is advocated.

Richard C. Henry

1999-03-18

301

The Cosmic Background Radiation  

E-print Network

We summarise the current status of cosmic microwave background spectrum and anisotropy measurements, and their theoretical interpretation. This is the update of the mini-review for the 1997 web-version of the Review of Particle Properties.

George Smoot; Douglas Scott

1997-11-08

302

The GLAST Background Model  

SciTech Connect

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.

Ormes, J. F. [University of Denver (United States); Atwood, W. [University of California at Santa Cruz (United States); Burnett, T. [University of Washington (United States); Grove, E. [Naval Research Laboratory (United States); Longo, F. [Instituto Nazionale di Fisica Nucleare (INFN)-Pisa (Italy); McEnery, J.; Ritz, S. [NASA Goddard Space Flight Center (United States); Mizuno, T. [Hiroshima University (Japan)

2007-07-12

303

The GLAST Background Model  

SciTech Connect

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.

Ormes, J.F.; /Denver U.; Atwood, W.; /UC, Santa Cruz; Burnett, T.; /Washington U., Seattle; Grove, E.; /Naval Research Lab, Wash., D.C.; Longo, F.; /INFN, Pisa; McEnery, J.; /NASA, Goddard; Mizuno, T.; /Hiroshima U.; Ritz, S.; /NASA, Goddard

2007-10-17

304

Introduction 1 Background 1  

E-print Network

squirrel (Sciurus carolinensis). The grey squirrel was introduced to Britain in the late 19 th century populations in Northern Ireland. Background The red squirrel (Sciurus vulgaris) was once ubiquitous

305

Background Studies for EXIST  

NASA Technical Reports Server (NTRS)

We present results from a study of the trapped proton and electron background for several orbital inclinations and altitudes. This study includes time dependent effects. In addition we describe a 3 component cosmic background model developed at the University of Southampton, UK. The three components are cosmic diffuse gamma rays, atmospheric albedo gamma rays, and cosmic ray protons. We present examples of how this model was applied to BATSE and discuss its application to EXIST.

Wilson, Colleen A.; Pendleton, G. N.; Fishman, G. J.

2004-01-01

306

Sequential genetic modification of the hprt locus in human ESCs combining gene targeting and recombinase-mediated cassette exchange.  

PubMed

Genetic modification of human embryonic stem cells (hESCs) will be an essential tool to allow full exploitation of these cells in regenerative medicine and in the study of hESC biology. Here we report multiple sequential modifications of an endogenous gene (hprt) in hESCs. A selectable marker flanked by heterospecific lox sites was first introduced by homologous recombination (HR) into the hprt gene. In a subsequent step, exchange of the selectable marker with another cassette was achieved by recombinase-mediated cassette exchange (RMCE). We show that 100% of the recovered clones were the result of RMCE using a promoter trap strategy at the hprt locus. hprt-targeted H1 cells maintained a diploid karyotype and expressed hESC surface markers before and after RMCE. Finally, we report a double replacement strategy using two sequential gene targeting steps resulting in the targeted correction of an hprt-mutated hESC line. PMID:18386992

Di Domenico, Alexandra I; Christodoulou, Ioannis; Pells, Steve C; McWhir, Jim; Thomson, Alison J

2008-06-01

307

Energy use of televisions and video cassette recorders in the U.S.  

SciTech Connect

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.

Meier, Alan; Rosen, Karen

1999-03-01

308

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA  

PubMed Central

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

2012-01-01

309

In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer.  

PubMed

Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer. PMID:17989704

White, S J; Papadakis, E D; Rogers, C A; Johnson, J L; Biessen, E A L; Newby, A C

2008-03-01

310

Photorhabdus virulence cassettes confer injectable insecticidal activity against the wax moth.  

PubMed

Two recently sequenced genomes of the insect-pathogenic bacterium Photorhabdus and a large Serratia entomophila plasmid, pADAP, have phage-related loci containing putative toxin effector genes, designated the "Photorhabdus virulence cassettes" (PVCs). In S. entomophila, the single plasmid PVC confers antifeeding activity on larvae of a beetle. Here, we show that recombinant Escherichia coli expressing PVC-containing cosmids from Photorhabdus has injectable insecticidal activity against larvae of the wax moth. Electron microscopy showed that the structure of the PVC products is similar to the structure of the antibacterial R-type pyocins. However, unlike these bacteriocins, the PVC products of Photorhabdus have no demonstrable antibacterial activity. Instead, injection of Photorhabdus PVC products destroys insect hemocytes, which undergo dramatic actin cytoskeleton condensation. Comparison of the genomic organizations of several PVCs showed that they have a conserved phage-like structure with a variable number of putative anti-insect effectors encoded at one end. Expression of these putative effectors directly inside cultured cells showed that they are capable of rearranging the actin cytoskeleton. Together, these data show that the PVCs are functional homologs of the S. entomophila antifeeding genes and encode physical structures that resemble bacteriocins. This raises the interesting hypothesis that the PVC products are bacteriocin-like but that they have been modified to attack eukaryotic host cells. PMID:16513755

Yang, G; Dowling, A J; Gerike, U; ffrench-Constant, R H; Waterfield, N R

2006-03-01

311

On Background Independence  

E-print Network

This paper concerns what Background Independence itself is (as opposed to some particular physical theory that is background independent). The notions presented mostly arose from a layer-by-layer analysis of the facets of the Problem of Time in Quantum Gravity. Part of this coincides with two relational postulates which are thus identified as classical precursors of two of the facets of the Problem of Time. These are furthemore tied to the forms of each of the GR Hamiltonian and momentum constraints. Other aspects of Background Independence include the algebraic closure of these constraints, expressing physics in terms of beables, foliation independence as implemented by refoliation invariance, the reconstruction of spacetime from space. The final picture is that Background Independence - a philosophically desirable and physically implementable feature for a theory to have - has the facets of the Problem of Time among its consequences. Thus these arise naturally and are problems to be resolved, as opposed to avoided `by making one's physics background-dependent in order not to have these problems'. This serves as a selection criterion that limits the use of a number of model arenas and physical theories.

Edward Anderson

2013-10-05

312

Structural Comparison of Three Types of Staphylococcal Cassette Chromosome mec Integrated in the Chromosome in Methicillin-Resistant Staphylococcus aureus  

Microsoft Academic Search

The b-lactam resistance gene mecA of Staphylococcus aureus is carried by a novel mobile genetic element, designated staphylococcal cassette chromosome mec (SCCmec), identified in the chromosome of a Japanese methicillin-resistant S. aureus (MRSA) strain. We now report identification of two additional types of mecA- carrying genetic elements found in the MRSA strains isolated in other countries of the world. There

TERUYO ITO; YUKI KATAYAMA; KAZUMI ASADA; NAMIKO MORI; KANAE TSUTSUMIMOTO; CHUNTIMA TIENSASITORN; KEIICHI HIRAMATSU

2001-01-01

313

Expression levels of domestic cDNA cassettes integrated in the nuclear genomes of various Chlamydomonas reinhardtii strains.  

PubMed

We attempted to overexpress three types of expression cassettes, each of which contained a different open reading frame (ORF) of domestic Chlamydomonas cDNAs. Each ORF was strongly driven by an artificial hybrid promoter. We used two wild-type Chlamydomonas strains (i.e., CC-124 and CC-125) and two mutant strains [i.e., UV-mutated (UVM) 4 and UVM11] that have been reported to have a high potency for expressing nondomestic nuclear transgenes. We found that the 1-deoxy-d-xylulose-5-phosphatesynthase (DXS1), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR1), and squalene synthase (SQS) cassettes were not readily overexpressed in the wild-type strains at levels where the products were clearly detectable by Western blotting using a monoclonal antibody. In contrast, Western blot-positive SQS cassette transformants were frequently detected in the UVM4 and UVM11 strains, i.e., at an approximately 4.5 times higher frequency than that in the CC-124 wild-type strain. Moreover, transformants that accumulated large amounts of the SQS protein were obtained frequently in the UVM4 and UVM11 strains, i.e., the frequency was approximately 2.2 times higher than that in the CC-124 strain. However, a position effect of the integrated expression cassette was obviously detected not only in the wild-type but also in UVM strains. This suggests that the epigenetic repression mechanism of transgenic genes was not completely knocked out, even in the UVM strains. Further improved Chlamydomonas strains are essential to facilitate high-throughput screening of transformants that express nuclear transgenes at a high level. PMID:24342172

Kong, Fantao; Yamasaki, Tomohito; Ohama, Takeshi

2014-05-01

314

GRAVIMETRIC DETERMINATION OF AIRBORNE DUST BY USING A FILTER CARTRIDGE INSIDE A CLOSED-FACE, 37MM POLYSTYRENE CASSETTE  

Microsoft Academic Search

The current practice for the determination of personal exposures to airborne dusts involves the aspiration of known quantities of air through membrane filters held in filter holders. The two-piece, 37-mm polystyrene cassette is the most typically used filter holder. Two potential major errors exist with filter-based air-sampling methods. The first error is caused by potential sample loss on the inside

Mark A. Puskar; Scott M. Fergon; James M. Harkins; Lawrence H. Hecker

1992-01-01

315

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

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

Cohen, Aina E.; Ellis, Paul J.; Miller, Mitchell D.; Deacon, Ashley M.; Phizackerley, R. Paul

2014-01-01

316

Supersymmetric heterotic string backgrounds  

E-print Network

We present the main features of the solution of the gravitino and dilatino Killing spinor equations derived in hep-th/0510176 and hep-th/0703143 which have led to the classification of geometric types of all type I backgrounds. We then apply these results to the supersymmetric backgrounds of the heterotic string. In particular, we solve the gaugino Killing spinor equation together with the other two Killing spinor equations of the theory. We also use our results to classify all supersymmetry conditions of ten-dimensional gauge theory.

Ulf Gran; George Papadopoulos; Diederik Roest

2007-06-29

317

Cosmological Gravitational Wave Backgrounds  

E-print Network

An overview is presented of possible cosmologically distant sources of gravitational wave backgrounds, especially those which might produce detectable backgrounds in the LISA band between 0.1 and 100 mHz. Examples considered here include inflation-amplified vacuum fluctuations in inflaton and graviton fields, bubble collisions in first-order phase transitions, Goldstone modes of classical self-ordering scalars, and cosmic strings and other gauge defects. Characteristic scales and basic mechanisms are reviewed and spectra are estimated for each of these sources. The unique impact of a LISA detection on fundamental physics and cosmology is discussed.

Craig J. Hogan

1998-09-28

318

Gene disruption in Escherichia coli: Tc R and Km R cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant  

Microsoft Academic Search

Two cassettes with tetracycline-resistance (TcR) and kanamycin-resistance (KmR) determinants have been developed for the construction of insertion and deletion mutants of cloned genes in Escherichia coli. In both cassettes, the resistance determinants are flanked by the short direct repeats (FRT sites) required for site-specific recombination mediated by the yeast Flp recombinase. In addition, a plasmid with temperature-sensitive replication for temporal

Peter P. Cherepanov; Wilfried Wackernagel

1995-01-01

319

Cosmic background radiation  

Microsoft Academic Search

We summarise the current status of cosmic microwave background spectrum and\\u000aanisotropy measurements, and their theoretical interpretation. This is the\\u000aupdate of the mini-review for the 1997 web-version of the Review of Particle\\u000aProperties.

George Smoot; Douglas Scott

2000-01-01

320

PANDEMIC INFLUENZA background briefing  

E-print Network

PANDEMIC INFLUENZA background briefing Biomedicine Forum 5 November 2008 compiled by David Evans, Dave Carr, David Lynn and Phil Green Transmission electron micrograph of Influenza A virus (Wellcome influenza!' Page 2 #12;Consequences of an influenza pandemic THE PANDEMIC THREAT DEATH If the next pandemic

Rambaut, Andrew

321

David Smith Academic background  

E-print Network

David Smith Academic background Ph.D. in Mathematics (Algebra), Université de Sherbrooke, Canada project program (I. Assem, F. Bergeron, C. Reutenauer, D. Smith) $132,000 ($44,000 per year for 3 years. Schiffler and D. Smith, Friezes, strings and cluster variables, to appear in Glasgow Mathematcal Journal. 2

322

INDIAN BACKGROUNDS Patuxent Wildlife  

E-print Network

of Sport Fisheries and Wildlife Circular 138 #12;#12;INDIAN BACKGROUNDS of the Patuxent Wildlife Research of the Interior Fish and Wildlife Service Bureau of Sport Fisheries and Wildlife Circular 138 #12; Exhibit the Indian hunted with spear and arrow to supply the necessities of life--food, clothing, and shelter

323

Local microwave background radiation  

E-print Network

An inquiry on a possible local origin for the Microwave Background Radiation is made. Thermal MBR photons are contained in a system called {\\it magnetic bottle} which is due to Earth magnetic field and solar wind particles, mostly electrons. Observational tests are anticipated.

Domingos S. L. Soares

2006-07-11

324

The lytic cassette of mycobacteriophage Ms6 encodes an enzyme with lipolytic activity.  

PubMed

dsDNA bacteriophages use the dual system endolysin-holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His(6)-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His(6)-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C(4)-C(18)), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C(16) and C(18)). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 degrees C and pH 7.5-8.0. Activity was increased in the presence of Ca(2+) and Mn(2+). To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage. PMID:18451045

Gil, Filipa; Catalão, Maria João; Moniz-Pereira, José; Leandro, Paula; McNeil, Michael; Pimentel, Madalena

2008-05-01

325

Unexpected diversity of staphylococcal cassette chromosome mec type IV in methicillin-resistant Staphylococcus aureus strains.  

PubMed

Staphylococcal cassette chromosome mec (SCCmec) is a large mobile genetic element which is used frequently for subtyping of methicillin-resistant Staphylococcus aureus (MRSA) strains. MRSA SCCmec type IV not only predominates among community-acquired MRSA (CA-MRSA) strains but also is associated with several genetic lineages of hospital-acquired MRSA (HA-MRSA) and with other species. The objective of this study was to investigate the diversity of MRSA strains classified as SCCmec type IV by using a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay as well as spa typing and pulsed-field gel electrophoresis (PFGE). Sixty-two primer pairs and 63 probes were designed to interrogate each open reading frame (ORF) of SCCmec type IV sequences. A set of 131 MRSA SCCmec type IV isolates were classified into 79 subtypes by this method. There was considerable concordance between SCCmec type IV subtyping, spa typing, and PFGE patterns for clinical isolates, and the stability of SCCmec type IV subtyping was comparable to that of the other two methods. Using an in-house computer program, we showed that a subset of 20 genetic markers could achieve the same level of discrimination between isolates as the full set of 62, with a Simpson's index of diversity of 0.975. SCCmec type IV has a much higher level of diversity than previously suggested. The application of the mPCR/RLB hybridization assay to MRSA SCCmec type IV subtyping can improve the discriminatory power and throughput of MRSA typing and has the potential to enhance rapid infection control surveillance and outbreak detection. PMID:20686095

Liu, Ying; Kong, Fanrong; Xiao, Meng; Wang, Qinning; O'Sullivan, Matthew; Sintchenko, Vitali; Ma, Lin; Gilbert, Gwendolyn L

2010-10-01

326

POLYFLOW theoretical background  

NASA Astrophysics Data System (ADS)

In the scope of the 1990-04 lecture series on computational fluid dynamics, a finite element program is described. POLYFLOW was designed for the analysis of industrial processes dominated by nonlinear viscous phenomena and viscoelastic effects. It is based on the general principles of continuum mechanics, together with phenomenological or kinetic theoretical models for describing the rheological behavior of the fluid. The theoretical background, necessary to understand the models, and numerical techniques used in POLYFLOW, are provided. The governing equations, their finite element formulations and the solution procedures are summarized.

Crochet, M. J.

327

The Iron Line Background  

E-print Network

We investigate the presence of iron line emission among faint X-ray sources identified in the 1Ms Chandra Deep Field South and in the 2Ms Chandra Deep Field North. Individual source spectra are stacked in seven redshift bins over the range z=0.5-4. We find that iron line emission is an ubiquitous property of X-ray sources up to z~3. The measured line strengths are in good agreement with those expected by simple pre-Chandra estimates based on X-ray background synthesis models. The average rest frame equivalent width of the iron line does not show significant changes with redshift.

Marcella Brusa; Roberto Gilli; Andrea Comastri

2005-01-25

328

Novel 30 kDa protein possessing ATP-binding and chaperone activities.  

PubMed Central

A 30 kDa protein was purified from pig liver cytosol by using ATP-Sepharose and Green A column chromatography. The partial amino acid sequences of the protein (95 amino acid residues) had no similarity with any proteins recorded in data banks. The protein was able to form a stable complex with unfolded dihydrofolate reductase (DHFR). The spontaneous refolding of chemically denatured DHFR was arrested by the 30 kDa protein. This inhibition presumably results from the formation of a stable complex between the 30 kDa protein and DHFR. Bound DHFR could be released from the protein with ATP. The protein also showed protease resistance in an ATP-dependent manner. Incubation of the 30 kDa protein with 5 mM ATP resulted in its resistance to V8 protease or to trypsin treated with 1-chloro-4-phenyl-3-L-toluene-p-sulphonamidobutan-2-one. Divalent cations enhanced the ATP-protection effect. CD analysis of the 30 kDa protein showed that ATP induced an increase in the beta-pleated sheet content and a decrease in the alpha-helix content of the 30 kDa protein. These results suggest that the 30 kDa protein, a novel cytosolic protein, might have an affinity for ATP, a chaperonin activity, and and an ATP-protection effect against some proteases in vivo. PMID:9291133

Itoh, H; Tashima, Y

1997-01-01

329

In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis  

Microsoft Academic Search

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is in- volved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet un- derstood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal

Wolfgang M. J. Obermann; Holger Sondermann; Alicia A. Russo; Nikola P. Pavletich; F. Ulrich Hartl

1998-01-01

330

A large conformational change in the putative ATP pyrophosphatase PF0828 induced by ATP binding.  

PubMed

ATP pyrophosphatases (ATP PPases) are widely distributed in archaea and eukaryotes. They share an HUP domain at the N-terminus with a conserved PP-motif that interacts with the phosphates of ATP. The PF0828 protein from Pyrococcus furiosus is a member of the ATP PPase superfamily and it also has a 100-residue C-terminal extension that contains a strictly conserved EGG(E/D)xE(T/S) motif, which has been named the EGT-motif. Here, crystal structures of PF0828 alone and in complex with ATP or AMP are reported. The HUP domain contains a central five-stranded ?-sheet that is surrounded by four helices, as in other related structures. The C-terminal extension forms a separate domain, named the EGT domain, which makes tight interactions with the HUP domain, bringing the EGT-motif near to the PP-motif and defining the putative active site of PF0828. Both motifs interact with the phosphate groups of ATP. A loop in the HUP domain undergoes a large conformational change to recognize the adenine base of ATP. In solution and in the crystal PF0828 is a dimer formed by the side-by-side arrangement of the HUP domains of the two monomers. The putative active site is located far from the dimer interface. PMID:22102225

Forouhar, Farhad; Saadat, Nabila; Hussain, Munif; Seetharaman, Jayaraman; Lee, Insun; Janjua, Haleema; Xiao, Rong; Shastry, Ritu; Acton, Thomas B; Montelione, Gaetano T; Tong, Liang

2011-11-01

331

Cosmic Microwave Background Polarization  

E-print Network

Cosmic microwave background (CMB) anisotropy is our richest source of cosmological information; the standard cosmological model was largely established thanks to study of the temperature anisotropies. By the end of the decade, the Planck satellite will close this important chapter and move us deeper into the new frontier of polarization measurements. Numerous ground--based and balloon--borne experiments are already forging into this new territory. Besides providing new and independent information on the primordial density perturbations and cosmological parameters, polarization measurements offer the potential to detect primordial gravity waves, constrain dark energy and measure the neutrino mass scale. A vigorous experimental program is underway worldwide and heading towards a new satellite mission dedicated to CMB polarization.

James G. Bartlett

2006-01-25

332

A comparative proteomic study identified LRPPRC and MCM7 as putative actors in imatinib mesylate cross-resistance in Lucena cell line  

PubMed Central

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

2012-01-01

333

The multidrug transporters—proteins of an ancient immune system  

Microsoft Academic Search

The multidrug resistance proteins, discovered as membrane transporters producing chemotherapy-resistance in cancer, are functioning as complex cellular defence systems through recognition and energy-dependent removal of a large variety of toxic agents. The multidrug transporters belong to the ATP-binding cassette (ABC) transporters, present both in prokaryotes and eukaryotes and built from a combination of characteristic membrane-spanning helices and cytoplasmic ATP-binding domains.

Balázs Sarkadi; Marianna Müller; Zsolt Holló

1996-01-01

334

Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli.  

PubMed

The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc(R)) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc(R). A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems. PMID:24203710

Li, Xin-Tian; Thomason, Lynn C; Sawitzke, James A; Costantino, Nina; Court, Donald L

2013-12-01

335

Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli  

PubMed Central

The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (TcR) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for TcR. A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems. PMID:24203710

Li, Xin-tian; Thomason, Lynn C.; Sawitzke, James A.; Costantino, Nina; Court, Donald L.

2013-01-01

336

Genotyping single nucleotide polymorphisms in human genomic DNA with an automated and self-contained PCR cassette.  

PubMed

Point-of-care devices can lower costs through reduced reagent costs, shifting diagnostics from centralized laboratories to local clinics or hospitals, rapidly informing on the spot medical decision making, and enabling personalized treatment options. We have previously described a self-contained miniaturized device that uses an array of gel-based reaction units that can simultaneously detect multiple biomarkers and/or multiple patients in one PCR cassette and can be stored for up to 7 months. In this article, we document the ability of cassette PCR to detect single nucleotide polymorphisms (SNPs) in human genomic DNA from buccal swabs. Swab processing takes 8 minutes, and PCR is completed in just more than an hour. To demonstrate potential for genotyping, we used allele-specific PCR and melt curve analysis to detect major and minor alleles of two SNPs in the fibroblast growth factor receptor 2 gene (FGFR2) that are linked with breast cancer. After allele-specific PCR, seamless melt curve analysis and the presence or absence of melt peaks from melt curve analysis identifies the FGFR2 SNP genotypes for each patient. The near point-of-care/point-of-need genotyping methods reported here can be applied for detecting and assessing risks of diseases such as cancer and to detect SNPs that alter drug metabolism and hence response to therapy. PMID:24998937

Manage, Dammika P; Ma, Lucy; Lauzon, Jana; Howell, Anita; Belch, Andrew R; Mackey, John R; Pilarski, Linda M

2014-09-01

337

Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette[W][OPEN  

PubMed Central

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

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

338

Integrable Background Geometries  

NASA Astrophysics Data System (ADS)

This work has its origins in an attempt to describe systematically the integrable geometries and gauge theories in dimensions one to four related to twistor theory. In each such dimension, there is a nondegenerate integrable geometric structure, governed by a nonlinear integrable differential equation, and each solution of this equation determines a background geometry on which, for any Lie group G, an integrable gauge theory is defined. In four dimensions, the geometry is selfdual conformal geometry and the gauge theory is selfdual Yang-Mills theory, while the lower-dimensional structures are nondegenerate (i.e., non-null) reductions of this. Any solution of the gauge theory on a k-dimensional geometry, such that the gauge group H acts transitively on an ?-manifold, determines a (k+?)-dimensional geometry (k+??4) fibering over the k-dimensional geometry with H as a structure group. In the case of an ?-dimensional group H acting on itself by the regular representation, all (k+?)-dimensional geometries with symmetry group H are locally obtained in this way. This framework unifies and extends known results about dimensional reductions of selfdual conformal geometry and the selfdual Yang-Mills equation, and provides a rich supply of constructive methods. In one dimension, generalized Nahm equations provide a uniform description of four pole isomonodromic deformation problems, and may be related to the {SU}(?) Toda and dKP equations via a hodograph transformation. In two dimensions, the {Diff}(S^1) Hitchin equation is shown to be equivalent to the hyperCR Einstein-Weyl equation, while the {SDiff}(?^2) Hitchin equation leads to a Euclidean analogue of Plebanski's heavenly equations. In three and four dimensions, the constructions of this paper help to organize the huge range of examples of Einstein-Weyl and selfdual spaces in the literature, as well as providing some new ! ones. The nondegenerate reductions have a long ancestry. More ! recently , degenerate or null reductions have attracted increased interest. Two of these reductions and their gauge theories (arguably, the two most significant) are also described.

Calderbank, David M. J.

2014-03-01

339

MAR Background Report MAR Background Report: Indigenous Protest in Brazil  

E-print Network

MAR Background Report MAR Background Report: Indigenous Protest in Brazil Hundreds of indigenous. According to MAR data, several violent incidents against landowners, miners and others have been observed groups worldwide #12;MAR Background Report About the Minorities at Risk Project The Minorities at Risk

Milchberg, Howard

340

MAR Background Report MAR Background Report: The Revolution in Bahrain  

E-print Network

MAR Background Report MAR Background Report: The Revolution in Bahrain Unrest in Tunisia, Egypt forces. MAR data also reports that social services in Shi'a neighborhoods are inferior to those in Sunni of ethnic groups worldwide #12;MAR Background Report In Bahrain, the freedom of press and expression

Milchberg, Howard

341

Capturing Video Using the SONY GV-HD700 Cassette Deck In the AV Production Room (Room 261), Mann Library has a SONY GV-HD700 Digital HD Video  

E-print Network

Capturing Video Using the SONY GV-HD700 Cassette Deck In the AV Production Room (Room 261), Mann Library has a SONY GV-HD700 Digital HD Video Cassette Deck. It is attached to the left side Mac Pro Creation Station, and can be used to "capture", or convert video from miniDV tapes to digital files. 1

Angenent, Lars T.

342

Background issues for defensive interceptors.  

National Technical Information Service (NTIS)

Mean nuclear backgrounds are large, but are arguably amenable to frame-to-frame subtraction. Striated backgrounds on the sensors for defensive interceptors could, however, cause clutter leak-through, which could make detection and track difficult. Nominal...

G. H. Canavan

1991-01-01

343

Internal and External Radioactive Backgrounds  

E-print Network

Chapter 3 Internal and External Radioactive Backgrounds New physics is often discovered by pushing of the low energies involved. There are many radioactive elements that have decays at lower energies which;Chapter 3: Internal and External Radioactive Backgrounds 104 the rate of background. High-energy neutrino

344

REPORT NO. 5 background material  

E-print Network

REPORT NO. 5 background material for the development of radiation protection standards July 1964 Staff Report of the FEDERAL RADIATION COUNCIL #12;REPORT NO. 5 background material for the development INTRODUCTION This report contains background material used in the development of guidance for Federal agencies

345

Isolated Cerebellar Variant of Adrenoleukodystrophy with a de novo Adenosine Triphosphate-Binding Cassette D1 (ABCD1) Gene Mutation  

PubMed Central

X-linked adrenoleukodystrophy (X-ALD) shows a wide range of phenotypic expression, but clinical presentation as an isolated lesion of the cerebellar white matter and dentate nuclei has not been reported. We report an unusual presentation of X-ALD only with an isolated lesion of the cerebellar white matter and dentate nuclei. The proband, a 37-year-old man presented with bladder incontinence, slurred speech, dysmetria in all limbs, difficulties in balancing, and gait ataxia. Brain magnetic resonance imaging showed an isolated signal change of white matter around the dentate nucleus in cerebellum. With high level of very long chain fatty acid, gene study showed a de novo mutation in exon 1 at nucleotide position c.277_296dup20 (p.Ala100Cysfs*10) of the adenosine triphosphate-binding cassette D1 gene. It is advised to consider X-ALD as a differential diagnosis in patients with isolated cerebellar degeneration symptoms. PMID:24954351

Kang, Joon Won; Lee, Sang Mi; Koo, Kyo Yeon; Lee, Young-Mock; Nam, Hyo Suk; Quan, Zhejiu

2014-01-01

346

The extragenic spacer length between the 5' and 3' ends of the transgene expression cassette affects transgene silencing from plasmid-based vectors.  

PubMed

In quiescent tissues, minicircle DNA vectors provide at least 10 times higher sustained levels of transgene expression compared to that achieved with a canonical plasmid containing the same expression cassette. It is not known if there is a specific DNA sequence or structure that is needed for DNA silencing. To directly address this question, we substituted the bacterial plasmid DNA with various lengths of extragenic spacer DNAs between the 5' and 3' ends of the transgene expression cassette and determined the expression profiles using two different reporter expression cassettes. Both the human alphoid repeat (AR) and randomly generated DNA sequences of ?1 kb in length resulted in transgene silencing while shorter spacers, ?500 bp exhibited similar transgene expression patterns to conventional minicircle DNA vectors. In contrast, when the ?1 kb random DNA (RD) sequences were expressed as part of the 3'-untranslated region (UTR) transgene silencing was not observed. These data suggest that the length and not the sequence or origin of the extragenic DNA flanking the expression cassette is responsible for plasmid-mediated transgene silencing. This has implications for the design of nonviral vectors for gene transfer applications as well as providing insights into how genes are regulated. PMID:22565847

Lu, Jiamiao; Zhang, Feijie; Xu, Siqun; Fire, Andrew Z; Kay, Mark A

2012-11-01

347

Adenosine Triphosphate Binding Cassette (ABC) Transporters Are Expressed and Regulated During Terminal Keratinocyte Differentiation: A Potential Role for ABCA7 in Epidermal Lipid Reorganization  

Microsoft Academic Search

Central aspects of the cellular lipid trafficking mechanisms that occur during keratinocyte differentiation are still not well understood. In the past years, evidence has accumulated to suggest that members of the superfamily of adenosine triphosphate binding cassette (ABC) transporters are critically involved in the transmembrane transport of cellular lipids. To test the hypothesis that ABC molecules are potentially involved in

Danuta Kielar; Wolfgang E Kaminski; Gerhard Liebisch; Armin Piehler; Jürgen J Wenzel; Christoph Möhle; Susanne Heimerl; Thomas Langmann; Sven O Friedrich; Alfred Böttcher; Stefan Barlage; Wolfgang Drobnik; Gerd Schmitz

2003-01-01

348

Ultrathin wear-resistant coatings for the tape bearing surface of thin-film magnetic heads for digital compact cassette  

NASA Astrophysics Data System (ADS)

The introduction of the digital compact cassette (DCC) system into the consumer market imposed some severe requirements on the durability of the thin-film heads used in this system. The reliability of the head's record and playback functions has to be maintained under a wide variety of climatic conditions and tape types, as a consequence of the system's backward compatibility with the analog compact cassette. This paper describes the results of several accelerated wear tests as well as of durability tests against tape, which were set-up to find the best material and process conditions for a wear-resistant coating on the tape bearing surface of a DCC head. This ultrathin coating (less than 75 nm) appeared to be the best solution as a safeguard against severe pole-tip recession, which would cause an unacceptable distance loss in the recording system. The first material which showed a very high wear resistance was CrN. As CrN fulfils all requirements for wear and corrosion resistance in the home application, this material is presently used in all DCC home decks. However, under extremely abrasive conditions, such as may be encountered in a car environment (e.g. cold start at -20 C), CrN is surpassed by an even more wear-resistant material, called SPL (super protective layer). Therefore, SPL is preferred as a coating on thin-film heads for car stereo (as well as portable) DCC apparatus. Both materials are characterized by their specific wear performance, which has a superior impact on the electrical (record and playback) properties of the head during its lifetime.

Zieren, V.; Dejongh, M.; Vangroenou, A. Broese; Vanzon, J. B. A.; Lasinski, P.; Theunissen, G. S. A. M.

1994-03-01

349

Background  

E-print Network

This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Embryonic diapause in humans: time to consider?

Grazyna E Ptak; Jacek A Modlinski; Grazyna E Ptak; Jacek A Modlinski; Pasqualino Loi

2013-01-01

350

Background  

E-print Network

Obligate intracellular bacteria commonly have much reduced genome sizes compared to their nearest free-living relatives. One reason for this is reductive evolution: the loss of genes rendered non-essential due to the intracellular habitat. This can occur because of the presence of orthologous genes in the host, combined with the ability of the bacteria to import the protein or metabolite products of the host genes. In this article we take a look at three such bacteria whose genomes have been fully sequenced. Buchnera is an endosymbiont of the pea aphid, Acyrthosiphon pisum, the relationship between these two organisms being so essential that neither can reproduce in the absence of the other. Rickettsia prowazekii is the causative agent of louse-borne typhus in humans and Mycobacterium leprae infection of humans leads to leprosy. Both of these human pathogens have fastidious growth requirements, which has made them very difficult to

Comp Funct Genom; Jo Wixon

351

Background  

E-print Network

OCA is a group of autosomal recessive disorders characterized by hypopigmentation and abnormalities related to ocular development. Mutations in genes regulating melanin-biosynthesis cause four classical types of OCA (OCA 1-4). The clinical spectrum of OCA often depends on the pigmentation threshold of a patient, highlighting the importance of ethnic- specific SNPs. We aimed to understand the molecular bases of OCA in India, where it is one of the four major causes of childhood blindness. Materials and methods Blood samples were collected from OCA patients and family members, mostly from eastern and southern India. Seven pigmentation related genes were screened for variations. Relevant non-synonymous changes in tyrosinase (TYR) were functionally validated. Eighteen SNPs from three OCA genes were genotyped in 552 normal individuals covering various ethnic groups of India. Results Our data suggest that defects in TYR cause albinism in 58 % (36/62) of the cases [1] (and unpublished data; see Figure 1). Functional assays with missense mutations proved that none of mutants are enzymatically active and are retained in the endoplasmic reticulum [1]. Screening of the remaining cases (43%) revealed OCA2 to be the second common locus followed by SLC45A2 [2] (Figure 1). Evaluation of SNPs in TYR, OCA2 and SLC45A2 in normal population suggested definitive bias for some of the SNPs towards specific populations.

Kunal Ray; Mainak Sengupta; Moumita Chaki; Maitreyee Mondal; Swapan Samanta

352

Background  

Cancer.gov

Extensive evidence has demonstrated that 24-hour dietary recalls provide the highest quality, least biased dietary data. Traditional 24-hour recalls, however, are expensive and impractical for large-scale research because they rely on trained interviewers and multiple administrations to estimate usual intakes. As a result, researchers often make use of food frequency questionnaires, which are less expensive but contain substantial error.

353

Background  

Cancer.gov

The discovery that proteins and peptides are "leaked" by tumors into clinically accessible bodily fluids such as blood has led to the possibility of diagnosing cancer at an early stage or monitoring response to treatment by collecting these fluids and testing for the presence of cancer-related biomarkers. Prostate-specific antigen (PSA) and cancer antigen 125 (CA-125) are examples of blood-borne cancer protein biomarkers that are currently being used in the clinic.

354

FAMILY BACKGROUND OF RURAL YOUTH.  

ERIC Educational Resources Information Center

FAMILY BACKGROUNDS OF RURAL YOUTH ARE DISCUSSED. THE BACKGROUND PROVIDED BY THE FAMILY HAS IMPLICATIONS FOR THE ADJUSTMENT OF RURAL YOUTH IN AN URBANIZED, HIGHLY TECHNICAL SOCIETY. THE BASIC ECOLOGICAL CONDITIONS OF RURAL AREAS INFLUENCE THE RATE OF SOCIAL CHANGE, THE IMPORTANCE OF THE FAMILY AS A SOCIAL UNIT, AND THE ORIENTATION TOWARD LEGAL…

COPP, JAMES H.

355

Lattice QCD in Background Fields  

SciTech Connect

Electromagnetic properties of hadrons can be computed by lattice simulations of QCD in background fields. We demonstrate new techniques for the investigation of charged hadron properties in electric fields. Our current calculations employ large electric fields, motivating us to analyze chiral dynamics in strong QED backgrounds, and subsequently uncover surprising non-perturbative effects present at finite volume.

William Detmold, Brian Tiburzi, Andre Walker-Loud

2009-06-01

356

Backgrounds and characteristics of arsonists  

Microsoft Academic Search

The aim of this study was to gain more insight in the backgrounds and characteristics of arsonists. For this, the psychiatric, psychological, personal, and criminal backgrounds of all arsonists (n=25), sentenced to forced treatment in the maximum security forensic hospital “De Kijvelanden”, were compared to the characteristics of a control group of patients (n=50), incarcerated at the same institution for

Wim Labree; Henk Nijman; Hjalmar van Marle; Eric Rassin

2010-01-01

357

Low background counting at the LBNL low background facility  

NASA Astrophysics Data System (ADS)

The Low Background Facility (LBF) at the Lawrence Berkeley National Laboratory (LBNL) in Berkeley, California provides low background gamma spectroscopy services to end-users in two unique facilities: locally within a carefully-constructed, low background laboratory space; and a satellite underground station (600 m.w.e) in Oroville, CA. 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 and anthropogenic products, as well as active screening via neutron activation analysis for specific applications. A general overview of the facilities, services, and capabilities will be discussed. Recent activities will also be presented, including the recent installation of a 3? muon veto at the surface facility, cosmogenic activation studies of TeO2 for CUORE, and environmental monitoring of Fukushima fallout.

Thomas, K. J.; Smith, A. R.; Chan, Y. D.; Norman, E. B.; Wang, B. S.; Hurley, D. L.

2013-08-01

358

Meghan Miller Background and Significance  

E-print Network

1 Meghan Miller Background and Significance Autistic spectrum disorders (ASDs) represent variable pathological symptoms from one individual to the next, representing a continual spectrum rather within the autism spectrum include Asperger, Fragile X, Angelman, Rett, Williams, Prader

Gleeson, Joseph G.

359

Nongeometric fluxes as supergravity backgrounds  

SciTech Connect

We consider examples of D=4 string theory vacua which, although globally nongeometric, admit a local description in terms of D=10 supergravity backgrounds. We analyze such backgrounds and find that the supersymmetry spinors vary nontrivially along the internal manifold, reproducing the interpolating supergravity solutions found by Frey and Grana. Finally, we propose a simple, local expression for nongeometric fluxes in terms of the internal spinors of the compactification.

Marchesano, Fernando [ASC, Ludwig-Maximilians-Universitaet, Theresienstrasse 37, 80333 Munich (Germany); Schulgin, Waldemar [Max Planck Institut fuer Physik, Foehringer Ring 6, 80805 Munich (Germany)

2007-08-15

360

Recombinations in Staphylococcal Cassette Chromosome mec Elements Compromise the Molecular Detection of Methicillin Resistance in Staphylococcus aureus  

PubMed Central

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. PMID:24972080

Hill-Cawthorne, Grant A.; Hudson, Lyndsey O.; El Ghany, Moataz Fouad Abd; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S.

2014-01-01

361

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus.  

PubMed

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. PMID:24972080

Hill-Cawthorne, Grant A; Hudson, Lyndsey O; El Ghany, Moataz Fouad Abd; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S; Clark, Taane G; Pain, Arnab

2014-01-01

362

Detection and Organ-Specific Ablation of Neuroendocrine Cells by Synaptophysin Locus-Based BAC Cassette in Transgenic Mice  

PubMed Central

The role of cells of the diffuse neuroendocrine system in development and maintenance of individual organs and tissues remains poorly understood. Here we identify a regulatory region sufficient for accurate in vivo expression of synaptophysin (SYP), a common marker of neuroendocrine differentiation, and report generation of Tg(Syp-EGFPloxP-DTA)147Ayn (SypELDTA) mice suitable for flexible organ-specific ablation of neuroendocrine cells. These mice express EGFP and diphtheria toxin fragment A (DTA) in SYP positive cells before and after Cre-loxP mediated recombination, respectively. As a proof of principle, we have crossed SypELDTA mice with EIIA-Cre and PB-Cre4 mice. EIIA-Cre mice express Cre recombinase in a broad range of tissues, while PB-Cre4 mice specifically express Cre recombinase in the prostate epithelium. Double transgenic EIIA-Cre; SypELDTA embryos exhibited massive cell death in SYP positive cells. At the same time, PB-Cre4; SypELDTA mice showed a substantial decrease in the number of neuroendocrine cells and associated prostate hypotrophy. As no increase in cell death and/or Cre-loxP mediated recombination was observed in non-neuroendocrine epithelium cells, these results suggest that neuroendocrine cells play an important role in prostate development. High cell type specificity of Syp locus-based cassette and versatility of generated mouse model should assure applicability of these resources to studies of neuroendocrine cell functions in various tissues and organs. PMID:23630575

Cheng, Chieh-Yang; Zhou, Zongxiang; Nikitin, Alexander Yu.

2013-01-01

363

A Novel Staphylococcal Cassette Chromosomal Element, SCCfusC, Carrying fusC and speG in Fusidic Acid-Resistant Methicillin-Resistant Staphylococcus aureus  

PubMed Central

A high prevalence of fusC (16/46, 59%) was found in fusidic acid-resistant methicillin-resistant Staphylococcus aureus isolates collected from 2008 to 2010. Nucleotide sequencing of fusC and flanking regions revealed a novel staphylococcal cassette chromosome (SCC) structure, SCCfusC, which was integrated into rlmH and located upstream from SCCmec. The SCCfusC element contained speG, which may contribute to the polyamine resistance. PMID:24277045

Lin, Yu-Tzu; Tsai, Jui-Chang; Chen, Hsiao-Jan; Hung, Wei-Chun; Hsueh, Po-Ren

2014-01-01

364

ATP-Dependent Binding Cassette Transporter G Family Member 16 Increases Plant Tolerance to Abscisic Acid and Assists in Basal Resistance against Pseudomonas syringae DC3000.  

PubMed

Plants have been shown previously to perceive bacteria on the leaf surface and respond by closing their stomata. The virulent bacterial pathogen Pseudomonas syringae pv tomato DC3000 (PstDC3000) responds by secreting a virulence factor, coronatine, which blocks the functioning of guard cells and forces stomata to reopen. After it is inside the leaf, PstDC3000 has been shown to up-regulate abscisic acid (ABA) signaling and thereby suppress salicylic acid-dependent resistance. Some wild plants exhibit resistance to PstDC3000, but the mechanisms by which they achieve this resistance remain unknown. Here, we used genome-wide association mapping to identify an ATP-dependent binding cassette transporter gene (ATP-dependent binding cassette transporter G family member16) in Arabidopsis (Arabidopsis thaliana) that contributes to wild plant resistance to PstDC3000. Through microarray analysis and ?-glucuronidase reporter lines, we showed that the gene is up-regulated by ABA, bacterial infection, and coronatine. We also used a green fluorescent protein fusion protein and found that transporter is more likely to localize on plasma membranes than in cell walls. Transferred DNA insertion lines exhibited consistent defective tolerance of exogenous ABA and reduced resistance to infection by PstDC3000. Our conclusion is that ATP-dependent binding cassette transporter G family member16 is involved in ABA tolerance and contributes to plant resistance against PstDC3000. This is one of the first examples, to our knowledge, of ATP-dependent binding cassette transporter involvement in plant resistance to infection by a bacterial pathogen. It also suggests a possible mechanism by which plants reduce the deleterious effects of ABA hijacking during pathogen attack. Collectively, these results improve our understanding of basal resistance in Arabidopsis and offer unique ABA-related targets for improving the innate resistance of plants to bacterial infection. PMID:25146567

Ji, Hao; Peng, Yanhui; Meckes, Nicole; Allen, Sara; Stewart, C Neal; Traw, M Brian

2014-10-01

365

Local Variants of Staphylococcal Cassette Chromosome mec in Sporadic Methicillin-Resistant Staphylococcus aureus and Methicillin-Resistant Coagulase-Negative Staphylococci: Evidence of Horizontal Gene Transfer?  

Microsoft Academic Search

The mecA gene in Staphylococcus aureus is located on the genetic element staphylococcal cassette chromosome (SCC). Different SCCmecs have been classified according to their putative recombinase genes (ccrA and ccrB) and overall genetic composition. Clinical isolates of coagulase-negative staphylococci (CoNS; n 39) and S. aureus (n 20) from Norway, India, Italy, Finland, the United States, and the United Kingdom were

Anne-Merethe Hanssen; Gry Kjeldsen; Johanna U. Ericson Sollid

2004-01-01

366

Ovary-drip transformation: a simple method for directly generating vector- and marker-free transgenic maize ( Zea mays L.) with a linear GFP cassette transformation  

Microsoft Academic Search

The presence of selectable marker genes and vector backbone sequences has affected the safe assessment of transgenic plants.\\u000a In this study, the ovary-drip method for directly generating vector- and selectable marker-free transgenic plants was described,\\u000a by which maize was transformed with a linear GFP cassette (Ubi-GFP-nos). The key features of this method center on the complete removal of the styles

Aifu Yang; Qiao Su; Lijia An

2009-01-01

367

Identification of the O antigen polymerase (rfc) gene in Escherichia coli O4 by insertional mutagenesis using a nonpolar chloramphenicol resistance cassette.  

PubMed Central

Computer analysis of the O4 polysaccharide gene cluster of Escherichia coli revealed the presence of two open reading frames (ORFs) encoding strongly hydrophobic polypeptides. O antigen polymerase, which is encoded by the rfc gene, is a potential membrane protein and therefore should be hydrophobic. To identify the rfc gene, these two ORFs were subjected to insertional mutagenesis. A chloramphenicol resistance cassette was designed which, when properly inserted, does not cause a polar effect in downstream genes. Each of two ORFs, cloned into a plasmid vector, was inactivated with this cassette. Two types of mutants bearing chromosomal insertions of the cassettes in each ORF were constructed by homologous recombination. These mutants were characterized by PCR, Southern blotting, and transverse-alternating-field electrophoresis. Only one class of mutants exhibited the expected O polymerase-deficient phenotype; they produced O4-specific, semirough lipopolysaccharide. Therefore, this ORF was identified as the rfc gene. The chromosomal rfc mutation was complemented in trans by the rfc gene expressed from a plasmid vector. PMID:8550424

Lukomski, S; Hull, R A; Hull, S I

1996-01-01

368

Looking for Cosmic Neutrino Background  

NASA Astrophysics Data System (ADS)

Since the discovery of neutrino oscillation in atmospheric neutrinos by the Super-Kamiokande experiment in 1998, study of neutrinos has been one of exciting fields in high-energy physics. All the mixing angles were measured. Quests for 1) measurements of the remaining parameters, the lightest neutrino mass, the CP violating phase(s), and the sign of mass splitting between the mass eigenstates m3 and m1, and 2) better measurements to determine whether the mixing angle theta23 is less than pi/4, are in progress in a well-controlled manner. Determining the nature of neutrinos, whether they are Dirac or Majorana particles is also in progress with continuous improvement. On the other hand, although the ideas of detecting cosmic neutrino background have been discussed since 1960s, there has not been a serious concerted effort to achieve this goal. One of the reasons is that it is extremely difficult to detect such low energy neutrinos from the Big Bang. While there has been tremendous accumulation of information on Cosmic Microwave Background since its discovery in 1965, there is no direct evidence for Cosmic Neutrino Background. The importance of detecting Cosmic Neutrino Background is that, although detailed studies of Big Bang Nucleosynthesis and Cosmic Microwave Background give information of the early Universe at ~a few minutes old and ~300 k years old, respectively, observation of Cosmic Neutrino Background allows us to study the early Universe at ˜ 1 sec old. This article reviews progress made in the past 50 years on detection methods of Cosmic Neutrino Background.

Yanagisawa, Chiaki

2014-06-01

369

Detector Background at Muon Colliders  

SciTech Connect

Physics goals of a Muon Collider (MC) can only be reached with appropriate design of the ring, interaction region (IR), high-field superconducting magnets, machine-detector interface (MDI) and detector. Results of the most recent realistic simulation studies are presented for a 1.5-TeV MC. It is shown that appropriately designed IR and MDI with sophisticated shielding in the detector have a potential to substantially suppress the background rates in the MC detector. The main characteristics of backgrounds are studied.

Mokhov, N.V.; Striganov, S.I.; /Fermilab

2011-09-01

370

Background music and cognitive performance.  

PubMed

The present experiment employed standardized test batteries to assess the effects of fast-tempo music on cognitive performance among 56 male and female university students. A linguistic processing task and a spatial processing task were selected from the Criterion Task Set developed to assess verbal and nonverbal performance. Ten excerpts from Mozart's music matched for tempo were selected. Background music increased the speed of spatial processing and the accuracy of linguistic processing. The findings suggest that background music can have predictable effects on cognitive performance. PMID:20865993

Angel, Leslie A; Polzella, Donald J; Elvers, Greg C

2010-06-01

371

Mathematical background of Parrondo's paradox  

NASA Astrophysics Data System (ADS)

Parrondo's paradox states that there are losing gambling games which, when being combined stochastically or in a suitable deterministic way, give rise to winning games. Here we investigate the probabilistic background. We show how the properties of the equilibrium distributions of the Markov chains under consideration give rise to the paradoxical behavior, and we provide methods how to find the best a priori strategies.

Behrends, Ehrhard

2004-05-01

372

Educational Attainment and Family Background  

Microsoft Academic Search

This paper analyses the effect of aspects of family background, such as family income and parental education, on the educational attainment of persons born from 1967 to 1972. Family income is measured at different periods of a child's life to separate long-term versus short-term effects of family income on educational choices. We find that permanent income matters to a certain

Arild Aakvik; Kjell Gunnar Salvanes; Kjell Vaage

2005-01-01

373

Hurricanes and Tropical Meteorology Background  

E-print Network

- 1 - Hurricanes and Tropical Meteorology Background: Over the last 20 years, hurricane research at AOML has focused on improved scientific understanding of hurricanes and of tropical meteorology scientific goals for AOMLs hurricane research derive from the U.S. Weather Research Programs (USWRP

374

Wormhole on the Lobachevsky background.  

National Technical Information Service (NTIS)

The exact spherical symmetric static solution of Rosen like equations of the bi metric theory is investigated. The background metric is not flat, but curved, with the Lobachevsky spatial sections and 'cosmic time' c(sup 2) d t(sup 2). There are two branch...

M. N. Tentyukov

1994-01-01

375

Shark Fact or Fiction? Background  

E-print Network

Shark Fact or Fiction? Background: This is a fun classroom activity based on the basic biology of sharks. This goes well with the enclosed Project Shark Awareness PowerPoint and should be used in conjunction with the presentation. Materials: Shark Fact of Fiction activity sheet and answer key

Watson, Craig A.

376

A Little Background Music, Please.  

ERIC Educational Resources Information Center

Background music could be used to provide a pleasant beginning for the school day, to help keep students quiet and relaxed in the school cafeteria at lunchtime, and to provide a midafternoon lift for bored and tired children. The most effective music pleases children without overly exciting them through jarring rhythms and loud dynamics. (nine…

Giles, Martha Mead

1991-01-01

377

Teacher Pensions: A Background Paper  

ERIC Educational Resources Information Center

Pensions are an important but comparatively unexamined component of human resource policies in education. In an increasingly competitive world where employees are more mobile than ever, pension policies that were designed in the last century may be out of step with the needs of both individuals and schools. This background paper aims to foster…

Hansen, Janet S.

2008-01-01

378

Teaching about Natural Background Radiation  

ERIC Educational Resources Information Center

Ambient gamma dose rates in air were measured at different locations (indoors and outdoors) to demonstrate the ubiquitous nature of natural background radiation in the environment and to show that levels vary from one location to another, depending on the underlying geology. The effect of a lead shield on a gamma radiation field was also…

Al-Azmi, Darwish; Karunakara, N.; Mustapha, Amidu O.

2013-01-01

379

Modeling Dense Stellar Systems: Background  

E-print Network

I provide some background about recent efforts made in modeling dense stellar systems, within the context of the MODEST initiative. During the last four years, we have seen more than fifteen MODEST workshops, with an attendance between twenty and a hundred participants, and topics ranging from very specialized discussions to rather general overviews.

Piet Hut

2006-10-07

380

Quantization by cosmic background radiation  

Microsoft Academic Search

It is suggested that various modes in the cosmic background radiation field may account for the discrete properties exhibited by small systems. In particular, this view is applied to the 1, 2-, and 3-D oscillators and the hydrogen atom, systems which were treated by Schrodinger in his first papers on quantum mechanics. The usual energy formulas for the above systems

James T. Dehn

1989-01-01

381

Fifty Percent Law: Background Paper.  

ERIC Educational Resources Information Center

This paper provides background information about a statute that affects the fiscal operation of California community colleges. The Fifty Percent Law (Education Code 84362) requires "there shall be expended each fiscal year for payment of salaries of classroom instructors by a community college district, 50 percent of the district's current expense…

Community Coll. League of California, Sacramento.

382

The Cosmic Microwave Background Radiation  

NSDL National Science Digital Library

This online article, from Cosmic Horizons: Astronomy at the Cutting Edge, provides an overview of how scientists are working to explain the origin of the universe. Specifically, it discusses the two major theories about the origin of the universe (Big Bang and Steady State), the search for microwave background radiation, and the discovery of the first observational evidence to support the Big Bang theory.

383

Plasmodium falciparum Expressing Domain Cassette 5 Type PfEMP1 (DC5-PfEMP1) Bind PECAM1  

PubMed Central

Members of the Plasmodium falciparum Erythrocyte Membrane protein 1 (PfEMP1) family expressed on the surface of malaria-infected erythrocytes mediate binding of the parasite to different receptors on the vascular lining. This process drives pathologies, and severe childhood malaria has been associated with the expression of particular subsets of PfEMP1 molecules. PfEMP1 are grouped into subtypes based on upstream sequences and the presence of semi-conserved PfEMP1 domain compositions named domain cassettes (DCs). Earlier studies have indicated that DC5-containing PfEMP1 (DC5-PfEMP1) are more likely to be expressed in children with severe malaria disease than in children with uncomplicated malaria, but these PfEMP1 subtypes only dominate in a relatively small proportion of the children with severe disease. In this study, we have characterised the genomic sequence characteristic for DC5, and show that two genetically different parasite lines expressing DC5-PfEMP1 bind PECAM1, and that anti-DC5-specific antibodies inhibit binding of DC5-PfEMP1-expressing parasites to transformed human bone marrow endothelial cells (TrHBMEC). We also show that antibodies against each of the four domains characteristic for DC5 react with native PfEMP1 expressed on the surface of infected erythrocytes, and that some of these antibodies are cross-reactive between the two DC5-containing PfEMP1 molecules tested. Finally, we confirm that anti-DC5 antibodies are acquired early in life by individuals living in malaria endemic areas, that individuals having high levels of these antibodies are less likely to develop febrile malaria episodes and that the antibody levels correlate positively with hemoglobin levels. PMID:23874884

Berger, Sanne S.; Turner, Louise; Wang, Christian W.; Petersen, Jens E. V.; Kraft, Maria; Lusingu, John P. A.; Mmbando, Bruno; Marquard, Andrea M.; Bengtsson, Dominique B. A. C.; Hviid, Lars; Nielsen, Morten A.; Theander, Thor G.; Lavstsen, Thomas

2013-01-01

384

Acid-soluble internal capsules for closed-face cassette elemental sampling and analysis of workplace air.  

PubMed

Airborne particles that are collected using closed-face filter cassettes (CFCs), which are used widely in the sampling of workplace aerosols, can deposit in places other than on the filter and thereby may not be included in the ensuing analysis. A technique for ensuring that internal non-filter deposits are included in the analysis is to collect airborne particles within an acid-soluble internal capsule that, following sampling, can be dissolved along with the filter for subsequent elemental analysis. An interlaboratory study (ILS) was carried out to evaluate the use of cellulosic CFC capsule inserts for their suitability in the determination of trace elements in airborne samples. The ILS was performed in accordance with an applicable ASTM International standard practice, ASTM E691, which describes statistical procedures for investigating interlaboratory precision. Performance evaluation materials consisted of prototype cellulose acetate capsules attached to mixed-cellulose ester filters. Batches of capsules were dosed with Pb-containing materials (standard aqueous solutions, and certified reference material soil and paint). Also, aerosol samples containing nine target analyte elements (As, Cd, Co, Cr, Cu, Fe, Pb, Mn, and Ni) were generated using a multiport sampler; various concentrations and sampling times were employed to yield samples fortified at desired loading levels. Triplicates of spiked capsules at three different loadings were conveyed to each volunteer laboratory; loading levels were unknown to the participants. The laboratories were asked to prepare the samples by acid dissolution and to analyze aliquots of extracted samples by atomic spectrometry in accordance with applicable ASTM International Standards. Participants were asked to report their results in units of ?g of each target element per sample. For the elements investigated, inter-laboratory precision and recovery estimates from the participating laboratories demonstrated the utility of the cellulosic capsule inserts for the measurement of sampled trace elements. PMID:23548078

Harper, Martin; Ashley, Kevin

2013-01-01

385

Acid-Soluble Internal Capsules for Closed-Face Cassette Elemental Sampling and Analysis of Workplace Air  

PubMed Central

Airborne particles that are collected using closed-face filter cassettes (CFCs), which are used widely in the sampling of workplace aerosols, can deposit in places other than on the filter and thereby may not be included in the ensuing analysis. A technique for ensuring that internal non-filter deposits are included in the analysis is to collect airborne particles within an acid-soluble internal capsule that, following sampling, can be dissolved along with the filter for subsequent elemental analysis. An interlaboratory study (ILS) was carried out to evaluate the use of cellulosic CFC capsule inserts for their suitability in the determination of trace elements in airborne samples. The ILS was performed in accordance with an applicable ASTM International standard practice, ASTM E691, which describes statistical procedures for investigating interlaboratory precision. Performance evaluation materials consisted of prototype cellulose acetate capsules attached to mixed-cellulose ester filters. Batches of capsules were dosed with Pb-containing materials (standard aqueous solutions, and certified reference material soil and paint). Also, aerosol samples containing nine target analyte elements (As, Cd, Co, Cr, Cu, Fe, Pb, Mn, and Ni) were generated using a multiport sampler; various concentrations and sampling times were employed to yield samples fortified at desired loading levels. Triplicates of spiked capsules at three different loadings were conveyed to each volunteer laboratory; loading levels were unknown to the participants. The laboratories were asked to prepare the samples by acid dissolution and to analyze aliquots of extracted samples by atomic spectrometry in accordance with applicable ASTM International Standards. Participants were asked to report their results in units of ?g of each target element per sample. For the elements investigated, interlaboratory precision and recovery estimates from the participating laboratories demonstrated the utility of the cellulosic capsule inserts for the measurement of sampled trace elements. PMID:23548078

Harper, Martin; Ashley, Kevin

2013-01-01

386

Engineered human Tmpk fused with truncated cell-surface markers: versatile cell-fate control safety cassettes.  

PubMed

Cell-fate control gene therapy (CFCGT)-based strategies can augment existing gene therapy and cell transplantation approaches by providing a safety element in the event of deleterious outcomes. Previously, we described a novel enzyme/prodrug combination for CFCGT. Here, we present results employing novel lentiviral constructs harboring sequences for truncated surface molecules (CD19 or low-affinity nerve growth factor receptor) directly fused to that CFCGT cDNA (TmpkF105Y). This confers an enforced one-to-one correlation between cell marking and eradication functions. In-vitro analysis demonstrated the full functionality of the fusion product. Next, low-dose 3'-azido-3'-deoxythymidine (AZT) administration to non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with transduced clonal K562 cells suppressed tumor growth; furthermore, one integrated vector on average was sufficient to mediate cytotoxicity. Further, in a murine xenogeneic leukemia-lymphoma model we also demonstrated in-vivo control over transduced Raji cells. Finally, in a proof-of-principle study to examine the utility of this cassette in combination with a therapeutic cDNA, we integrated this novel CFCGT fusion construct into a lentivector designed for treatment of Fabry disease. Transduction with this vector restored enzyme activity in Fabry cells and retained AZT sensitivity. In addition, human Fabry patient CD34(+) cells showed high transduction efficiencies and retained normal colony-generating capacity when compared with the non-transduced controls. These collective results demonstrated that this novel and broadly applicable fusion system may enhance general safety in gene- and cell-based therapies. PMID:22241175

Scaife, M; Pacienza, N; Au, B C Y; Wang, J C M; Devine, S; Scheid, E; Lee, C-J; Lopez-Perez, O; Neschadim, A; Fowler, D H; Foley, R; Medin, J A

2013-01-01

387

Novel Pseudo-Staphylococcal Cassette Chromosome mec Element (?SCCmec57395) in Methicillin-Resistant Staphylococcus pseudintermedius CC45  

PubMed Central

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I–pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to ?-lactams (mecA; blaZ), aminoglycosides [aac(6?)-Ie–aph(2?)-Ia; aph(3?)-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (?SCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ?SCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ?SCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ?SCCmec57395 element amplified by long-range PCR revealed the presence of ?SCCmec57395 in the 33 additional isolates of MRSP CC45. The ?SCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand. PMID:23979735

Chanchaithong, Pattrarat; Prapasarakul, Nuvee; Rossano, Alexandra; Blum, Shlomo E.; Elad, Daniel; Schwendener, Sybille

2013-01-01

388

Conductivity in an anisotropic background  

SciTech Connect

By using the gauge/gravity duality, we investigate the dual field theories of the anisotropic backgrounds, which are exact solutions of Einstein-Maxwell-dilaton theory with a Liouville potential. When we turn on the bulk gauge field fluctuation A{sub x} with a nontrivial dilaton coupling, the AC conductivity of this dual field theory is proportional to the frequency with an exponent depending on parameters of the anisotropic background. In some parameter regions, we find that this conductivity can have the negative exponent like the strange metal. In addition, we also investigate another U(1) gauge field fluctuation, which is not coupled with a dilaton field. We classify all possible conductivities of this system and find that the exponent of the conductivity is always positive.

Lee, Bum-Hoon [Department of Physics, Sogang University, Seoul 121-742 (Korea, Republic of); Center for Quantum Spacetime (CQUeST), Sogang University, Seoul 121-742 (Korea, Republic of); Nam, Siyoung [Department of Physics, Sogang University, Seoul 121-742 (Korea, Republic of); Pang, Da-Wei; Park, Chanyong [Center for Quantum Spacetime (CQUeST), Sogang University, Seoul 121-742 (Korea, Republic of)

2011-03-15

389

Quantization by cosmic background radiation  

NASA Astrophysics Data System (ADS)

It is suggested that various modes in the cosmic background radiation field may account for the discrete properties exhibited by small systems. In particular, this view is applied to the 1, 2-, and 3-D oscillators and the hydrogen atom, systems which were treated by Schrodinger in his first papers on quantum mechanics. The usual energy formulas for the above systems are derived using this point of view, together with some indication of how transition probabilities might also be calculated. A connection between de Broglie's associated wave and a free mass moving in the cosmic background is also discussed. Analogs of the uncertainty and correspondence principles are briefly mentioned as are some of the implications this view might have for interpreting quantum theory. In this view particles and waves are separate, interacting entities and not complementary aspects of the same thing.

Dehn, James T.

1989-05-01

390

Superspace geometry for supermembrane backgrounds  

Microsoft Academic Search

We construct part of the superspace vielbein and tensor gauge field in terms of the component fields of 11-dimensional on-shell supergravity. The result can be utilized to describe supermembranes and corresponding matrix models for Dirichlet particles in non-trivial supergravity backgrounds to second order in anticommuting coordinates. We exhibit the ?-invariance of the corresponding supermembrane action, which at this order holds

Bernard de Wit; Kasper Peeters; Jan Plefka

1998-01-01

391

Emergent Supersymmetry in Warped Backgrounds  

NASA Astrophysics Data System (ADS)

We show that quantum mechanical supersymmetries are emerged in Kaluza-Klein spectrum of linearized gravity in several warped backgrounds as a consequence of higher-dimensional general coordinate invariance. These emergent supersymmetries play an essential role for the spectral structure of braneworld gravity. We show that for the case of braneworld models with two codimension-1 branes the spectral pattern is completely determined only through the supersymmetries.

Nagasawa, Tomoaki; Ohya, Satoshi; Sakamoto, Kazuki; Sakamoto, Makoto

2011-07-01

392

Backgrounds and characteristics of arsonists.  

PubMed

The aim of this study was to gain more insight in the backgrounds and characteristics of arsonists. For this, the psychiatric, psychological, personal, and criminal backgrounds of all arsonists (n=25), sentenced to forced treatment in the maximum security forensic hospital "De Kijvelanden", were compared to the characteristics of a control group of patients (n=50), incarcerated at the same institution for other severe crimes. Apart from DSM-IV Axis I and Axis II disorders, family backgrounds, level of education, treatment history, intelligence (WAIS scores), and PCL-R scores were included in the comparisons. Furthermore, the apparent motives for the arson offences were explored. It was found that arsonists had more often received psychiatric treatment, prior to committing their index offence, and had a history of severe alcohol abuse more often in comparison to the controls. The arsonists turned out to be less likely to suffer from a major psychotic disorder. Both groups did not differ significantly on the other variables, among which the PCL-R total scores and factor scores. Exploratory analyses however, did suggest that arsonists may differentiate from non-arsonists on three items of the PCL-R, namely impulsivity (higher scores), superficial charm (lower scores), and juvenile delinquency (lower scores). Although the number of arsonists with a major psychotic disorder was relatively low (28%), delusional thinking of some form was judged to play a role in causing arson crimes in about half of the cases (52%). PMID:20434774

Labree, Wim; Nijman, Henk; van Marle, Hjalmar; Rassin, Eric

2010-01-01

393

The Sunyaev-Zeldovich Background  

E-print Network

The cosmic background due to the Sunyaev-Zeldovich (SZ) effect is expected to be the largest signal at mm and cm wavelengths at a resolution of a few arcminutes. We investigate some simple statistics of SZ maps and their scaling with the normalization of the matter power spectrum, sigma_8, as well as the effects of the unknown physics of the intracluster medium on these statistics. We show that the SZ background provides a significant background for SZ cluster searches, with the onset of confusion occurring around 10^{14} h^{-1} solar masses in a cosmology-dependent way, where confusion is defined as typical errors in recovered flux larger than 20%. The confusion limit, corresponds to the mass at which there are roughly ten clusters per square degree, with this number nearly independent of cosmology and cluster gas physics. Typical errors grow quickly as lower mass objects are included in the catalog. We also point out that there is nothing in particular about the rms of the filtered map that makes it especially well-suited for capturing aspects of the SZ effect, and other indicators of the one-point SZ probability distribution function are at least as well suited for the task. For example, the full width at half maximum of the one point probability distribution has a field-to-field scatter that is about 60% that of the rms. The simplest statistics of SZ maps are largely unaffected by cluster physics such aspreheating, although the impact of preheating is clear by eye in the maps.Studies aimed at learning about the physics of the intracluster medium will apparently require more specialized statistical indicators.

Gilbert Holder; Ian G. McCarthy; Arif Babul

2007-02-27

394

High background photon counting lidar  

NASA Technical Reports Server (NTRS)

Photon counting with lidar returns is usually limited to low light levels, while wide dynamic range is achieved by counting for long times. The broad emission spectrum of inexpensive high-power semiconductor lasers makes receiver filters pass too much background light for traditional photon counting in daylight. Very high speed photon counting is possible, however, at more than 500 MHz which allows the construction of eyesafe lidar operating in the presence of bright clouds. Detector improvements are possible to count to 20 GHz producing a single shot dynamic range of ten decades.

Lentz, W. J.

1992-01-01

395

Mobile insertion cassette elements found in small non-transmissible plasmids in Proteeae may explain qnrD mobilization.  

PubMed

qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36-60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae. PMID:24504382

Guillard, Thomas; Grillon, Antoine; de Champs, Christophe; Cartier, Céline; Madoux, Janick; Berçot, Béatrice; Lebreil, Anne-Laure; Lozniewski, Alain; Riahi, Jacques; Vernet-Garnier, Véronique; Cambau, Emmanuelle

2014-01-01

396

Mobile Insertion Cassette Elements Found in Small Non-Transmissible Plasmids in Proteeae May Explain qnrD Mobilization  

PubMed Central

qnrD is a plasmid mediated quinolone resistance gene from unknown origin, recently described in Enterobacteriaceae. It encodes a pentapeptide repeat protein 36–60% different from the other Qnr (A, B, C, S and VC). Since most qnrD-positive strains were described as strains belonging to Proteus or Providencia genera, we hypothesized that qnrD originated in Proteeae before disseminating to other enterobacterial species. We screened 317 strains of Proteeae for qnrD and its genetic support by PCR. For all the seven qnrD-positive strains (4 Proteus mirabilis, 1 Proteus vulgaris and 2 Providencia rettgeri) the gene was carried onto a small non-transmissible plasmid, contrarily to other qnr genes that are usually carried onto large multi-resistant plasmids. Nucleotide sequences of the qnrD-bearing plasmids were 96% identical. Plasmids contained 3 ORFs apart from qnrD and belonged to an undescribed incompatibility group. Only one plasmid, in P. vulgaris, was slightly different with a 1,568-bp insertion between qnrD and its promoter, leading to absence of quinolone resistance. We sought for similar plasmids in 15 reference strains of Proteeae, but which were tested negative for qnrD, and found a 48% identical plasmid (pVERM) in Providencia vermicola. In order to explain how qnrD could have been inserted into such native plasmid, we sought for gene mobilization structures. qnrD was found to be located within a mobile insertion cassette (mic) element which sequences are similar to one mic also found in pVERM. Our conclusions are that (i) the small non-transmissible qnrD-plasmids described here may result from the recombination between an as-yet-unknown progenitor of qnrD and pVERM, (ii) these plasmids are maintained in Proteeae being a qnrD reservoir (iii) the mic element may explain qnrD mobilization from non-transmissible plasmids to mobilizable or conjugative plasmids from other Enterobacteriaceae, (iv) they can recombined with larger multiresistant plasmids conjugated in Proteeae. PMID:24504382

Guillard, Thomas; Grillon, Antoine; de Champs, Christophe; Cartier, Celine; Madoux, Janick; Bercot, Beatrice; Lebreil, Anne-Laure; Lozniewski, Alain; Riahi, Jacques; Vernet-Garnier, Veronique; Cambau, Emmanuelle

2014-01-01

397

Reversal of antifungal resistance mediated by ABC efflux pumps from Candida albicans functionally expressed in yeast  

Microsoft Academic Search

The enhanced efflux of antifungal drugs through ATP-binding cassette (ABC) transporters constitutes a major cause of clinical multidrug resistance (MDR). The inhibition of drug efflux pumps by specific compounds is considered to be a feasible strategy to overcome clinical antifungal resistance. Therefore, several blockers of mammalian and yeast ABC drug pumps, including FK506, propafenones, as well as the antifungal drug

Manuela Schuetzer-Muehlbauer; Birgit Willinger; Ralf Egner; Gerhard Ecker; Karl Kuchler

2003-01-01

398

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

Microsoft Academic Search

Metabolism by phase II enzymes and transport from intestinal cells back into the lumen by ATP binding cassette (ABC) transporters limits the bioavailability of the flavanone hesperetin, the aglycone of hesperidin. This study investigates to what extent other flavonoids modulate the metabolism and transport of hesperetin by characterizing the effect of co-administrating a series of flavonoids using Caco-2 cell monolayers

Walter Brand; Beatriz Padilla; Bladeren van P. J; Gary Williamson; Ivonne M. C. M. Rietjens

2010-01-01

399

Bis(monoacylglycero)phosphate accumulation in macrophages induces intracellular cholesterol redistribution, attenuates LXR/ABCA1/ABCG1 pathway and impairs  

E-print Network

Bis(monoacylglycero)phosphate accumulation in macrophages induces intracellular cholesterol1, ATP-binding cassette G1; BMP, bis (monacylglycero)phosphate; CE, cholesterol ester; ER signature phospholipid bis(monoacylglycero)phosphate (BMP) has been involved in the regulation of cellular

Boyer, Edmond

400

The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil  

Microsoft Academic Search

During pathogenesis, fungal pathogens are exposed to a variety of fungitoxic compounds. This may be particularly relevant to Botrytis cinerea, a plant pathogen that has a broad host range and, consequently, is subjected to exposure to many plant defense compounds. In practice, the pathogen is controlled with fungicides belonging to different chemical groups. ATP-binding cassette (ABC) transporters might provide protection

H. Schoonbeek; G. Del Sorbo; Waard De M. A

2001-01-01

401

Can ABCF2 protein expression predict the prognosis of uterine cancer?  

Microsoft Academic Search

Uterine cervical and endometrial cancers are common malignant solid neoplasms for which there are no useful prognostic markers. In this study, we evaluate the relationship between ATP-binding cassette superfamily F2 (ABCF2) expression and clinical factors including clinical stage, histologic type, grade and prognosis in uterine cervical and endometrial cancer. Two hundred and sixty seven cervical and 103 endometrial cancers were

S Nishimura; H Tsuda; Y Miyagi; A Hirasawa; A Suzuki; F Kataoka; H Nomura; T Chiyoda; K Banno; T Fujii; N Susumu; D Aoki

2008-01-01

402

Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation  

Microsoft Academic Search

MAJOR histocompatibility complex (MHC) class I molecules bind and deliver peptides derived from endogenously synthesized proteins to the cell surface for survey by cytotoxic T lymphocytes. It is believed that endogenous antigens are generally degraded in the cytosol, the resulting peptides being translocated into the endoplasmic reticulum where they bind to MHC class I molecules. Transporters containing an ATP-binding cassette

Frank Momburg; Vianney Ortiz-Navarrete; Jacques Neefjes; Els Goulmy; Yvonne van de Wal; Hergen Spits; Simon J. Powis; Geoffrey W. Butcher; Jonathan C. Howard; Peter Walden; Günter J. Hämmerling

1992-01-01

403

A Comparative Electron Paramagnetic Resonance Study of the Nucleotide-Binding Domains' Catalytic Cycle in the Assembled Maltose  

E-print Network

and in the vanadate-trapped intermediate and move back toward the apo-state after ATP hydrolysis. The distance between the catalytic cycle, and show an unforeseen potential interaction between MalK and the transmembrane subunit MalG. INTRODUCTION ATP-binding cassette (ABC) transporters are membrane pro- teins that mediate the uptake or export

Steinhoff, Heinz-Jürgen

404

Common and Rare ABCA1 Variants Affecting Plasma HDL Cholesterol  

Microsoft Academic Search

Mutations in ABCA1, a member of the ATP-binding cassette family, have been shown to underlie Tangier disease (TD) and familial hypoalphalipoproteinemia (FHA), which are genetic disorders that are characterized by depressed concentrations of plasma high density lipoprotein (HDL) cholesterol. An important question is whether common variants within the coding sequence of ABCA1 can affect plasma HDL cholesterol in the general

Jian Wang; John R. Burnett; Kue Young; Bernard Zinman; Anthony J. G. Hanley; Philip W. Connelly; Stewart B. Harris; Robert A. Hegele

2009-01-01

405

Human and Rat ABC Transporter Efflux of Bisphenol A and Bisphenol A Glucuronide: Interspecies Comparison and Implications for Pharmacokinetic Assessment  

EPA Science Inventory

Significant interspecies differences exist between human and rodent with respect to absorption, distribution, and excretion of bisphenol A (BPA) and its primary metabolite, BPA-glucuronide (BPA-G). ATP-Binding Cassette (ABC) transporter enzymes play important roles in these physi...

406

The role of the photoreceptor ABC transporter ABCA4 in lipid transport and Stargardt macular degeneration  

Microsoft Academic Search

ABCA4 is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters that is expressed in rod and cone photoreceptors of the vertebrate retina. ABCA4, also known as the Rim protein and ABCR, is a large 2273 amino acid glycoprotein organized as two tandem halves, each containing a single membrane spanning segment followed sequentially by a large exocytoplasmic

Robert S. Molday; Ming Zhong; Faraz Quazi

2009-01-01

407

Universittsmedizin Gttingen Publikationen und Hochschulschriften 2011  

E-print Network

of SLP-76 at tyrosine 173 is required for activation of T and mast cells. EMBO J, 30: 3160- 72. 15 (2011) Fc gamma receptor IIb modulates the molecular Grb2 interaction network in activated B cells. CELL) Exosomal evasion of humoral immunotherapy in aggressive B-cell lymphoma modulated by ATP-binding cassette

Gollisch, Tim

408

Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec (SCCmec-SCCcad/ars/cop) in the Neonatal Sepsis-Associated Staphylococcus capitis Pulsotype NRCS-A  

PubMed Central

Multiresistant Staphylococcus capitis pulsotype NRCS-A has been reported to be a major pathogen causing nosocomial bacteremia in preterm infants. We report that the NRCS-A strain CR01 harbors a novel 60.9-kb composite staphylococcal cassette chromosome mec (SCCmec) element, composed of an SCCmec with strong homologies to Staphylococcus aureus ST398 SCCmec and of an SCCcad/ars/cop harboring resistance genes for cadmium, arsenic, and copper. Whole-genome-based comparisons of published S. capitis strains suggest that strain CR01 acquired the two elements independently. PMID:24060879

Rasigade, J.-P.; Lemriss, H.; Butin, M.; Ginevra, C.; Lemriss, S.; Goering, R. V.; Ibrahimi, A.; Picaud, J. C.; El Kabbaj, S.; Vandenesch, F.; Laurent, F.

2013-01-01

409

Novel Organization of the Arginine Catabolic Mobile Element and Staphylococcal Cassette Chromosome mec Composite Island and Its Horizontal Transfer between Distinct Staphylococcus aureus Genotypes  

PubMed Central

In this study, 425 methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered in the Dutch-German Euregio were investigated for the presence of the arginine catabolic mobile element (ACME). Sequence analysis by whole-genome sequencing revealed an entirely new organization of the ACME-staphylococcal cassette chromosome mec composite island (SCCmec-CI), with truncated ACME type II located downstream of SCCmec. An identical nucleotide sequence of ACME-SCCmec-CI was found in two distinct MRSA lineages (t064-ST8 and t002-ST5), which has not been reported previously in S. aureus. PMID:24002094

Sabat, Artur J.; Kock, Robin; Akkerboom, Viktoria; Hendrix, Ron; Skov, Robert L.; Becker, Karsten

2013-01-01

410