Sample records for abc transport protein

  1. [The ABC transporters of Saccharomyces cerevisiae].

    PubMed

    Wawrzycka, Donata

    2011-01-01

    The ABC transporters (ATP Binding Cassette) compose one of the bigest protein family with the great medical, industrial and economical impact. They are found in all organism from bacteria to man. ABC proteins are responsible for resistance of microorganism to antibiotics and fungicides and multidrug resistance of cancer cells. Mutations in ABC transporters genes cause seriuos deseases like cystic fibrosis, adrenoleucodystrophy or ataxia. Transport catalized by ABC proteins is charged with energy from the ATP hydrolysis. The ABC superfamily contains transporters, canals, receptors. Analysis of the Saccharomyces cerevisiae genome allowed to distinguish 30 potential ABC proteins which are classified into 6 subfamilies. The structural and functional similarity of the yeast and human ABC proteins allowes to use the S. cerevisiae as a model organism for ABC transporters characterisation. In this work the present state of knowleadge on yeast S. cerevisiae ABC proteins was summarised.

  2. Harnessing Drug Resistance: Using ABC Transporter Proteins To Target Cancer Cells

    PubMed Central

    Leitner, Heather M.; Kachadourian, Remy; Day, Brian J.

    2007-01-01

    The ATP-binding cassette (ABC) class of proteins is one of the most functionally diverse transporter families found in biological systems. Although the abundance of ABC proteins varies between species, they are highly conserved in sequence and often demonstrate similar functions across prokaryotic and eukaryotic organisms. Beginning with a brief summary of the events leading to our present day knowledge of ABC transporters, the purpose of this review is to discuss the potential for utilizing ABC transporters as a means for cellular glutathione (GSH) modulation. GSH is one of the most abundant thiol antioxidants in cells. It is involved in cellular division, protein and DNA synthesis, maintenance of cellular redox status and xenobiotic metabolism. Cellular GSH levels are often altered in many disease states including cancer. Over the past two decades there has been considerable emphasis on methods to sensitize cancer cells to chemotherapeutics and ionization radiation therapy by GSH depletion. We contend that ABC transporters, particularly multi-drug resistant proteins (MRPs), may be used as therapeutic targets for applications aimed at modulation of GSH levels. This review will emphasize MRP-mediated modulation of intracellular GSH levels as a potential alternative and adjunctive approach for cancer therapy. PMID:17585883

  3. Interaction of ABC multidrug transporters with anticancer protein kinase inhibitors: substrates and/or inhibitors?

    PubMed

    Hegedus, Csilla; Ozvegy-Laczka, Csilla; Szakács, Gergely; Sarkadi, Balázs

    2009-05-01

    Protein kinase inhibitors (PKI) are becoming key agents in modern cancer chemotherapy, and combination of PKIs with classical chemotherapeutic drugs may help to overcome currently untreatable metastatic cancers. Since chemotherapy resistance is a recurrent problem, mechanisms of resistance should be clarified in order to help further drug development. Here we suggest that in addition to PKI resistance based on altered target structures, the active removal of these therapeutic agents by the MDR-ABC transporters should also be considered as a major cause of clinical resistance. We discuss the occurring systemic and cellular mechanisms, which may hamper PKI efficiency, and document the role of selected MDR-ABC transporters in these phenomena through their interactions with these anticancer agents. Moreover, we suggest that PKI interactions with ABC transporters may modulate overall drug metabolism, including the fate of diverse, chemically or target-wise unrelated drugs. These effects are based on multiple forms of MDR-ABC transporter interaction with PKIs, as these compounds may be both substrates and/or inhibitors of an ABC transporter. We propose that these interactions should be carefully considered in clinical application, and a combined MDR-ABC transporter and PKI effect may bring a major advantage in future drug development.

  4. Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Giulliani, S. E.; Frank, A. E.; Collart, F. R.

    2008-12-08

    We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. The assay also identified additional candidate binding ligands for several of the amino acid-binding proteins in the validation set. We extended this approach to additional targets and demonstrated the ability of the FTS assay to unambiguously identify preferential binding for several homologues of amino acid-binding proteins with known specificity andmore » to functionally annotate proteins of unknown binding specificity. The assay is implemented in a microwell plate format and provides a rapid approach to validate an anticipated function or to screen proteins of unknown function. The ABC-type transporter family is ubiquitous and transports a variety of biological compounds, but the current annotation of the ligand-binding proteins is limited to mostly generic descriptions of function. The results illustrate the feasibility of the FTS assay to improve the functional annotation of binding proteins associated with ABC-type transporters and suggest this approach that can also be extended to other protein families.« less

  5. Evolutionary Trajectories of Entomopathogenic Fungi ABC Transporters.

    PubMed

    Baral, Bikash

    2017-01-01

    The ABC protein superfamily-also called traffic ATPases-are energy-dependent ubiquitous proteins, representing one of the crucial and the largest family in the fungal genomes. The ATP-binding cassette endows a characteristic 200-250 amino acids and is omnipresent in all organisms ranging from prokaryotes to eukaryotes. Unlike in bacteria with nutrient import functions, ABC transporters in fungal entomopathogens serve as effective efflux pumps that are largely involved in the shuttle of metabolites across the biological membranes. Thus, the search for ABC proteins may prove of immense importance in elucidating the functional and molecular mechanism at the host-pathogen (insect-fungus) interface. Their sequence homology, domain topology, and functional traits led to the actual identification of nine different families in fungal entomopathogens. Evolutionary relationships within the ABC superfamily are discussed, concentrating on computational approaches for comparative identification of ABC transporters in insect-pathogenic fungi (entomopathogens) with those of animals, plants, and their bacterial orthologs. Ancestors of some fungal candidates have duplicated extensively in some phyla, while others were lost in one lineage or the other, and predictions for the cause of their duplications and/or loss in some phyla are made. ABC transporters of fungal insect-pathogens serve both defensive and offensive functions effective against land-dwelling and ground foraging voracious insects. This study may help to unravel the molecular cascades of ABC proteins to illuminate the means through which insects cope with fungal infection and fungal-related diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Detergent-free purification of ABC (ATP-binding-cassette) transporters.

    PubMed

    Gulati, Sonali; Jamshad, Mohammed; Knowles, Timothy J; Morrison, Kerrie A; Downing, Rebecca; Cant, Natasha; Collins, Richard; Koenderink, Jan B; Ford, Robert C; Overduin, Michael; Kerr, Ian D; Dafforn, Timothy R; Rothnie, Alice J

    2014-07-15

    ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.

  7. The ABC protein turned chloride channel whose failure causes cystic fibrosis

    NASA Astrophysics Data System (ADS)

    Gadsby, David C.; Vergani, Paola; Csanády, László

    2006-03-01

    CFTR chloride channels are encoded by the gene mutated in patients with cystic fibrosis. These channels belong to the superfamily of ABC transporter ATPases. ATP-driven conformational changes, which in other ABC proteins fuel uphill substrate transport across cellular membranes, in CFTR open and close a gate to allow transmembrane flow of anions down their electrochemical gradient. New structural and biochemical information from prokaryotic ABC proteins and functional information from CFTR channels has led to a unifying mechanism explaining those ATP-driven conformational changes.

  8. Catalytic and transport cycles of ABC exporters.

    PubMed

    Al-Shawi, Marwan K

    2011-09-07

    ABC (ATP-binding cassette) transporters are arguably the most important family of ATP-driven transporters in biology. Despite considerable effort and advances in determining the structures and physiology of these transporters, their fundamental molecular mechanisms remain elusive and highly controversial. How does ATP hydrolysis by ABC transporters drive their transport function? Part of the problem in answering this question appears to be a perceived need to formulate a universal mechanism. Although it has been generally hoped and assumed that the whole superfamily of ABC transporters would exhibit similar conserved mechanisms, this is proving not to be the case. Structural considerations alone suggest that there are three overall types of coupling mechanisms related to ABC exporters, small ABC importers and large ABC importers. Biochemical and biophysical characterization leads us to the conclusion that, even within these three classes, the catalytic and transport mechanisms are not fully conserved, but continue to evolve. ABC transporters also exhibit unusual characteristics not observed in other primary transporters, such as uncoupled basal ATPase activity, that severely complicate mechanistic studies by established methods. In this chapter, I review these issues as related to ABC exporters in particular. A consensus view has emerged that ABC exporters follow alternating-access switch transport mechanisms. However, some biochemical data suggest that alternating catalytic site transport mechanisms are more appropriate for fully symmetrical ABC exporters. Heterodimeric and asymmetrical ABC exporters appear to conform to simple alternating-access-type mechanisms.

  9. The interaction of gut microbes with host ABC transporters

    PubMed Central

    Mercado-Lubo, Regino

    2010-01-01

    ATP binding cassette (ABC) transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, secretion and toxicity of xenobiotics. In addition to their essential function in drug resistance, there is also emerging evidence documenting the important role ABC transporters play in tissue defense. In this respect, the gastrointestinal tract represents a critical vanguard of defense against oral exposure of drugs while at the same time functions as a physical barrier between the lumenal contents (including bacteria) and the intestinal epithelium. Given emerging evidence suggesting that multidrug resistance protein (MDR) plays an important role in host-bacterial interactions in the gastrointestinal tract, this review will discuss the interplay between MDR of the intestinal epithelial cell barrier and gut microbes in health and disease. In particular, we will explore host-microbe interactions involving three apically restricted ABC transporters of the intestinal epithelium; P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2) and cystic fibrosis transmembrane regulator (CFTR). PMID:21327038

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

    PubMed

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

    2018-04-01

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

  11. Characterization of SiaA, a streptococcal heme-binding protein associated with a heme ABC transport system.

    PubMed

    Sook, Brian R; Block, Darci R; Sumithran, Suganya; Montañez, Griselle E; Rodgers, Kenton R; Dawson, John H; Eichenbaum, Zehava; Dixon, Dabney W

    2008-02-26

    Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The lipoprotein-anchored heme binding protein (HBP) of this transporter is SiaA (HtsA). In the current study, resonance Raman (rR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopies were used to determine the coordination state and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggest that the heme is six-coordinate and low-spin in both oxidation states of the protein, with methionine and histidine as axial ligands. SiaA has a pKa of 9.7 +/- 0.1, attributed to deprotonation of the axial histidine. Guanidinium titration studies show that the ferric state is less stable than the ferrous state, with DeltaG(H2O) values for the oxidized and reduced proteins of 7.3 +/- 0.8 and 16.0 +/- 3.6 kcal mol-1, respectively. The reductive and oxidative midpoint potentials determined via spectroelectrochemistry are 83 +/- 3 and 64 +/- 3 mV, respectively; the irreversibility of heme reduction suggests that redox cycling of the heme is coupled to a kinetically sluggish change in structure or conformation. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

  12. ABC Transporters Involved in Export of Cell Surface Glycoconjugates

    PubMed Central

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-01-01

    Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

  13. Transcriptome-Based Identification of ABC Transporters in the Western Tarnished Plant Bug Lygus hesperus

    PubMed Central

    Hull, J. Joe; Chaney, Kendrick; Geib, Scott M.; Fabrick, Jeffrey A.; Brent, Colin S.; Walsh, Douglas; Lavine, Laura Corley

    2014-01-01

    ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic clearance. While ABC transporters have been extensively studied in vertebrates, less is known concerning this superfamily in insects, particularly hemipteran pests. We used RNA-Seq transcriptome sequencing to identify 65 putative ABC transporter sequences (including 36 full-length sequences) from the eight ABC subfamilies in the western tarnished plant bug (Lygus hesperus), a polyphagous agricultural pest. Phylogenetic analyses revealed clear orthologous relationships with ABC transporters linked to insecticide/xenobiotic clearance and indicated lineage specific expansion of the L. hesperus ABCG and ABCH subfamilies. The transcriptional profile of 13 LhABCs representative of the ABCA, ABCB, ABCC, ABCG, and ABCH subfamilies was examined across L. hesperus development and within sex-specific adult tissues. All of the transcripts were amplified from both reproductively immature and mature adults and all but LhABCA8 were expressed to some degree in eggs. Expression of LhABCA8 was spatially localized to the testis and temporally timed with male reproductive development, suggesting a potential role in sexual maturation and/or spermatozoa protection. Elevated expression of LhABCC5 in Malpighian tubules suggests a possible role in xenobiotic clearance. Our results provide the first transcriptome-wide analysis of ABC transporters in an agriculturally important hemipteran pest and, because ABC transporters are known to be important mediators of insecticidal resistance, will provide the basis for future biochemical and toxicological studies on the role of this protein family in insecticide resistance in Lygus species. PMID:25401762

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

    PubMed Central

    Andreoletti, Pierre; Raas, Quentin; Gondcaille, Catherine; Cherkaoui-Malki, Mustapha; Trompier, Doriane; Savary, Stéphane

    2017-01-01

    The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85  Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues. PMID:28737695

  15. Structure of a Type-1 Secretion System ABC Transporter.

    PubMed

    Morgan, Jacob L W; Acheson, Justin F; Zimmer, Jochen

    2017-03-07

    Type-1 secretion systems (T1SSs) represent a widespread mode of protein secretion across the cell envelope in Gram-negative bacteria. The T1SS is composed of an inner-membrane ABC transporter, a periplasmic membrane-fusion protein, and an outer-membrane porin. These three components assemble into a complex spanning both membranes and providing a conduit for the translocation of unfolded polypeptides. We show that ATP hydrolysis and assembly of the entire T1SS complex is necessary for protein secretion. Furthermore, we present a 3.15-Å crystal structure of AaPrtD, the ABC transporter found in the Aquifex aeolicus T1SS. The structure suggests a substrate entry window just above the transporter's nucleotide binding domains. In addition, highly kinked transmembrane helices, which frame a narrow channel not observed in canonical peptide transporters, are likely involved in substrate translocation. Overall, the AaPrtD structure supports a polypeptide transport mechanism distinct from alternating access. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Transcriptomic insights on the ABC transporter gene family in the salmon louse Caligus rogercresseyi.

    PubMed

    Valenzuela-Muñoz, Valentina; Sturm, Armin; Gallardo-Escárate, Cristian

    2015-04-09

    ATP-binding cassette (ABC) protein family encode for membrane proteins involved in the transport of various biomolecules through the cellular membrane. These proteins have been identified in all taxa and present important physiological functions, including the process of insecticide detoxification in arthropods. For that reason the ectoparasite Caligus rogercresseyi represents a model species for understanding the molecular underpinnings involved in insecticide drug resistance. llumina sequencing was performed using sea lice exposed to 2 and 3 ppb of deltamethrin and azamethiphos. Contigs obtained from de novo assembly were annotated by Blastx. RNA-Seq analysis was performed and validated by qPCR analysis. From the transcriptome database of C. rogercresseyi, 57 putative members of ABC protein sequences were identified and phylogenetically classified into the eight subfamilies described for ABC transporters in arthropods. Transcriptomic profiles for ABC proteins subfamilies were evaluated throughout C. rogercresseyi development. Moreover, RNA-Seq analysis was performed for adult male and female salmon lice exposed to the delousing drugs azamethiphos and deltamethrin. High transcript levels of the ABCB and ABCC subfamilies were evidenced. Furthermore, SNPs mining was carried out for the ABC proteins sequences, revealing pivotal genomic information. The present study gives a comprehensive transcriptome analysis of ABC proteins from C. rogercresseyi, providing relevant information about transporter roles during ontogeny and in relation to delousing drug responses in salmon lice. This genomic information represents a valuable tool for pest management in the Chilean salmon aquaculture industry.

  17. Classification of a Haemophilus influenzae ABC Transporter HI1470/71 through Its Cognate Molybdate Periplasmic Binding Protein, MolA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C.

    2014-10-02

    molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB{sub 2}C{sub 2} (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 {angstrom} resolution, respectively. The MolA-binding protein binds molybdate and tungstate, but not other oxyanions such as sulfate and phosphate, making it the first class III molybdate-binding protein structurally solved. The {approx}100 {mu}M binding affinity for tungstate and molybdate is significantly lower than observed for the class II ModA molybdate-binding proteins that have nanomolar to low micromolar affinity for molybdate.more » The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.« less

  18. ABC-B transporter genes in Dirofilaria immitis.

    PubMed

    Bourguinat, Catherine; Che, Hua; Mani, Thangadurai; Keller, Kathy; Prichard, Roger K

    2016-08-01

    Dirofilaria immitis is a filarial nematode causing infection and heartworm disease in dogs and other canids, cats, and occasionally in humans. Prevention with macrocyclic lactones (ML) is recommended during the mosquito transmission season. Recently, ML resistance has been reported. ABC-B transporter genes are thought to be involved in the mechanism of ML resistance in other nematodes. This study aimed to identify all the ABC-B transporter genes in D. immitis using as a reference the nDi.2.2 D. immitis whole genome, which is not completely annotated. Using bioinformatic tools and PCR amplification on pooled D. immitis genomic DNA and on pooled cDNA, nine ABC transporter genes including one pseudogene were characterized. Bioinformatic and phylogenetic analyses allowed identification of three P-glycoproteins (Pgps) (Dim-pgp-3 Dim-pgp-10, Dim-pgp-11), of two ABC-B half transporter genes (one ortholog of Cel-haf-4 and Cel-haf-9; and one ortholog of Cel-haf-1 and Cel-haf-3), of one ABC half transporter gene (ortholog of Cel-haf-5) that contained an ABC-C motif, and of one additional half transporter that would require functional study for characterization. The number of ABC-B transporter genes identified was lower than in Caenorhabditis elegans and Haemonchus contortus. Further studies are needed to understand their possible role in ML resistance in D. immitis. These ABC transporters constitute a base for ML resistance investigation in D. immitis and advance our understanding of the molecular biology of this parasite. Copyright © 2016. Published by Elsevier Ltd.

  19. Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling.

    PubMed

    Jones, P M; George, A M

    2005-04-30

    Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein.

  20. Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective

    PubMed Central

    Greene, Nicholas P.; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

    2018-01-01

    The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking. PMID:29892271

  1. Antibiotic Resistance Mediated by the MacB ABC Transporter Family: A Structural and Functional Perspective.

    PubMed

    Greene, Nicholas P; Kaplan, Elise; Crow, Allister; Koronakis, Vassilis

    2018-01-01

    The MacB ABC transporter forms a tripartite efflux pump with the MacA adaptor protein and TolC outer membrane exit duct to expel antibiotics and export virulence factors from Gram-negative bacteria. Here, we review recent structural and functional data on MacB and its homologs. MacB has a fold that is distinct from other structurally characterized ABC transporters and uses a unique molecular mechanism termed mechanotransmission. Unlike other bacterial ABC transporters, MacB does not transport substrates across the inner membrane in which it is based, but instead couples cytoplasmic ATP hydrolysis with transmembrane conformational changes that are used to perform work in the extra-cytoplasmic space. In the MacAB-TolC tripartite pump, mechanotransmission drives efflux of antibiotics and export of a protein toxin from the periplasmic space via the TolC exit duct. Homologous tripartite systems from pathogenic bacteria similarly export protein-like signaling molecules, virulence factors and siderophores. In addition, many MacB-like ABC transporters do not form tripartite pumps, but instead operate in diverse cellular processes including antibiotic sensing, cell division and lipoprotein trafficking.

  2. Sensitive and Specific Fluorescent Probes for Functional Analysis of the Three Major Types of Mammalian ABC Transporters

    PubMed Central

    Lebedeva, Irina V.; Pande, Praveen; Patton, Wayne F.

    2011-01-01

    An underlying mechanism for multi drug resistance (MDR) is up-regulation of the transmembrane ATP-binding cassette (ABC) transporter proteins. ABC transporters also determine the general fate and effect of pharmaceutical agents in the body. The three major types of ABC transporters are MDR1 (P-gp, P-glycoprotein, ABCB1), MRP1/2 (ABCC1/2) and BCRP/MXR (ABCG2) proteins. Flow cytometry (FCM) allows determination of the functional expression levels of ABC transporters in live cells, but most dyes used as indicators (rhodamine 123, DiOC2(3), calcein-AM) have limited applicability as they do not detect all three major types of ABC transporters. Dyes with broad coverage (such as doxorubicin, daunorubicin and mitoxantrone) lack sensitivity due to overall dimness and thus may yield a significant percentage of false negative results. We describe two novel fluorescent probes that are substrates for all three common types of ABC transporters and can serve as indicators of MDR in flow cytometry assays using live cells. The probes exhibit fast internalization, favorable uptake/efflux kinetics and high sensitivity of MDR detection, as established by multidrug resistance activity factor (MAF) values and Kolmogorov-Smirnov statistical analysis. Used in combination with general or specific inhibitors of ABC transporters, both dyes readily identify functional efflux and are capable of detecting small levels of efflux as well as defining the type of multidrug resistance. The assay can be applied to the screening of putative modulators of ABC transporters, facilitating rapid, reproducible, specific and relatively simple functional detection of ABC transporter activity, and ready implementation on widely available instruments. PMID:21799851

  3. Investigation of the quaternary structure of an ABC transporter in living cells using spectrally resolved resonance energy transfer

    NASA Astrophysics Data System (ADS)

    Singh, Deo Raj

    Forster resonance energy transfer (FRET) has become an important tool to study proteins inside living cells. It has been used to explore membrane protein folding and dynamics, determine stoichiometry and geometry of protein complexes, and measure the distance between two molecules. In this dissertation, we use a method based on FRET and optical micro-spectroscopy (OptiMiS) technology, developed in our lab, to probe the structure of dynamic (as opposed to static) protein complexes in living cells. We use this method to determine the association stoichiometry and quaternary structure of an ABC transporter in living cells. Specifically, the transporter we investigate originates from the pathogen Pseudomonas aeruginosa, which is a Gram-negative bacterium with several virulence factors, lipopolysaccharides being one of them. This pathogen coexpresses two unique forms of lipopolysaccharides on its surface, the A- and B-bands. The A-band polysaccharides, synthesized in the cytoplasm, are translocated into the periplasm through an ATP-binding-cassette (ABC) transporter consisting of a transmembranar protein, Wzm, and a nucleotide-binding protein, Wzt. In P. aeruginosa, all of the biochemical studies of A-band LPS are concentrated on the stages of the synthesis and ligation of polysaccharides (PSs), leaving the export stage involving ABC transporter unexplored. The mode of PS export through ABC transporters is still unknown. This difficulty is due to the lack of information about sub-unit composition and structure of this bi-component ABC transporter. Using the FRET-OptiMiS combination method developed by our lab, we found that Wzt forms a rhombus-shaped homo-tetramer which becomes a square upon co-expression with Wzm, and that Wzm forms a square-shaped homo-tetramer both in the presence and absence of Wzt. Based on these results, we propose a structural model for the double-tetramer complex formed by the bi-component ABC transporter in living cells. An understanding of the

  4. ATP-binding cassette (ABC) transporters in caprine preantral follicles: gene and protein expression.

    PubMed

    Guerreiro, Denise Damasceno; de Lima, Laritza Ferreira; Mbemya, Gildas Tetaping; Maside, Carolina Mielgo; Miranda, André Marrocos; Tavares, Kaio César Simiano; Alves, Benner Geraldo; Faustino, Luciana Rocha; Smitz, Johan; de Figueiredo, José Ricardo; Rodrigues, Ana Paula Ribeiro

    2018-06-01

    The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.

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

    PubMed

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

    2014-07-29

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

  6. Functional Dependence between Septal Protein SepJ from Anabaena sp. Strain PCC 7120 and an Amino Acid ABC-Type Uptake Transporter.

    PubMed

    Escudero, Leticia; Mariscal, Vicente; Flores, Enrique

    2015-08-01

    In the diazotrophic filaments of heterocyst-forming cyanobacteria, two different cell types, the CO2-fixing vegetative cells and the N2-fixing heterocysts, exchange nutrients, including some amino acids. In the model organism Anabaena sp. strain PCC 7120, the SepJ protein, composed of periplasmic and integral membrane (permease) sections, is located at the intercellular septa joining adjacent cells in the filament. The unicellular cyanobacterium Synechococcus elongatus strain PCC 7942 bears a gene, Synpcc7942_1024 (here designated dmeA), encoding a permease homologous to the SepJ permease domain. Synechococcus strains lacking dmeA or lacking dmeA and expressing Anabaena sepJ were constructed. The Synechococcus dmeA mutant showed a significant 22 to 32% decrease in the uptake of aspartate, glutamate, and glutamine, a phenotype that could be partially complemented by Anabaena sepJ. Synechococcus mutants of an ATP-binding-cassette (ABC)-type transporter for polar amino acids showed >98% decreased uptake of glutamate irrespective of the presence of dmeA or Anabaena sepJ in the same strain. Thus, Synechococcus DmeA or Anabaena SepJ is needed to observe full (or close to full) activity of the ABC transporter. An Anabaena sepJ deletion mutant was significantly impaired in glutamate and aspartate uptake, which also in this cyanobacterium requires the activity of an ABC-type transporter for polar amino acids. SepJ appears therefore to generally stimulate the activity of cyanobacterial ABC-type transporters for polar amino acids. Conversely, an Anabaena mutant of three ABC-type transporters for amino acids was impaired in the intercellular transfer of 5-carboxyfluorescein, a SepJ-related property. Our results unravel possible functional interactions in transport elements important for diazotrophic growth. Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular molecular exchange in

  7. Transcriptome-based identification of ABC transporters in the western tarnished plant bug lygus hesperus

    USDA-ARS?s Scientific Manuscript database

    ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate diverse physiological functions by coupling ATP hydrolysis with substrate transport across lipid membranes. In insects, these proteins play roles in metabolism, development, eye pigmentation, and xenobiotic cle...

  8. Placental ABC Transporters: Biological Impact and Pharmaceutical Significance.

    PubMed

    Joshi, Anand A; Vaidya, Soniya S; St-Pierre, Marie V; Mikheev, Andrei M; Desino, Kelly E; Nyandege, Abner N; Audus, Kenneth L; Unadkat, Jashvant D; Gerk, Phillip M

    2016-12-01

    The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy.

  9. Placental ABC Transporters: Biological Impact and Pharmaceutical Significance

    PubMed Central

    Joshi, Anand A.; Vaidya, Soniya S.; St-Pierre, Marie V.; Mikheev, Andrei M.; Desino, Kelly E.; Nyandege, Abner N.; Audus, Kenneth L.; Unadkat, Jashvant D.; Gerk, Phillip M.

    2017-01-01

    The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy. PMID:27644937

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

    PubMed

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

    2017-04-01

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

  11. ABC transporters and immunity: mechanism of self-defense.

    PubMed

    Hinz, Andreas; Tampé, Robert

    2012-06-26

    The transporter associated with antigen processing (TAP) is a prototype of an asymmetric ATP-binding cassette (ABC) transporter, which uses ATP binding and hydrolysis to translocate peptides from the cytosol to the lumen of the endoplasmic reticulum (ER). Here, we review molecular details of peptide binding and ATP binding and hydrolysis as well as the resulting allosteric cross-talk between the nucleotide-binding domains and the transmembrane domains that drive translocation of the solute across the ER membrane. We also discuss the general molecular architecture of ABC transporters and demonstrate the importance of structural and functional studies for a better understanding of the role of the noncanonical site of asymmetric ABC transporters. Several aspects of peptide binding and specificity illustrate details of peptide translocation by TAP. Furthermore, this ABC transporter forms the central part of the major histocompatibility complex class I (MHC I) peptide-loading machinery. Hence, TAP is confronted with a number of viral factors, which prevent antigen translocation and MHC I loading in virally infected cells. We review how these viral factors have been used as molecular tools to decipher mechanistic aspects of solute translocation and discuss how they can help in the structural analysis of TAP.

  12. ABC transporters affect the elimination and toxicity of CdTe quantum dots in liver and kidney cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Mingli; Yin, Huancai; Bai, Pengli

    This paper aimed to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters on the efflux and the toxicity of nanoparticles in liver and kidney cells. In this study, we synthesized CdTe quantum dots (QDs) that were monodispersed and emitted green fluorescence (maximum peak at 530 nm). Such QDs tended to accumulate in human hepatocellular carcinoma cells (HepG2), human kidney cells 2 (HK-2), and Madin-Darby canine kidney (MDCK) cells, and cause significant toxicity in all the three cell lines. Using specific inhibitors and inducers of P-glycoprotein (Pgp) and multidrug resistance associated proteins (Mrps), the cellular accumulation and subsequent toxicity ofmore » QDs in HepG2 and HK-2 cells were significantly affected, while only slight changes appeared in MDCK cells, corresponding well with the functional expressions of ABC transporters in cells. Moreover, treatment of QDs caused concentration- and time- dependent induction of ABC transporters in HepG2 and HK-2 cells, but such phenomenon was barely found in MDCK cells. Furthermore, the effects of CdTe QDs on ABC transporters were found to be greater than those of CdCl{sub 2} at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd{sup 2+} and specific properties of QDs. Overall, these results indicated a strong dependence between the functional expressions of ABC transporters and the efflux of QDs, which could be an important reason for the modulation of QDs toxicity by ABC transporters. - Highlights: • ABC transporters contributed actively to the cellular efflux of CdTe quantum dots. • ABC transporters affected the cellular toxicity of CdTe quantum dots. • Treatment of CdTe quantum dots induced the gene expression of ABC transporters. • Free Cd{sup 2+} should be partially involved in the effects of QDs on ABC transporters. • Cellular efflux of quantum dots could be an important modulator for its toxicity.« less

  13. Diversity in ABC transporters: Type I, II and III importers

    PubMed Central

    Rice, Austin J.; Park, Aekyung

    2014-01-01

    ATP-binding cassette transporters are multi-subunit membrane pumps that transport substrates across membranes. While significant in the transport process, transporter architecture exhibits a range of diversity that we are only beginning to recognize. This divergence may provide insight into the mechanisms of substrate transport and homeostasis. Until recently, ABC importers have been classified into two types, but with the emergence of energy-coupling factor (ECF) transporters there are potentially three types of ABC importers. In this review, we summarize an expansive body of research on the three types of importers with an emphasis on the basics that underlie ABC importers, such as structure, subunit composition and mechanism. PMID:25155087

  14. ALS3 encodes a phloem-localized ABC transporter-like protein that is required for aluminum tolerance in Arabidopsis.

    PubMed

    Larsen, Paul B; Geisler, Matt J B; Jones, Carol A; Williams, Kelly M; Cancel, Jesse D

    2005-02-01

    Aluminum (Al) toxicity in acid soils is a worldwide agricultural problem that severely limits crop productivity through inhibition of root growth. Previously, Arabidopsis mutants with increased Al sensitivity were isolated in order to identify genes important for Al tolerance in plants. One mutant, als3, exhibited extreme root growth inhibition in the presence of Al, suggesting that this mutation negatively impacts a gene required for Al tolerance. Map-based cloning of the als3-1 mutation resulted in the isolation of a novel gene that encodes a previously undescribed ABC transporter-like protein, which is highly homologous to a putative bacterial metal resistance protein, ybbM. Northern analysis for ALS3 expression revealed that it is found in all organs examined, which is consistent with the global nature of Al sensitivity displayed by als3, and that expression increases in roots following Al treatment. Based on GUS fusion and in situ hybridization analyses, ALS3 is primarily expressed in leaf hydathodes and the phloem throughout the plant, along with the root cortex following Al treatment. Immunolocalization indicates that ALS3 predominantly accumulates in the plasma membrane of cells that express ALS3. From our results, it appears that ALS3 encodes an ABC transporter-like protein that is required for Al resistance/tolerance and may function to redistribute accumulated Al away from sensitive tissues in order to protect the growing root from the toxic effects of Al.

  15. The Yeast Plasma Membrane ATP Binding Cassette (ABC) Transporter Aus1

    PubMed Central

    Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L.; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

    2011-01-01

    The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter. PMID:21521689

  16. ABC transporter Cdr1p harbors charged residues in the intracellular loop and nucleotide-binding domain critical for protein trafficking and drug resistance.

    PubMed

    Shah, Abdul Haseeb; Banerjee, Atanu; Rawal, Manpreet Kaur; Saxena, Ajay Kumar; Mondal, Alok Kumar; Prasad, Rajendra

    2015-08-01

    The ABC transporter Cdr1 protein of Candida albicans, which plays a major role in antifungal resistance, has two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). The 12 transmembrane helices of TMDs that are interconnected by extracellular and intracellular loops (ICLs) mainly harbor substrate recognition sites where drugs bind while cytoplasmic NBDs hydrolyze ATP which powers drug efflux. The coupling of ATP hydrolysis to drug transport requires proper communication between NBDs and TMDs typically accomplished by ICLs. This study examines the role of cytoplasmic ICLs of Cdr1p by rationally predicting the critical residues on the basis of their interatomic distances. Among nine pairs that fall within a proximity of <4 Å, an ion pair between K577 of ICL1 and E315 of NBD1 was found to be critical. The substitution, swapping and changing of the length or charge of K577 or E315 by directed mutagenesis led to a misfolded, non-rescuable protein entrapped in intracellular structures. Furthermore, the equipositional ionic pair-forming residues from ICL3 and NBD2 (R1260 and E1014) did not impact protein trafficking. These results point to a new role for ICL/NBD interacting residues in PDR ABC transporters in protein folding and trafficking. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Effect of β-elemene on the kinetics of intracellular transport of d-luciferin potassium salt (ABC substrate) in doxorubicin-resistant breast cancer cells and the associated molecular mechanism.

    PubMed

    Tang, Chao-Yuan; Zhu, Li-Xin; Yu, Jian-Dong; Chen, Zhi; Gu, Man-Cang; Mu, Chao-Feng; Liu, Qi; Xiong, Yang

    2018-07-30

    In order to explore the mechanism of the reversing multidrug resistance (MDR) phenotypes by β-elemene (β-ELE) in doxorubicin (DOX)-resistant breast cancer cells (MCF-7/DOX), both the functionality and quantity of the ABC transporters in MCF-7/DOX were studied. Bioluminescence imaging (BLI) was used to study the efflux of d-luciferin potassium salt, the substrate of ATP-binding cassette transporters (ABC transporters), in MCF-7/DOX cells treated by β-ELE. At the same time three major ABC transport proteins and genes-related MDR, P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1) as well as breast cancer resistance protein (BCRP, ABCG2) were analyzed by q-PCR and Western blot. To investigate the efflux functionality of ABC transporters, MCF-7/DOX Fluc cell line with stably-overexpressed luciferase was established. BLI was then used to real-time monitor the efflux kinetics of d-luciferin potassium salt before and after MCF-7/DOX Fluc cells being treated with β-ELE or not. The results showed that the efflux of d-luciferin potassium salt from MCF-7/DOX Fluc was lessened when pretreated with β-ELE, which means that β-ELE may dampen the functionality of ABC transporters, thus decrease the efflux of d-fluorescein potassium or other chemotherapies which also serve as the substrates of ABC transporters. As the effect of β-ELE on the expression of ABC transporters, the results of q-PCR and Western blot showed that gene and protein expression of ABC transporters such as P-gp, MRP, and BCRP were down-regulated after the treatment of β-ELE. To verify the efficacy of β-ELE on reversing MDR, MCF-7/DOX cells were treated with the combination of DOX and β-ELE. MTT assay showed that β-ELE increased the inhibitory effect of DOX on the proliferation of MCF-7/DOX, and the IC 50 of the combination group was much lower than that of the single DOX or β-ELE treatment. In all, β-ELE may reverse MDR through the substrates of ABC transporters

  18. ATP binding cassette (ABC) transporters: expression and clinical value in glioblastoma.

    PubMed

    Dréan, Antonin; Rosenberg, Shai; Lejeune, François-Xavier; Goli, Larissa; Nadaradjane, Aravindan Arun; Guehennec, Jérémy; Schmitt, Charlotte; Verreault, Maïté; Bielle, Franck; Mokhtari, Karima; Sanson, Marc; Carpentier, Alexandre; Delattre, Jean-Yves; Idbaih, Ahmed

    2018-03-08

    ATP-binding cassette transporters (ABC transporters) regulate traffic of multiple compounds, including chemotherapeutic agents, through biological membranes. They are expressed by multiple cell types and have been implicated in the drug resistance of some cancer cells. Despite significant research in ABC transporters in the context of many diseases, little is known about their expression and clinical value in glioblastoma (GBM). We analyzed expression of 49 ABC transporters in both commercial and patient-derived GBM cell lines as well as from 51 human GBM tumor biopsies. Using The Cancer Genome Atlas (TCGA) cohort as a training dataset and our cohort as a validation dataset, we also investigated the prognostic value of these ABC transporters in newly diagnosed GBM patients, treated with the standard of care. In contrast to commercial GBM cell lines, GBM-patient derived cell lines (PDCL), grown as neurospheres in a serum-free medium, express ABC transporters similarly to parental tumors. Serum appeared to slightly increase resistance to temozolomide correlating with a tendency for an increased expression of ABCB1. Some differences were observed mainly due to expression of ABC transporters by microenvironmental cells. Together, our data suggest that the efficacy of chemotherapeutic agents may be misestimated in vitro if they are the targets of efflux pumps whose expression can be modulated by serum. Interestingly, several ABC transporters have prognostic value in the TCGA dataset. In our cohort of 51 GBM patients treated with radiation therapy with concurrent and adjuvant temozolomide, ABCA13 overexpression is associated with a decreased progression free survival in univariate (p < 0.01) and multivariate analyses including MGMT promoter methylation (p = 0.05) suggesting reduced sensitivity to temozolomide in ABCA13 overexpressing GBM. Expression of ABC transporters is: (i) detected in GBM and microenvironmental cells and (ii) better reproduced in GBM

  19. Surface charge-specific interactions between polymer nanoparticles and ABC transporters in Caco-2 cells

    NASA Astrophysics Data System (ADS)

    Bhattacharjee, Sourav; van Opstal, Edward J.; Alink, Gerrit M.; Marcelis, Antonius T. M.; Zuilhof, Han; Rietjens, Ivonne M. C. M.

    2013-06-01

    The surface charge-dependent transport of polymeric nanoparticles (PNPs) across Caco-2 monolayers grown on transwell culture systems as an in vitro model for intestinal transport was tested. The transport of well-characterized, monodisperse, and fluorescent tri-block copolymer nanoparticles (TCNPs/size 45 nm) and polystyrene nanoparticles (PSNPs/size 50 nm), with different surface charges (positive and negative), was quantified. The positive PNPs showed a higher intracellular uptake and flux across the Caco-2 monolayers than the negative PNPs. Multidrug resistance/P-glycoprotein (MDR1/P-gp), a specific ATP-binding cassette (ABC) transporter, was found to play a major role in the cellular efflux of positive PNPs, whereas the multidrug resistance protein 1 took part in the efflux of negative PNPs from Caco-2 cells. The positive PNPs also caused an increased cellular uptake and apical to basolateral transport of the carcinogen PhIP across the Caco-2 monolayer. The flavonoid quercetin, which is known to interact with ABC transporters, promoted the intracellular uptake of different PNPs and interfered with the normal distribution patterns of PNPs in the transwell system. These results indicate that PNPs display surface charge-specific interactions with ABC transporters and can even affect the bioavailability of toxic food-borne compounds (like pro-carcinogens).

  20. Structural basis for lipopolysaccharide extraction by ABC transporter LptB2FG.

    PubMed

    Luo, Qingshan; Yang, Xu; Yu, Shan; Shi, Huigang; Wang, Kun; Xiao, Le; Zhu, Guangyu; Sun, Chuanqi; Li, Tingting; Li, Dianfan; Zhang, Xinzheng; Zhou, Min; Huang, Yihua

    2017-05-01

    After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB 2 FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB 2 FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB 2 FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

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

    NASA Astrophysics Data System (ADS)

    Flechsig, Holger

    2016-02-01

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

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

    PubMed

    Chen, Lin; Duan, Kangmin

    2016-05-01

    Pseudomonas aeruginosa is an important human pathogen whose infections are difficult to treat due to its high intrinsic resistance to many antibiotics. Here, we show that the disruption of PA4456, encoding the ATP binding component of a putative ATP-binding cassette (ABC) transporter, increased the bacterium's susceptible to tetracycline and other antibiotics or toxic chemicals. Fluorescence spectroscopy and antibiotic accumulation tests showed that the interruption of the ABC transporter caused increased intracellular accumulation of tetracycline, demonstrating a role of the ABC transporter in tetracycline expulsion. Site-directed mutagenesis proved that the conserved residues of E170 in the Walker B motif and H203 in the H-loop, which are important for ATP hydrolysis, were essential for the function of PA4456. Through a genome-wide search, the PhoPQ two-component system was identified as a regulator of the computationally predicted PA4456-4452 operon that encodes the ABC transporter system. A >5-fold increase of the expression of this operon was observed in the phoQ mutant. The results obtained also show that the expression of the phzA1B1C1D1E1 operon and the production of pyocyanin were significantly higher in the ABC transporter mutant, signifying a connection between the ABC transporter and pyocyanin production. These results indicated that the PhoPQ-regulated ABC transporter is associated with intrinsic resistance to antibiotics and other adverse compounds in P. aeruginosa, probably by extruding them out of the cell. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Identification of ABC Transporter Genes of Fusarium graminearum with Roles in Azole Tolerance and/or Virulence

    PubMed Central

    Döll, Katharina; Karlovsky, Petr; Deising, Holger B.; Wirsel, Stefan G. R.

    2013-01-01

    Fusarium graminearum is a plant pathogen infecting several important cereals, resulting in substantial yield losses and mycotoxin contamination of the grain. Triazole fungicides are used to control diseases caused by this fungus on a worldwide scale. Our previous microarray study indicated that 15 ABC transporter genes were transcriptionally upregulated in response to tebuconazole treatment. Here, we deleted four ABC transporter genes in two genetic backgrounds of F. graminearum representing the DON (deoxynivalenol) and the NIV (nivalenol) trichothecene chemotypes. Deletion of FgABC3 and FgABC4 belonging to group I of ABC-G and to group V of ABC-C subfamilies of ABC transporters, respectively, considerably increased the sensitivity to the class I sterol biosynthesis inhibitors triazoles and fenarimol. Such effects were specific since they did not occur with any other fungicide class tested. Assessing the contribution of the four ABC transporters to virulence of F. graminearum revealed that, irrespective of their chemotypes, deletion mutants of FgABC1 (ABC-C subfamily group V) and FgABC3 were impeded in virulence on wheat, barley and maize. Phylogenetic context and analyses of mycotoxin production suggests that FgABC3 may encode a transporter protecting the fungus from host-derived antifungal molecules. In contrast, FgABC1 may encode a transporter responsible for the secretion of fungal secondary metabolites alleviating defence of the host. Our results show that ABC transporters play important and diverse roles in both fungicide resistance and pathogenesis of F. graminearum. PMID:24244413

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

    PubMed Central

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

    2016-01-01

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

  5. GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

    PubMed Central

    Zhao, Jinlei

    2014-01-01

    Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments. PMID:24957625

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

    PubMed

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

    2016-05-31

    The ATP-binding cassette (ABC) transporter gene superfamily is ubiquitous among extant organisms and prominently represented in plants. ABC transporters act to transport compounds across cellular membranes and are involved in a diverse range of biological processes. Thus, the applicability to biotechnology is vast, including cancer resistance in humans, drug resistance among vertebrates, and herbicide and other xenobiotic resistance in plants. In addition, plants appear to harbor the highest diversity of ABC transporter genes compared with any other group of organisms. This study applied transcriptome analysis to survey the kingdom-wide ABC transporter diversity in plants and suggest biotechnology applications of this diversity. We utilized sequence similarity-based informatics techniques to infer the identity of ABC transporter gene candidates from 1295 phylogenetically-diverse plant transcriptomes. A total of 97,149 putative (approximately 25 % were full-length) ABC transporter gene members were identified; each RNA-Seq library (plant sample) had 88 ± 30 gene members. As expected, simpler organisms, such as algae, had fewer unique members than vascular land plants. Differences were also noted in the richness of certain ABC transporter subfamilies. Land plants had more unique ABCB, ABCC, and ABCG transporter gene members on average (p < 0.005), and green algae, red algae, and bryophytes had significantly more ABCF transporter gene members (p < 0.005). Ferns had significantly fewer ABCA transporter gene members than all other plant groups (p < 0.005). We present a transcriptomic overview of ABC transporter gene members across all major plant groups. An increase in the number of gene family members present in the ABCB, ABCC, and ABCD transporter subfamilies may indicate an expansion of the ABC transporter superfamily among green land plants, which include all crop species. The striking difference between the number of ABCA subfamily transporter

  7. A Silent ABC Transporter Isolated from Streptomyces rochei F20 Induces Multidrug Resistance

    PubMed Central

    Fernández-Moreno, Miguel A.; Carbó, Lázaro; Cuesta, Trinidad; Vallín, Carlos; Malpartida, Francisco

    1998-01-01

    In the search for heterologous activators for actinorhodin production in Streptomyces lividans, 3.4 kb of DNA from Streptomyces rochei F20 (a streptothricin producer) were characterized. Subcloning experiments showed that the minimal DNA fragment required for activation was 0.4 kb in size. The activation is mediated by increasing the levels of transcription of the actII-ORF4 gene. Sequencing of the minimal activating fragment did not reveal any clues about its mechanism; nevertheless, it was shown to overlap the 3′ end of two convergent genes, one of whose translated products (ORF2) strongly resembles that of other genes belonging to the ABC transporter superfamily. Computer-assisted analysis of the 3.4-kb DNA sequence showed the 3′ terminus of an open reading frame (ORF), i.e., ORFA, and three complete ORFs (ORF1, ORF2, and ORFB). Searches in the databases with their respective gene products revealed similarities for ORF1 and ORF2 with ATP-binding proteins and transmembrane proteins, respectively, which are found in members of the ABC transporter superfamily. No similarities for ORFA and ORFB were found in the databases. Insertional inactivation of ORF1 and ORF2, their transcription analysis, and their cloning in heterologous hosts suggested that these genes were not expressed under our experimental conditions; however, cloning of ORF1 and ORF2 together (but not separately) under the control of an expressing promoter induced resistance to several chemically different drugs: oleandomycin, erythromycin, spiramycin, doxorubicin, and tetracycline. Thus, this genetic system, named msr, is a new bacterial multidrug ABC transporter. PMID:9696745

  8. ATP-induced conformational changes of nucleotide-binding domains in an ABC transporter. Importance of the water-mediated entropic force.

    PubMed

    Hayashi, Tomohiko; Chiba, Shuntaro; Kaneta, Yusuke; Furuta, Tadaomi; Sakurai, Minoru

    2014-11-06

    ATP binding cassette (ABC) proteins belong to a superfamily of active transporters. Recent experimental and computational studies have shown that binding of ATP to the nucleotide binding domains (NBDs) of ABC proteins drives the dimerization of NBDs, which, in turn, causes large conformational changes within the transmembrane domains (TMDs). To elucidate the active substrate transport mechanism of ABC proteins, it is first necessary to understand how the NBD dimerization is driven by ATP binding. In this study, we selected MalKs (NBDs of a maltose transporter) as a representative NBD and calculated the free-energy change upon dimerization using molecular mechanics calculations combined with a statistical thermodynamic theory of liquids, as well as a method to calculate the translational, rotational, and vibrational entropy change. This combined method is applied to a large number of snapshot structures obtained from molecular dynamics simulations containing explicit water molecules. The results suggest that the NBD dimerization proceeds with a large gain of water entropy when ATP molecules bind to the NBDs. The energetic gain arising from direct NBD-NBD interactions is canceled by the dehydration penalty and the configurational-entropy loss. ATP hydrolysis induces a loss of the shape complementarity between the NBDs, which leads to the dissociation of the dimer, due to a decrease in the water-entropy gain and an increase in the configurational-entropy loss. This interpretation of the NBD dimerization mechanism in concert with ATP, especially focused on the water-mediated entropy force, is potentially applicable to a wide variety of the ABC transporters.

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

    PubMed Central

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-01-01

    AIM: To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development. METHODS: Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1/Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of host-microbiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that

  10. Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane.

    PubMed

    van der Heide, T; Poolman, B

    2000-06-20

    An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein-lipid interactions.

  11. Drug resistance-associated changes in sphingolipids and ABC transporters occur in different regions of membrane domains.

    PubMed

    Hinrichs, John W J; Klappe, Karin; van Riezen, Manon; Kok, Jan W

    2005-11-01

    We have recently shown that two ATP binding cassette (ABC) transporters are enriched in Lubrol-resistant noncaveolar membrane domains in multidrug-resistant human cancer cells [Hinrichs, J. W. J., K. Klappe, I. Hummel, and J. W. Kok. 2004. ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells. J. Biol. Chem. 279: 5734-5738]. Here, we show that aminophospholipids are relatively enriched in Lubrol-resistant membrane domains compared with Triton X-100-resistant membrane domains, whereas sphingolipids are relatively enriched in the latter. Moreover, Lubrol-resistant membrane domains contain more protein and lipid mass. Based on these results, we postulate a model for detergent-insoluble glycosphingolipid-enriched membrane domains consisting of a Lubrol-insoluble/Triton X-100-insoluble region and a Lubrol-insoluble/Triton X-100-soluble region. The latter region contains most of the ABC transporters as well as lipids known to be necessary for their efflux activity. Compared with drug-sensitive cells, the detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in drug-resistant cells differ specifically in sphingolipid content and not in protein, phospholipid, or cholesterol content. In drug-resistant cells, sphingolipids with specific fatty acids (especially C24:1) are enriched in these membrane domains. Together, these data show that multidrug resistance-associated changes in both sphingolipids and ABC transporters occur in DIGs, but in different regions of these domains.

  12. ABC-F Proteins Mediate Antibiotic Resistance through Ribosomal Protection.

    PubMed

    Sharkey, Liam K R; Edwards, Thomas A; O'Neill, Alex J

    2016-03-22

    Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to anin vitrotranslation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosomein vitro To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection.IMPORTANCEAntimicrobial resistance ranks among the greatest threats currently facing human health. Elucidation of the mechanisms by which microorganisms resist the effect of antibiotics is central to understanding the biology of this phenomenon and has the potential to inform the development of new drugs capable of blocking or circumventing resistance. Members of the ABC-F family, which includelsa(A),msr(A),optr(A), andvga(A), collectively yield resistance to a broader range of clinically significant antibiotic classes than any other family of resistance determinants, although their mechanism of action has been controversial since their discovery 25 years ago. Here we present the first direct evidence that proteins of the ABC-F family act to protect the bacterial ribosome from antibiotic-mediated inhibition. Copyright © 2016 Sharkey et al.

  13. A lower isoelectric point increases signal sequence-mediated secretion of recombinant proteins through a bacterial ABC transporter.

    PubMed

    Byun, Hyunjong; Park, Jiyeon; Kim, Sun Chang; Ahn, Jung Hoon

    2017-12-01

    Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. Pseudomonas fluorescens has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF in vivo when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. ABC transporter activity linked to radiation resistance and molecular subtype in pediatric medulloblastoma

    PubMed Central

    2013-01-01

    Background Resistance to radiation treatment remains a major clinical problem for patients with brain cancer. Medulloblastoma is the most common malignant brain tumor of childhood, and occurs in the cerebellum. Though radiation treatment has been critical in increasing survival rates in recent decades, the presence of resistant cells in a substantial number of medulloblastoma patients leads to relapse and death. Methods Using the established medulloblastoma cell lines UW228 and Daoy, we developed a novel model system to enrich for and study radiation tolerant cells early after radiation exposure. Using fluorescence-activated cell sorting, dead cells and cells that had initiated apoptosis were removed, allowing surviving cells to be investigated before extensive proliferation took place. Results Isolated surviving cells were tumorigenic in vivo and displayed elevated levels of ABCG2, an ABC transporter linked to stem cell behavior and drug resistance. Further investigation showed another family member, ABCA1, was also elevated in surviving cells in these lines, as well as in early passage cultures from pediatric medulloblastoma patients. We discovered that the multi-ABC transporter inhibitors verapamil and reserpine sensitized cells from particular patients to radiation, suggesting that ABC transporters have a functional role in cellular radiation protection. Additionally, verapamil had an intrinsic anti-proliferative effect, with transient exposure in vitro slowing subsequent in vivo tumor formation. When expression of key ABC transporter genes was assessed in medulloblastoma tissue from 34 patients, levels were frequently elevated compared with normal cerebellum. Analysis of microarray data from independent cohorts (n = 428 patients) showed expression of a number of ABC transporters to be strongly correlated with certain medulloblastoma subtypes, which in turn are associated with clinical outcome. Conclusions ABC transporter inhibitors are already being

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

    PubMed

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

    2017-02-01

    The ATP-binding cassette (ABC) protein superfamily is one of the largest gene families and is highly conserved in all domains. The ABC proteins play roles in several biological processes, including multi-xenobiotic resistance (MXR), by functioning as transporters in the cellular membrane. They also mediate the cellular efflux of a wide range of substrates against concentration gradients. In this study, 37 ABC genes belonging to eight distinct subfamilies were identified in the marine copepod Paracyclopina nana and annotated based on a phylogenetic analysis. Also, the functions of P-glycoproteins (P-gp) and multidrug resistance-associated proteins (MRPs), conferring MXR, were verified using fluorescent substrates and specific inhibitors. The activities of MXR-mediated ABC proteins and their transcriptional level were examined in response to polyaromatic hydrocarbons (PAHs), main components of the water-accommodated fraction. This study increases the understanding of the protective role of MXR in response to PAHs over the comparative evolution of ABC gene families. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. An operon from Lactobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins.

    PubMed

    Varmanen, P; Rantanen, T; Palva, A

    1996-12-01

    A proline iminopeptidase gene (pepI) of an industrial Lactobacillus helveticus strain was cloned and found to be organized in an operon-like structure of three open reading frames (ORF1, ORF2 and ORF3). ORF1 was preceded by a typical prokaryotic promoter region, and a putative transcription terminator was found downstream of ORF3, identified as the pepI gene. Using primer-extension analyses, only one transcription start site, upstream of ORF1, was identifiable in the predicted operon. Although the size of mRNA could not be judged by Northern analysis either with ORF1-, ORF2- or pepI-specific probes, reverse transcription-PCR analyses further supported the operon structure of the three genes. ORF1, ORF2 and ORF3 had coding capacities for 50.7, 24.5 and 33.8 kDa proteins, respectively. The ORF3-encoded PepI protein showed 65% identity with the PepI proteins from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis. The ORF1-encoded protein had significant homology with several members of the ABC transporter family but, with two distinct putative ATP-binding sites, it would represent an unusual type among the bacterial ABC transporters. ORF2 encoded a putative integral membrane protein also characteristic of the ABC transporter family. The pepI gene was overexpressed in Escherichia coli. Purified PepI hydrolysed only di and tripeptides with proline in the first position. Optimum PepI activity was observed at pH 7.5 and 40 degrees C. A gel filtration analysis indicated that PepI is a dimer of M(r) 53,000. PepI was shown to be a metal-independent serine peptidase having thiol groups at or near the active site. Kinetic studies with proline-p-nitroanilide as substrate revealed Km and Vmax values of 0.8 mM and 350 mmol min-1 mg-1, respectively, and a very high turnover number of 135,000 s-1.

  17. Pharmacotherapy in pregnancy; effect of ABC and SLC transporters on drug transport across the placenta and fetal drug exposure.

    PubMed

    Staud, Frantisek; Cerveny, Lukas; Ceckova, Martina

    2012-11-01

    Pharmacotherapy during pregnancy is often inevitable for medical treatment of the mother, the fetus or both. The knowledge of drug transport across placenta is, therefore, an important topic to bear in mind when deciding treatment in pregnant women. Several drug transporters of the ABC and SLC families have been discovered in the placenta, such as P-glycoprotein, breast cancer resistance protein, or organic anion/cation transporters. It is thus evident that the passage of drugs across the placenta can no longer be predicted simply on the basis of their physical-chemical properties. Functional expression of placental drug transporters in the trophoblast and the possibility of drug-drug interactions must be considered to optimize pharmacotherapy during pregnancy. In this review we summarize current knowledge on the expression and function of ABC and SLC transporters in the trophoblast. Furthermore, we put this data into context with medical conditions that require maternal and/or fetal treatment during pregnancy, such as gestational diabetes, HIV infection, fetal arrhythmias and epilepsy. Proper understanding of the role of placental transporters should be of great interest not only to clinicians but also to pharmaceutical industry for future drug design and development to control the degree of fetal exposure.

  18. Functional Interaction between the Cytoplasmic ABC Protein LptB and the Inner Membrane LptC Protein, Components of the Lipopolysaccharide Transport Machinery in Escherichia coli

    PubMed Central

    Martorana, Alessandra M.; Benedet, Mattia; Maccagni, Elisa A.; Sperandeo, Paola; Villa, Riccardo; Dehò, Gianni

    2016-01-01

    ABSTRACT The assembly of lipopolysaccharide (LPS) in the outer leaflet of the outer membrane (OM) requires the transenvelope Lpt (lipopolysaccharide transport) complex, made in Escherichia coli of seven essential proteins located in the inner membrane (IM) (LptBCFG), periplasm (LptA), and OM (LptDE). At the IM, LptBFG constitute an unusual ATP binding cassette (ABC) transporter, composed by the transmembrane LptFG proteins and the cytoplasmic LptB ATPase, which is thought to extract LPS from the IM and to provide the energy for its export across the periplasm to the cell surface. LptC is a small IM bitopic protein that binds to LptBFG and recruits LptA via its N- and C-terminal regions, and its role in LPS export is not completely understood. Here, we show that the expression level of lptB is a critical factor for suppressing lethality of deletions in the C-terminal region of LptC and the functioning of a hybrid Lpt machinery that carries Pa-LptC, the highly divergent LptC orthologue from Pseudomonas aeruginosa. We found that LptB overexpression stabilizes C-terminally truncated LptC mutant proteins, thereby allowing the formation of a sufficient amount of stable IM complexes to support growth. Moreover, the LptB level seems also critical for the assembly of IM complexes carrying Pa-LptC which is otherwise defective in interactions with the E. coli LptFG components. Overall, our data suggest that LptB and LptC functionally interact and support a model whereby LptB plays a key role in the assembly of the Lpt machinery. IMPORTANCE The asymmetric outer membrane (OM) of Gram-negative bacteria contains in its outer leaflet an unusual glycolipid, the lipopolysaccharide (LPS). LPS largely contributes to the peculiar permeability barrier properties of the OM that prevent the entry of many antibiotics, thus making Gram-negative pathogens difficult to treat. In Escherichia coli the LPS transporter (the Lpt machine) is made of seven essential proteins (LptABCDEFG) that form a

  19. ABC Transporters and Isothiocyanates: Potential for Pharmacokinetic Diet–Drug Interactions

    PubMed Central

    Telang, Urvi; Ji, Yan; Morris, Marilyn E.

    2013-01-01

    Isothiocyanates, a class of anti-cancer agents, are derived from cruciferous vegetables such as broccoli, cabbage and watercress, and have demonstrated chemopreventive activity in a number of cancer models and epidemiologic studies. Due to public interest in cancer prevention and alternative therapies in cancer, the consumption of herbal supplements and vegetables containing these compounds is widespread and increasing. Isothiocyanates interact with ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein, MRP1, MRP2 and BCRP, and may influence the pharmacokinetics of substrates of these transporters. This review discusses the pharmacokinetic properties of isothiocyanates, their interactions with ABC transporters, and presents some data describing the potential for isothiocyanate-mediated diet–drug interactions. PMID:19623673

  20. ABCdb: an online resource for ABC transporter repertories from sequenced archaeal and bacterial genomes.

    PubMed

    Fichant, Gwennaele; Basse, Marie-Jeanne; Quentin, Yves

    2006-03-01

    The ATP-binding cassette (ABC) transporters are one of the major classes of active transporters. They are widespread in archaea, bacteria, and eukaryota, indicating that they have arisen early in evolution. They are involved in many essential physiological processes, but the majority import or export a wide variety of compounds across cellular membranes. These systems share a common architecture composed of four (exporters) or five (importers) domains. To identify and reconstruct functional ABC transporters encoded by archaeal and bacterial genomes, we have developed a bioinformatic strategy. Cross-reference to the transport classification system is used to predict the type of compound transported. A high quality of annotation is achieved by manual verification of the predictions. However, in order to face the rapid increase in the number of published genomes, we also include analyses of genomes issuing directly from the automated strategy. Querying the database (http://www-abcdb.biotoul.fr) allows to easily retrieve ABC transporter repertories and related data. Additional query tools have been developed for the analysis of the ABC family from both functional and evolutionary perspectives.

  1. The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo.

    PubMed

    Hyde, B B; Liesa, M; Elorza, A A; Qiu, W; Haigh, S E; Richey, L; Mikkola, H K; Schlaeger, T M; Shirihai, O S

    2012-07-01

    The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me-/- mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me-/- erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me-/- erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me-/- erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo.

  2. The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo

    PubMed Central

    Hyde, B B; Liesa, M; Elorza, A A; Qiu, W; Haigh, S E; Richey, L; Mikkola, H K; Schlaeger, T M; Shirihai, O S

    2012-01-01

    The mitochondrial transporter ATP binding cassette mitochondrial erythroid (ABC-me/ABCB10) is highly induced during erythroid differentiation by GATA-1 and its overexpression increases hemoglobin production rates in vitro. However, the role of ABC-me in erythropoiesis in vivo is unknown. Here we report for the first time that erythrocyte development in mice requires ABC-me. ABC-me−/− mice die at day 12.5 of gestation, showing nearly complete eradication of primitive erythropoiesis and lack of hemoglobinized cells at day 10.5. ABC-me−/− erythroid cells fail to differentiate because they exhibit a marked increase in apoptosis, both in vivo and ex vivo. Erythroid precursors are particularly sensitive to oxidative stress and ABC-me in the heart and its yeast ortholog multidrug resistance-like 1 have been shown to protect against oxidative stress. Thus, we hypothesized that increased apoptosis in ABC-me−/− erythroid precursors was caused by oxidative stress. Within this context, ABC-me deletion causes an increase in mitochondrial superoxide production and protein carbonylation in erythroid precursors. Furthermore, treatment of ABC-me−/− erythroid progenitors with the mitochondrial antioxidant MnTBAP (superoxide dismutase 2 mimetic) supports survival, ex vivo differentiation and increased hemoglobin production. Altogether, our findings demonstrate that ABC-me is essential for erythropoiesis in vivo. PMID:22240895

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

    PubMed

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

    2010-12-02

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

  4. Osmoregulated ABC-transport system of Lactococcus lactis senses water stress via changes in the physical state of the membrane

    PubMed Central

    van der Heide, Tiemen; Poolman, Bert

    2000-01-01

    An osmoregulated ABC transporter (OpuA) with novel structural features has been identified that responds to water stress. This glycine betaine transport system consists of an ATP-binding/hydrolyzing subunit (OpuAA) and a protein (OpuABC) that contains both the translocator and the substrate-binding domain. The components of OpuA have been overexpressed, purified, and functionally incorporated into liposomes with an ATP-regenerating system in the vesicle lumen. A transmembrane osmotic gradient (outside hyperosmotic relative to the inside) of both ionic and nonionic compounds was able to osmotically activate OpuA in the proteoliposomal system. Hypoosmotic medium conditions inhibited the basal activity of the system. The data show that OpuAA and OpuABC are sufficient for osmoregulated transport, indicating that OpuA can act both as osmosensor and osmoregulator. Strikingly, OpuA could also be activated by low concentrations of cationic and anionic amphipaths, which interact with the membrane. This result indicates that activation by a transmembrane osmotic gradient is mediated by changes in membrane properties/protein–lipid interactions. PMID:10860977

  5. Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

    PubMed

    Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

    2011-01-25

    Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11

  6. Effects of selected OATP and/or ABC transporter inhibitors on the brain and whole-body distribution of glyburide.

    PubMed

    Tournier, Nicolas; Saba, Wadad; Cisternino, Salvatore; Peyronneau, Marie-Anne; Damont, Annelaure; Goutal, Sébastien; Dubois, Albertine; Dollé, Frédéric; Scherrmann, Jean-Michel; Valette, Héric; Kuhnast, Bertrand; Bottlaender, Michel

    2013-10-01

    Glyburide (glibenclamide, GLB) is a widely prescribed antidiabetic with potential beneficial effects in central nervous system injury and diseases. In vitro studies show that GLB is a substrate of organic anion transporting polypeptide (OATP) and ATP-binding cassette (ABC) transporter families, which may influence GLB distribution and pharmacokinetics in vivo. In the present study, we used [(11)C]GLB positron emission tomography (PET) imaging to non-invasively observe the distribution of GLB at a non-saturating tracer dose in baboons. The role of OATP and P-glycoprotein (P-gp) in [(11)C]GLB whole-body distribution, plasma kinetics, and metabolism was assessed using the OATP inhibitor rifampicin and the dual OATP/P-gp inhibitor cyclosporine. Finally, we used in situ brain perfusion in mice to pinpoint the effect of ABC transporters on GLB transport at the blood-brain barrier (BBB). PET revealed the critical role of OATP on liver [(11)C]GLB uptake and its subsequent impact on [(11)C]GLB metabolism and plasma clearance. OATP-mediated uptake also occurred in the myocardium and kidney parenchyma but not the brain. The inhibition of P-gp in addition to OATP did not further influence [(11)C]GLB tissue and plasma kinetics. At the BBB, the inhibition of both P-gp and breast cancer resistance protein (BCRP) was necessary to demonstrate the role of ABC transporters in limiting GLB brain uptake. This study demonstrates that GLB distribution, metabolism, and elimination are greatly dependent on OATP activity, the first step in GLB hepatic clearance. Conversely, P-gp, BCRP, and probably multidrug resistance protein 4 work in synergy to limit GLB brain uptake.

  7. Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production.

    PubMed

    Steglich, Matthias; Hofmann, Julia D; Helmecke, Julia; Sikorski, Johannes; Spröer, Cathrin; Riedel, Thomas; Bunk, Boyke; Overmann, Jörg; Neumann-Schaal, Meina; Nübel, Ulrich

    2018-01-01

    We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p -cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile , with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile , we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291.

  8. Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production

    PubMed Central

    Steglich, Matthias; Hofmann, Julia D.; Helmecke, Julia; Sikorski, Johannes; Spröer, Cathrin; Riedel, Thomas; Bunk, Boyke; Overmann, Jörg; Neumann-Schaal, Meina; Nübel, Ulrich

    2018-01-01

    We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile, with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile, we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291. PMID:29867812

  9. Role of nuclear factor-erythroid 2-related factor 2 (Nrf2) in the transcriptional regulation of brain ABC transporters during acute acetaminophen (APAP) intoxication in mice.

    PubMed

    Ghanem, Carolina I; Rudraiah, Swetha; Bataille, Amy M; Vigo, María B; Goedken, Michael J; Manautou, José E

    2015-04-01

    Changes in expression of liver ABC transporters have been described during acute APAP intoxication. However, the effect of APAP on brain ABC transporters is poorly understood. The aim of this study was to evaluate the effect of APAP on brain ABC transporters expression and the role of the oxidative stress sensor Nrf2. Male C57BL/6J mice were administered APAP (400mg/kg) for analysis of brain mRNA and protein expression of Mrp1-6, Bcrp and P-gp. The results show induction of P-gp, Mrp2 and Mrp4 proteins, with no changes in Bcrp, Mrp1 or Mrp5-6. The protein values were accompanied by corresponding changes in mRNA levels. Additionally, brain Nrf2 nuclear translocation and expression of two Nrf2 target genes, quinone oxidoreductase 1 (Nqo1) and Hemoxygenase 1 (Ho-1), was evaluated at 6, 12 and 24h after APAP treatment. Nrf2 nuclear content increased by 58% at 12h after APAP along with significant increments in mRNA and protein expression of Nqo1 and Ho-1. Furthermore, APAP treated Nrf2 knockout mice did not increase mRNA or protein expression of Mrp2 and Mrp4 as observed in wildtypes. In contrast, P-gp induction by APAP was observed in both genotypes. In conclusion, acute APAP intoxication induces protein expression of brain P-gp, Mrp2 and Mrp4. This study also suggests that brain changes in Mrp2 and Mrp4 expression may be due to in situ Nrf2 activation by APAP, while P-gp induction is independent of Nrf2 function. The functional consequences of these changes in brain ABC transporters by APAP deserve further attention. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fuchs, Dominik; Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, D-69120 Heidelberg; Daniel, Volker

    2010-04-16

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

  11. The phytoestrogen genistein enhances multidrug resistance in breast cancer cell lines by translational regulation of ABC transporters.

    PubMed

    Rigalli, Juan Pablo; Tocchetti, Guillermo Nicolás; Arana, Maite Rocío; Villanueva, Silvina Stella Maris; Catania, Viviana Alicia; Theile, Dirk; Ruiz, María Laura; Weiss, Johanna

    2016-06-28

    Breast cancer is the most frequent malignancy in women. Multidrug resistance due to overexpression of ABC drug transporters is a common cause of chemotherapy failure and disease recurrence. Genistein (GNT) is a phytoestrogen present in soybeans and hormone supplements. We investigated the effect of GNT on the expression and function of ABC transporters in MCF-7 and MDA-MB-231 breast cancer cell lines. Results demonstrated an induction at the protein level of ABCC1 and ABCG2 and of ABCC1 in MCF-7 and MDA-MB-231, respectively. MCF-7 cells showed a concomitant increase in doxorubicin and mitoxantrone efflux and resistance, dependent on ABCG2 activity. ABCC1 induction by GNT in MDA-MB-231 cells modified neither drug efflux nor chemoresistance due to simultaneous acute inhibition of the transporter activity by GNT. All inductions took place at the translational level, as no increment in mRNA was observed and protein increase was prevented by cycloheximide. miR-181a, already demonstrated to inhibit ABCG2 translation, was down-regulated by GNT, explaining translational induction. Effects were independent of classical estrogen receptors. Results suggest potential nutrient-drug interactions that could threaten chemotherapy efficacy, especially in ABCG2-expressing tumors treated with substrates of this transporter. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Multiple ABC glucoside transporters mediate sugar-stimulated growth in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120.

    PubMed

    Nieves-Morión, Mercedes; Flores, Enrique

    2018-02-01

    Cyanobacteria are generally capable of photoautotrophic growth and are widely distributed on Earth. The model filamentous, heterocyst-forming strain Anabaena sp. PCC 7120 has long been considered as a strict photoautotroph but is now known to be able to assimilate fructose. We have previously described two components of ABC glucoside uptake transporters from Anabaena that are involved in uptake of the sucrose analog esculin: GlsC [a nucleotide-binding domain subunit (NBD)] and GlsP [a transmembrane component (TMD)]. Here, we created Anabaena mutants of genes encoding three further ABC transporter components needed for esculin uptake: GlsD (NBD), GlsQ (TMD) and GlsR (periplasmic substrate-binding protein). Phototrophic growth of Anabaena was significantly stimulated by sucrose, fructose and glucose. Whereas the glsC and glsD mutants were drastically hampered in sucrose-stimulated growth, the different gls mutants were generally impaired in sugar-dependent growth. Our results suggest the participation of Gls and other ABC transporters encoded in the Anabaena genome in sugar-stimulated growth. Additionally, Gls transporter components influence the function of septal junctions in the Anabaena filament. We suggest that mixotrophic growth is important in cyanobacterial physiology and may be relevant for the wide success of these organisms in diverse environments. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  13. Barley has two peroxisomal ABC transporters with multiple functions in β-oxidation

    PubMed Central

    Mendiondo, Guillermina M.; Medhurst, Anne; van Roermund, Carlo W.; Zhang, Xuebin; Devonshire, Jean; Scholefield, Duncan; Fernández, José; Axcell, Barry; Ramsay, Luke; Waterham, Hans R.; Waugh, Robbie; Theodoulou, Frederica L.; Holdsworth, Michael J.

    2014-01-01

    In oilseed plants, peroxisomal β-oxidation functions not only in lipid catabolism but also in jasmonate biosynthesis and metabolism of pro-auxins. Subfamily D ATP-binding cassette (ABC) transporters mediate import of β-oxidation substrates into the peroxisome, and the Arabidopsis ABCD protein, COMATOSE (CTS), is essential for this function. Here, the roles of peroxisomal ABCD transporters were investigated in barley, where the main storage compound is starch. Barley has two CTS homologues, designated HvABCD1 and HvABCD2, which are widely expressed and present in embryo and aleurone tissues during germination. Suppression of both genes in barley RNA interference (RNAi) lines indicated roles in metabolism of 2,4-dichlorophenoxybutyrate (2,4-DB) and indole butyric acid (IBA), jasmonate biosynthesis, and determination of grain size. Transformation of the Arabidopsis cts-1 null mutant with HvABCD1 and HvABCD2 confirmed these findings. HvABCD2 partially or completely complemented all tested phenotypes of cts-1. In contrast, HvABCD1 failed to complement the germination and establishment phenotypes of cts-1 but increased the sensitivity of hypocotyls to 100 μM IBA and partially complemented the seed size phenotype. HvABCD1 also partially complemented the yeast pxa1/pxa2Δ mutant for fatty acid β-oxidation. It is concluded that the core biochemical functions of peroxisomal ABC transporters are largely conserved between oilseeds and cereals but that their physiological roles and importance may differ. PMID:24913629

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

    PubMed

    Martin, Audrey; Daniel, Jaiyanth

    2018-02-05

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

  15. A bacterial-type ABC transporter is involved in aluminum tolerance in rice.

    PubMed

    Huang, Chao Feng; Yamaji, Naoki; Mitani, Namiki; Yano, Masahiro; Nagamura, Yoshiaki; Ma, Jian Feng

    2009-02-01

    Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall.

  16. Translocation of the ABC transporter ABCD4 from the endoplasmic reticulum to lysosomes requires the escort protein LMBD1.

    PubMed

    Kawaguchi, Kosuke; Okamoto, Takumi; Morita, Masashi; Imanaka, Tsuneo

    2016-07-26

    We previously demonstrated that ABCD4 does not localize to peroxisomes but rather, the endoplasmic reticulum (ER), because it lacks the NH2-terminal hydrophilic region required for peroxisomal targeting. It was recently reported that mutations in ABCD4 result in a failure to release vitamin B12 from lysosomes. A similar phenotype is caused by mutations in LMBRD1, which encodes the lysosomal membrane protein LMBD1. These findings suggested to us that ABCD4 translocated from the ER to lysosomes in association with LMBD1. In this report, it is demonstrated that ABCD4 interacts with LMBD1 and then localizes to lysosomes, and this translocation depends on the lysosomal targeting ability of LMBD1. Furthermore, endogenous ABCD4 was localized to both lysosomes and the ER, and its lysosomal localization was disturbed by knockout of LMBRD1. To the best of our knowledge, this is the first report demonstrating that the subcellular localization of the ABC transporter is determined by its association with an adaptor protein.

  17. Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts.

    PubMed

    Denecke, Shane; Fusetto, Roberto; Batterham, Philip

    2017-12-01

    ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC 50 values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi

    NASA Astrophysics Data System (ADS)

    Epis, Sara; Porretta, Daniele; Mastrantonio, Valentina; Urbanelli, Sandra; Sassera, Davide; De Marco, Leone; Mereghetti, Valeria; Montagna, Matteo; Ricci, Irene; Favia, Guido; Bandi, Claudio

    2014-12-01

    In insects, ABC transporters have been shown to contribute to defence/resistance to insecticides by reducing toxic concentrations in cells/tissues. Despite the extensive studies about this detoxifying mechanism, the temporal patterns of ABC transporter activation have been poorly investigated. Using the malaria vector Anopheles stephensi as a study system, we investigated the expression profile of ABC genes belonging to different subfamilies in permethrin-treated larvae at different time points (30 min to 48 h). Our results showed that the expression of ABCB and ABCG subfamily genes was upregulated at 1 h after treatment, with the highest expression observed at 6 h. Therefore, future investigations on the temporal dynamics of ABC gene expression will allow a better implementation of insecticide treatment regimens, including the use of specific inhibitors of ABC efflux pumps.

  19. Characterization of a novel domain ‘GATE’ in the ABC protein DrrA and its role in drug efflux by the DrrAB complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Han; Rahman, Sadia; Li, Wen

    2015-03-27

    A novel domain, GATE (Glycine-loop And Transducer Element), is identified in the ABC protein DrrA. This domain shows sequence and structural conservation among close homologs of DrrA as well as distantly-related ABC proteins. Among the highly conserved residues in this domain are three glycines, G215, G221 and G231, of which G215 was found to be critical for stable expression of the DrrAB complex. Other conserved residues, including E201, G221, K227 and G231, were found to be critical for the catalytic and transport functions of the DrrAB transporter. Structural analysis of both the previously published crystal structure of the DrrA homologmore » MalK and the modeled structure of DrrA showed that G215 makes close contacts with residues in and around the Walker A motif, suggesting that these interactions may be critical for maintaining the integrity of the ATP binding pocket as well as the complex. It is also shown that G215A or K227R mutation diminishes some of the atomic interactions essential for ATP catalysis and overall transport function. Therefore, based on both the biochemical and structural analyses, it is proposed that the GATE domain, located outside of the previously identified ATP binding and hydrolysis motifs, is an additional element involved in ATP catalysis. - Highlights: • A novel domain ‘GATE’ is identified in the ABC protein DrrA. • GATE shows high sequence and structural conservation among diverse ABC proteins. • GATE is located outside of the previously studied ATP binding and hydrolysis motifs. • Conserved GATE residues are critical for stability of DrrAB and for ATP catalysis.« less

  20. Inhibition of the Human ABC Efflux Transporters P-gp and ...

    EPA Pesticide Factsheets

    High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity.

  1. Molecular cloning and expression profile of an ATP-binding cassette (ABC) transporter gene from the hemipteran insect Nilaparvata lugens.

    PubMed

    Zha, W J; Li, S H; Zhou, L; Chen, Z J; Liu, K; Yang, G C; Hu, G; He, G C; You, A Q

    2015-03-30

    The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. In insects, ABC transporters have important functions in the transport of molecules, and are also involved in insecticide resistance, metabolism, and development. In this study, the Nilaparvata lugens Stal (Hemiptera: Delphacidae) ABCG (NlABCG) gene was identified and characterized. The complete mRNA sequence of NlABCG was 2608-bp long, with an open reading frame of 2064 bp encoding a protein comprised of 687 amino acids. The conserved regions include three N-glycosylation and 34 phosphorylation sites, as well as seven transmembrane domains. The amino acid identity with the closely related species Acyrthosiphon pisum was 42.8%. Developmental expression analysis using quantitative real-time reverse transcriptase PCR suggested that the NlABCG transcript was expressed at all developmental stages of N. lugens. The lowest expression of NlABCG was in the 1st instar, and levels increased with larval growth. The transcript profiles of NlABCG were analyzed in various tissues from a 5th instar nymph, and the highest expression was observed in the midgut. These results suggest that the sequence, characteristics, and expression of NlABCG are highly conserved, and basic information is provided for its functional analysis.

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

    PubMed Central

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

    2013-01-01

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

  3. Alzheimer's and ABC transporters--new opportunities for diagnostics and treatment.

    PubMed

    Pahnke, Jens; Langer, Oliver; Krohn, Markus

    2014-12-01

    Much has been said about the increasing number of demented patients and the main risk factor 'age'. Frustratingly, we do not know the precise pattern and all modulating factors that provoke the pathologic changes in the brains of affected elderly. We have to diagnose early to be able to stop the progression of diseases that irreversibly destroy brain substance. Familiar AD cases have mislead some researchers for almost 20 years, which has unfortunately narrowed the scientific understanding and has, thus, lead to insufficient funding of independent approaches. Therefore, basic researchers hardly have been able to develop causative treatments and clinicians still do not have access to prognostic and early diagnostic tools. During the recent years it became clear that insufficient Aβ export, physiologically facilitated by the ABC transporter superfamily at the brain's barriers, plays a fundamental role in disease initiation and progression. Furthermore, export mechanisms that are deficient in affected elderly are new targets for activation and, thus, treatment, but ideally also for prevention. In sporadic AD disturbed clearance of β-amyloid from the brain is so far the most important factor for its accumulation in the parenchyma and vessel walls. Here, we review findings about the contribution of ABC transporters and of the perivascular drainage/glymphatic system on β-amyloid clearance. We highlight their potential value for innovative early diagnostics using PET and describe recently described, effective ABC transporter-targeting agents as potential causative treatment for neurodegenerative proteopathies/dementias. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. The feoABC Locus of Yersinia pestis Likely Has Two Promoters Causing Unique Iron Regulation

    PubMed Central

    O'Connor, Lauren; Fetherston, Jacqueline D.; Perry, Robert D.

    2017-01-01

    The FeoABC ferrous transporter is a wide-spread bacterial system. While the feoABC locus is regulated by a number of factors in the bacteria studied, we have previously found that regulation of feoABC in Yersinia pestis appears to be unique. None of the non-iron responsive transcriptional regulators that control expression of feoABC in other bacteria do so in Y. pestis. Another unique factor is the iron and Fur regulation of the Y. pestis feoABC locus occurs during microaerobic but not aerobic growth. Here we show that this unique iron-regulation is not due to a unique aspect of the Y. pestis Fur protein but to DNA sequences that regulate transcription. We have used truncations, alterations, and deletions of the feoA::lacZ reporter to assess the mechanism behind the failure of iron to repress transcription under aerobic conditions. These studies plus EMSAs and DNA sequence analysis have led to our proposal that the feoABC locus has two promoters: an upstream P1 promoter whose expression is relatively iron-independent but repressed under microaerobic conditions and the known downstream Fur-regulated P2 promoter. In addition, we have identified two regions that bind Y. pestis protein(s), although we have not identified these protein(s) or their function. Finally we used iron uptake assays to demonstrate that both FeoABC and YfeABCD transport ferrous iron in an energy-dependent manner and also use ferric iron as a substrate for uptake. PMID:28785546

  5. Tungsten Transport Protein A (WtpA) in Pyrococcus furiosus: the First Member of a New Class of Tungstate and Molybdate Transporters

    PubMed Central

    Bevers, Loes E.; Hagedoorn, Peter-Leon; Krijger, Gerard C.; Hagen, Wilfred R.

    2006-01-01

    A novel tungstate and molybdate binding protein has been discovered from the hyperthermophilic archaeon Pyrococcus furiosus. This tungstate transport protein A (WtpA) is part of a new ABC transporter system selective for tungstate and molybdate. WtpA has very low sequence similarity with the earlier-characterized transport proteins ModA for molybdate and TupA for tungstate. Its structural gene is present in the genome of numerous archaea and some bacteria. The identification of this new tungstate and molybdate binding protein clarifies the mechanism of tungstate and molybdate transport in organisms that lack the known uptake systems associated with the ModA and TupA proteins, like many archaea. The periplasmic protein of this ABC transporter, WtpA (PF0080), was cloned and expressed in Escherichia coli. Using isothermal titration calorimetry, WtpA was observed to bind tungstate (dissociation constant [KD] of 17 ± 7 pM) and molybdate (KD of 11 ± 5 nM) with a stoichiometry of 1.0 mol oxoanion per mole of protein. These low KD values indicate that WtpA has a higher affinity for tungstate than do ModA and TupA and an affinity for molybdate similar to that of ModA. A displacement titration of molybdate-saturated WtpA with tungstate showed that the tungstate effectively replaced the molybdate in the binding site of the protein. PMID:16952940

  6. The yeast plasma membrane ATP binding cassette (ABC) transporter Aus1: purification, characterization, and the effect of lipids on its activity.

    PubMed

    Marek, Magdalena; Milles, Sigrid; Schreiber, Gabriele; Daleke, David L; Dittmar, Gunnar; Herrmann, Andreas; Müller, Peter; Pomorski, Thomas Günther

    2011-06-17

    The ATP binding cassette (ABC) transporter Aus1 is expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and is required for sterol uptake. These observations suggest that Aus1 promotes the translocation of sterols across membranes, but the precise transport mechanism has yet to be identified. In this study, an extraction and purification procedure was developed to characterize the Aus1 transporter. The detergent-solubilized protein was able to bind and hydrolyze ATP. Mutagenesis of the conserved lysine to methionine in the Walker A motif abolished ATP hydrolysis. Likewise, ATP hydrolysis was inhibited by classical inhibitors of ABC transporters. Upon reconstitution into proteoliposomes, the ATPase activity of Aus1 was specifically stimulated by phosphatidylserine (PS) in a stereoselective manner. We also found that Aus1-dependent sterol uptake, but not Aus1 expression and trafficking to the plasma membrane, was affected by changes in cellular PS levels. These results suggest a direct interaction between Aus1 and PS that is critical for the activity of the transporter.

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2013-01-01

    The ATP-binding cassette (ABC) protein superfamily constitutes one of the largest protein families known in plants. In this report, we performed a complete inventory of ABC protein genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with ABC protein members of Arabidopsis thaliana, we identified 135 putative ABC proteins with 1 or 2 NBDs in V. vinifera. Of these, 120 encode intrinsic membrane proteins, and 15 encode proteins missing TMDs. V. vinifera ABC proteins can be divided into 13 subfamilies with 79 “full-size,” 41 “half-size,” and 15 “soluble” putative ABC proteins. The main feature of the Vitis ABC superfamily is the presence of 2 large subfamilies, ABCG (pleiotropic drug resistance and white-brown complex homolog) and ABCC (multidrug resistance-associated protein). We identified orthologs of V. vinifera putative ABC transporters in different species. This work represents the first complete inventory of ABC transporters in V. vinifera. The identification of Vitis ABC transporters and their comparative analysis with the Arabidopsis counterparts revealed a strong conservation between the 2 species. This inventory could help elucidate the biological and physiological functions of these transporters in V. vinifera. PMID:24244377

  9. Alzheimer’s and ABC transporters - new opportunities for diagnostics and treatment

    PubMed Central

    Pahnke, Jens; Langer, Oliver; Krohn, Markus

    2014-01-01

    Much has been said about the increasing number of demented patients and the main risk factor ‘age’. Frustratingly, we do not know the precise pattern and all modulating factors that provoke the pathologic changes in the brains of affected elderly. We have to diagnose early to be able to stop the progression of diseases that irreversibly destroy brain substance. Familiar AD cases have mislead some researchers for almost 20 years, which has unfortunately narrowed the scientific understanding and has, thus, lead to insufficient funding of independent approaches. Therefore, basic researchers hardly have been able to develop causative treatments and clinicians still do not have access to prognostic and early diagnostic tools. During the recent years it became clear that insufficient Aβ export, physiologically facilitated by the ABC transporter superfamily at the brain’s barriers, plays a fundamental role in disease initiation and progression. Furthermore, export mechanisms that are deficient in affected elderly are new targets for activation and, thus, treatment, but ideally also for prevention. In sporadic AD disturbed clearance of β-amyloid from the brain is so far the most important factor for its accumulation in the parenchyma and vessel walls. Here, we review findings about the contribution of ABC transporters and of the perivascular drainage/glymphatic system on β-amyloid clearance. We highlight their potential value for innovative early diagnostics using PET and describe recently described, effective ABC transporter-targeting agents as potential causative treatment for neurodegenerative proteopathies/dementias. PMID:24746857

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

    PubMed

    Liu, Xiang; Li, Shangqi; Peng, Wenzhu; Feng, Shuaisheng; Feng, Jianxin; Mahboob, Shahid; Al-Ghanim, Khalid A; Xu, Peng

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Maltose Uptake by the Novel ABC Transport System MusEFGK2I Causes Increased Expression of ptsG in Corynebacterium glutamicum

    PubMed Central

    Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W.; Krämer, Reinhard

    2013-01-01

    The Gram-positive Corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. However, maltose uptake in C. glutamicum has not been investigated. Interestingly, the presence of maltose in the medium causes increased expression of ptsG in C. glutamicum by an unknown mechanism, although the ptsG-encoded glucose-specific EII permease of the phosphotransferase system itself is not required for maltose utilization. We identified the maltose uptake system as an ABC transporter encoded by musK (cg2708; ATPase subunit), musE (cg2705; substrate binding protein), musF (cg2704; permease), and musG (cg2703; permease) by combination of data obtained from characterization of maltose uptake and reanalyses of transcriptome data. Deletion of the mus gene cluster in C. glutamicum Δmus abolished maltose uptake and utilization. Northern blotting and reverse transcription-PCR experiments revealed that musK and musE are transcribed monocistronically, whereas musF and musG are part of an operon together with cg2701 (musI), which encodes a membrane protein of unknown function with no homologies to characterized proteins. Characterization of growth and [14C]maltose uptake in the musI insertion strain C. glutamicum IMcg2701 showed that musI encodes a novel essential component of the maltose ABC transporter of C. glutamicum. Finally, ptsG expression during cultivation on different carbon sources was analyzed in the maltose uptake-deficient strain C. glutamicum Δmus. Indeed, maltose uptake by the novel ABC transport system MusEFGK2I is required for the positive effect of maltose on ptsG expression in C. glutamicum. PMID:23543710

  13. Independent activity of the homologous small regulatory RNAs AbcR1 and AbcR2 in the legume symbiont Sinorhizobium meliloti.

    PubMed

    Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I

    2013-01-01

    The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5'-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia.

  14. Independent Activity of the Homologous Small Regulatory RNAs AbcR1 and AbcR2 in the Legume Symbiont Sinorhizobium meliloti

    PubMed Central

    Torres-Quesada, Omar; Millán, Vicenta; Nisa-Martínez, Rafael; Bardou, Florian; Crespi, Martín; Toro, Nicolás; Jiménez-Zurdo, José I.

    2013-01-01

    The legume symbiont Sinorhizobium meliloti expresses a plethora of small noncoding RNAs (sRNAs) whose function is mostly unknown. Here, we have functionally characterized two tandemly encoded S. meliloti Rm1021 sRNAs that are similar in sequence and structure. Homologous sRNAs (designated AbcR1 and AbcR2) have been shown to regulate several ABC transporters in the related α-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In Rm1021, AbcR1 and AbcR2 exhibit divergent unlinked regulation and are stabilized by the RNA chaperone Hfq. AbcR1 is transcribed in actively dividing bacteria, either in culture, rhizosphere or within the invasion zone of mature alfalfa nodules. Conversely, AbcR2 expression is induced upon entry into stationary phase and under abiotic stress. Only deletion of AbcR1 resulted into a discrete growth delay in rich medium, but both are dispensable for symbiosis. Periplasmic proteome profiling revealed down-regulation of the branched-chain amino acid binding protein LivK by AbcR1, but not by AbcR2. A double-plasmid reporter assay confirmed the predicted specific targeting of the 5′-untranslated region of the livK mRNA by AbcR1 in vivo. Our findings provide evidences of independent regulatory functions of these sRNAs, probably to fine-tune nutrient uptake in free-living and undifferentiated symbiotic rhizobia. PMID:23869210

  15. Characterization of acquired paclitaxel resistance of breast cancer cells and involvement of ABC transporters

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Němcová-Fürstová, Vlasta, E-mail: vlasta.furstova@

    Development of taxane resistance has become clinically very important issue. The molecular mechanisms underlying the resistance are still unclear. To address this issue, we established paclitaxel-resistant sublines of the SK-BR-3 and MCF-7 breast cancer cell lines that are capable of long-term proliferation in 100 nM and 300 nM paclitaxel, respectively. Application of these concentrations leads to cell death in the original counterpart cells. Both sublines are cross-resistant to doxorubicin, indicating the presence of the MDR phenotype. Interestingly, resistance in both paclitaxel-resistant sublines is circumvented by the second-generation taxane SB-T-1216. Moreover, we demonstrated that it was not possible to establish sublinesmore » of SK-BR-3 and MCF-7 cells resistant to this taxane. It means that at least the tested breast cancer cells are unable to develop resistance to some taxanes. Employing mRNA expression profiling of all known human ABC transporters and subsequent Western blot analysis of the expression of selected transporters, we demonstrated that only the ABCB1/PgP and ABCC3/MRP3 proteins were up-regulated in both paclitaxel-resistant sublines. We found up-regulation of ABCG2/BCRP and ABCC4 proteins only in paclitaxel-resistant SK-BR-3 cells. In paclitaxel-resistant MCF-7 cells, ABCB4/MDR3 and ABCC2/MRP2 proteins were up-regulated. Silencing of ABCB1 expression using specific siRNA increased significantly, but did not completely restore full sensitivity to both paclitaxel and doxorubicin. Thus we showed a key, but not exclusive, role for ABCB1 in mechanisms of paclitaxel resistance. It suggests the involvement of multiple mechanisms in paclitaxel resistance in tested breast cancer cells. - Highlights: • Expression of all ABC transporters in paclitaxel-resistant sublines of SK-BR-3 and MCF-7 cells was analyzed. • SK-BR-3 and MCF-7 cells are unable to develop resistance to some taxanes. • Some taxanes are able to overcome developed resistance to

  16. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

    PubMed Central

    Brzozowska, Natalia; Li, Kong M.; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S.

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−), Bcrp knockout (Abcg2−∕−), combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕−) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

  17. ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

    PubMed

    Brzozowska, Natalia; Li, Kong M; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S; Arnold, Jonathon C

    2016-01-01

    Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

  18. Protein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease.

    PubMed Central

    Yeung, A T; Mattes, W B; Grossman, L

    1986-01-01

    An examination has been made into the nature of the nucleoprotein complexes formed during the incision reaction catalyzed by the Escherichia coli UvrABC endonuclease when acting on a pyrimidine dimer-containing fd RF-I DNA species. The complexes of proteins and DNA form in unique stages. The first stage of binding involves an ATP-stimulated interaction of the UvrA protein with duplex DNA containing pyrimidine dimer sites. The UvrB protein significantly stabilizes the UvrA-pyrimidine dimer containing DNA complex which, in turn, provides a foundation for the binding of UvrC to activate the UvrABC endonuclease. The binding of one molecule of UvrC to each UvrAB-damaged DNA complex is needed to catalyze incision in the vicinity of pyrimidine dimer sites. The UvrABC-DNA complex persists after the incision event suggesting that the lack of UvrABC turnover may be linked to other activities in the excision-repair pathway beyond the initial incision reaction. PMID:3960727

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

    PubMed

    Adewoye, L O; Worobec, E A

    2000-08-08

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

  20. Interaction of BDE-47 and its Hydroxylated Metabolite 6-OH-BDE-47 with the Human ABC Efflux Transporters P-gp and BCRP: Considerations for Human Exposure and Risk Assessment

    EPA Science Inventory

    ATP binding cassette (ABC) transporters, including P-glycoprotein (P-gp; also known as MDR1, ABCB1) and breast cancer resistance protein (BCRP; also known as ABCG2), are membrane-bound proteins that mediate the cellular efflux of xenobiotics as an important defense against chemic...

  1. Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

    PubMed

    Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

    2016-10-18

    The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the

  2. The Role of the Photoreceptor ABC Transporter ABCA4 in Lipid Transport and Stargardt Macular Degeneration

    PubMed Central

    Molday, Robert S.; Zhong, Ming; Quazi, Faraz

    2009-01-01

    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 domain, a multispanning membrane domain and a nucleotide binding domain. Over 500 mutations in the gene encoding ABCA4 are associated with a spectrum of related autosomal recessive retinal degenerative diseases including Stargardt macular degeneration, cone-rod dystrophy and a subset of retinitis pigmentosa. Biochemical studies on the purified ABCA4 together with analysis of abca4 knockout mice and patients with Stargardt disease have implicated ABCA4 as a retinylidene-phosphatidylethanolamine transporter that facilitates the removal of potentially reactive retinal derivatives from photoreceptors following photoexcitation. Knowledge of the genetic and molecular basis for ABCA4 related retinal degenerative diseases is being used to develop rationale therapeutic treatments for this set of disorders. PMID:19230850

  3. Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters

    PubMed Central

    Doliwa, Christelle; Escotte-Binet, Sandie; Aubert, Dominique; Sauvage, Virginie; Velard, Frédéric; Schmid, Aline; Villena, Isabelle

    2013-01-01

    Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-RSDZ and ME-49-RSDZ, by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied. PMID:23707894

  4. Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

    PubMed Central

    Finkenwirth, Friedrich; Kirsch, Franziska

    2013-01-01

    Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

  5. Influence of multidrug resistance and drug transport proteins on chemotherapy drug metabolism.

    PubMed

    Joyce, Helena; McCann, Andrew; Clynes, Martin; Larkin, Annemarie

    2015-05-01

    Chemotherapy involving the use of anticancer drugs remains an important strategy in the overall management of patients with metastatic cancer. Acquisition of multidrug resistance remains a major impediment to successful chemotherapy. Drug transporters in cell membranes and intracellular drug metabolizing enzymes contribute to the resistance phenotype and determine the pharmacokinetics of anticancer drugs in the body. ATP-binding cassette (ABC) transporters mediate the transport of endogenous metabolites and xenobiotics including cytotoxic drugs out of cells. Solute carrier (SLC) transporters mediate the influx of cytotoxic drugs into cells. This review focuses on the substrate interaction of these transporters, on their biology and what role they play together with drug metabolizing enzymes in eliminating therapeutic drugs from cells. The majority of anticancer drugs are substrates for the ABC transporter and SLC transporter families. Together, these proteins have the ability to control the influx and the efflux of structurally unrelated chemotherapeutic drugs, thereby modulating the intracellular drug concentration. These interactions have important clinical implications for chemotherapy because ultimately they determine therapeutic efficacy, disease progression/relapse and the success or failure of patient treatment.

  6. The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin.

    PubMed

    Poudyal, Bandita; Sauer, Karin

    2018-02-01

    A hallmark of biofilms is their tolerance to killing by antimicrobial agents. In Pseudomonas aeruginosa , biofilm drug tolerance requires the c-di-GMP-responsive MerR transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm drug tolerance has not been elucidated. Here, we demonstrate that BrlR activates the expression of at least 7 ABC transport systems, including the PA1874-PA1875-PA1876-PA1877 (PA1874-77) operon, with chromatin immunoprecipitation and DNA binding assays confirming BrlR binding to the promoter region of PA1874-77. Insertional inactivation of the 7 ABC transport systems rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin or norfloxacin. Susceptibility was linked to drug accumulation, with BrlR contributing to norfloxacin accumulation in a manner dependent on multidrug efflux pumps and the PA1874-77 ABC transport system. Inactivation of the respective ABC transport system, furthermore, eliminated the recalcitrance of biofilms to killing by tobramycin but not norfloxacin, indicating that drug accumulation is not linked to biofilm drug tolerance. Our findings indicate for the first time that BrlR, a MerR-type transcriptional activator, activates genes encoding several ABC transport systems, in addition to multiple multidrug efflux pump genes. Moreover, our data confirm a BrlR target contributing to drug tolerance, likely countering the prevailing dogma that biofilm tolerance arises from a multiplicity of factors. Copyright © 2018 American Society for Microbiology.

  7. Secondary Metabolites from Plants Inhibiting ABC Transporters and Reversing Resistance of Cancer Cells and Microbes to Cytotoxic and Antimicrobial Agents

    PubMed Central

    Wink, Michael; Ashour, Mohamed L.; El-Readi, Mahmoud Zaki

    2012-01-01

    Fungal, bacterial, and cancer cells can develop resistance against antifungal, antibacterial, or anticancer agents. Mechanisms of resistance are complex and often multifactorial. Mechanisms include: (1) Activation of ATP-binding cassette (ABC) transporters, such as P-gp, which pump out lipophilic compounds that have entered a cell, (2) Activation of cytochrome p450 oxidases which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) Activation of glutathione transferase, which can conjugate xenobiotics. This review summarizes the evidence that secondary metabolites (SM) of plants, such as alkaloids, phenolics, and terpenoids can interfere with ABC transporters in cancer cells, parasites, bacteria, and fungi. Among the active natural products several lipophilic terpenoids [monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes] but also some alkaloids (isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids) function probably as competitive inhibitors of P-gp, multiple resistance-associated protein 1, and Breast cancer resistance protein in cancer cells, or efflux pumps in bacteria (NorA) and fungi. More polar phenolics (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) directly inhibit proteins forming several hydrogen and ionic bonds and thus disturbing the 3D structure of the transporters. The natural products may be interesting in medicine or agriculture as they can enhance the activity of active chemotherapeutics or pesticides or even reverse multidrug resistance, at least partially, of adapted and resistant cells. If these SM are applied in combination with a cytotoxic or antimicrobial agent, they may reverse resistance in a synergistic fashion. PMID:22536197

  8. Profiling of ABC transporters ABCB5, ABCF2 and nestin-positive stem cells in nevi, in situ and invasive melanoma.

    PubMed

    Setia, Namrata; Abbas, Ossama; Sousa, Yessica; Garb, Jane L; Mahalingam, Meera

    2012-08-01

    Distinct ABCB5 forms and ABCF2, members of the ATP-binding cassette (ABC) superfamily of transporters, are normally expressed in various tissues and cells, and enhanced expression of both has been demonstrated in select cancers. In melanoma cell lines, gene expression profiling of ABC transporters has revealed enhanced expression of melanocyte-specific ABCB5 and ABCF2 proteins. Given this, our primary aim was to ascertain immunohistochemical expression of the ABC transporters ABCB5 and ABCF2 and, the stem cell marker, nestin in a spectrum of benign and malignant nevomelanocytic proliferations, including nevi (n=30), in situ (n=31) and invasive (n=24) primary cutaneous melanomas to assess their role in the stepwise development of malignancy. In addition, their expression was compared with established melanoma prognosticators to ascertain their utility as independent prognosticators. A semiquantitative scoring system was utilized by deriving a cumulative score (based on percentage positivity cells and intensity of expression) and statistical analyses was carried out using analysis of variance with linear contrasts. Mean cumulative score in nevi, in situ and invasive melanoma were as follows: 3.8, 4.4 and 5.3 for ABCB5, respectively (P<0.005 for all), and 4.6, 4.6 and 5.3 for nestin, respectively (P=not significant for all). No appreciable expression of ABCF2 was noted in any of the groups. While ulcerated lesions of melanoma demonstrated lower levels of expression of ABCB5 and nestin than non-ulcerated lesions, and nestin expression was lower in lesions with mitoses >1, after controlling for the presence of ulceration and mitotic activity, the expression of both proteins did not significantly correlate with known melanoma prognosticators. The gradual increase in the expression of ABCB5 from benign nevus to in situ to invasive melanoma suggests that it plays a role in melanomagenesis. On the basis of our findings, a prospective study with follow-up data is required to

  9. ABC multidrug transporters: target for modulation of drug pharmacokinetics and drug-drug interactions.

    PubMed

    Marquez, Béatrice; Van Bambeke, Françoise

    2011-05-01

    Nine proteins of the ABC superfamily (P-glycoprotein, 7 MRPs and BCRP) are involved in multidrug transport. Being localised at the surface of endothelial or epithelial cells, they expel drugs back to the external medium (if located at the apical side [P-glycoprotein, BCRP, MRP2, MRP4 in the kidney]) or to the blood (if located at the basolateral side [MRP1, MRP3, MRP4, MRP5]), modulating thereby their absorption, distribution, and elimination. In the CNS, most transporters are oriented to expel drugs to the blood. Transporters also cooperate with Phase I/Phase II metabolism enzymes by eliminating drug metabolites. Their major features are (i) their capacity to recognize drugs belonging to unrelated pharmacological classes, and (ii) their redundancy, a single molecule being possibly substrate for different transporters. This ensures an efficient protection of the body against invasion by xenobiotics. Competition for transport is now characterized as a mechanism of interaction between co-administered drugs, one molecule limiting the transport of the other, potentially affecting bioavailability, distribution, and/or elimination. Again, this mechanism reinforces drug interactions mediated by cytochrome P450 inhibition, as many substrates of P-glycoprotein and CYP3A4 are common. Induction of the expression of genes coding for MDR transporters is another mechanism of drug interaction, which could affect all drug substrates of the up-regulated transporter. Overexpression of MDR transporters confers resistance to anticancer agents and other therapies. All together, these data justify why studying drug active transport should be part of the evaluation of new drugs, as recently recommended by the FDA.

  10. The ABC transporter Tba of Amycolatopsis balhimycina is required for efficient export of the glycopeptide antibiotic balhimycin.

    PubMed

    Menges, R; Muth, G; Wohlleben, W; Stegmann, E

    2007-11-01

    All known gene clusters for glycopeptide antibiotic biosynthesis contain a conserved gene supposed to encode an ABC-transporter. In the balhimycin-producer Amycolatopsis balhimycina this gene (tba) is localised between the prephenate dehydrogenase gene pdh and the peptide synthetase gene bpsA. Inactivation of tba in A. balhimycina by gene replacement did not interfere with growth and did not affect balhimycin resistance. However, in the supernatant of the tba mutant RM43 less balhimycin was accumulated compared to the wild type; and the intra-cellular balhimycin concentration was ten times higher in the tba mutant RM43 than in the wild type. These data suggest that the ABC transporter encoded in the balhimycin biosynthesis gene cluster is not involved in resistance but is required for the efficient export of the antibiotic. To elucidate the activity of Tba it was heterologously expressed in Escherichia coli with an N-terminal His-tag and purified by nickel chromatography. A photometric assay revealed that His(6)-Tba solubilised in dodecylmaltoside possesses ATPase activity, characteristic for ABC-transporters.

  11. Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway

    USDA-ARS?s Scientific Manuscript database

    Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the dou...

  12. Mutations in the Arabidopsis Peroxisomal ABC Transporter COMATOSE Allow Differentiation between Multiple Functions In Planta: Insights from an Allelic Series

    PubMed Central

    Dietrich, Daniela; Schmuths, Heike; Lousa, Carine De Marcos; Baldwin, Jocelyn M.; Baldwin, Stephen A.; Baker, Alison; Holdsworth, Michael J.

    2009-01-01

    COMATOSE (CTS), the Arabidopsis homologue of human Adrenoleukodystrophy protein (ALDP), is required for import of substrates for peroxisomal β-oxidation. A new allelic series and a homology model based on the bacterial ABC transporter, Sav1866, provide novel insights into structure-function relations of ABC subfamily D proteins. In contrast to ALDP, where the majority of mutations result in protein absence from the peroxisomal membrane, all CTS mutants produced stable protein. Mutation of conserved residues in the Walker A and B motifs in CTS nucleotide-binding domain (NBD) 1 resulted in a null phenotype but had little effect in NBD2, indicating that the NBDs are functionally distinct in vivo. Two alleles containing mutations in NBD1 outside the Walker motifs (E617K and C631Y) exhibited resistance to auxin precursors 2,4-dichlorophenoxybutyric acid (2,4-DB) and indole butyric acid (IBA) but were wild type in all other tests. The homology model predicted that the transmission interfaces are domain-swapped in CTS, and the differential effects of mutations in the conserved “EAA motif” of coupling helix 2 supported this prediction, consistent with distinct roles for each NBD. Our findings demonstrate that CTS functions can be separated by mutagenesis and the structural model provides a framework for interpretation of phenotypic data. PMID:19019987

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

    PubMed

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

    2017-09-20

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

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

    DOE PAGES

    Grossmann, Nina; Vakkasoglu, Ahmet S.; Hulpke, Sabine; ...

    2014-11-07

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

  15. Iowa ABC connections : [tech transfer summary].

    DOT National Transportation Integrated Search

    2015-06-01

    The Iowa Department of Transportation (DOT) and other organizations have : been developing accelerated bridge construction (ABC) concepts, details, and : processes, and Iowa has come to be viewed as a national leader in the area of : ABC. However, th...

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

    PubMed Central

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

    2014-01-01

    Expression of particular drug transporters in response to antibiotic pressure is a critical element in the development of bacterial multidrug resistance, and represents a serious concern for human health. To obtain a better understanding of underlying regulatory mechanisms, we have dissected the transcriptional activation of the ATP-binding cassette (ABC) transporter BmrC/BmrD of the Gram-positive model bacterium Bacillus subtilis. By using promoter-GFP fusions and live cell array technology, we demonstrate a temporally controlled transcriptional activation of the bmrCD genes in response to antibiotics that target protein synthesis. Intriguingly, bmrCD expression only occurs during the late-exponential and stationary growth stages, irrespective of the timing of the antibiotic challenge. We show that this is due to tight transcriptional control by the transition state regulator AbrB. Moreover, our results show that the bmrCD genes are co-transcribed with bmrB (yheJ), a small open reading frame immediately upstream of bmrC that harbors three alternative stem-loop structures. These stem-loops are apparently crucial for antibiotic-induced bmrCD transcription. Importantly, the antibiotic-induced bmrCD expression requires translation of bmrB, which implies that BmrB serves as a regulatory leader peptide. Altogether, we demonstrate for the first time that a ribosome-mediated transcriptional attenuation mechanism can control the expression of a multidrug ABC transporter. PMID:25217586

  17. Efficient extracellular production of type I secretion pathway-dependent Pseudomonas fluorescens lipase in recombinant Escherichia coli by heterologous ABC protein exporters.

    PubMed

    Eom, Gyeong Tae; Lee, Seung Hwan; Oh, Young Hoon; Choi, Ji Eun; Park, Si Jae; Song, Jae Kwang

    2014-10-01

    Heterologous ABC protein exporters, the apparatus of type I secretion pathway in Gram-negative bacteria, were used for extracellular production of Pseudomonas fluorescens lipase (TliA) in recombinant Escherichia coli. The effect of the expression of different ABC protein exporter gene clusters (P. fluorescens tliDEF, Pseudomonas aeruginosa aprDEF, Erwinia chrysanthemi prtDEF, and Serratia marcescens lipBCD genes) was examined on the secretion of TliA at growth temperatures of 20, 25, 30 and 35 °C. TliA secretion in recombinant E. coli XL10-Gold varied depending upon type of ABC protein exporter and culture temperature. E. coli expressing S. marcescens lipBCD genes showed the highest secretion level of TliA (122.8 U ml(-1)) when cultured at 25 °C. Thus, optimized culture conditions for efficient extracellular production of lipase in recombinant E. coli can be designed by changing the type of ABC protein exporter and the growth temperature.

  18. [Biological characteristics of an enteroinvasive Escherichia coli strain with tatABC deletion].

    PubMed

    Gong, Zhaolong; Ye, Changyun; Liu, Xiaobing; Zhang, Min; Zhuo, Qin

    2013-05-04

    To study the relationship between twin-arginine translocation system (Tat) system with the biological characteristics of enteroinvasive Escherichia coli (EIEC). Through homologous recombination, we constructed EIEC's tatABC gene deletion strain and complementary strain, and explored their impact on bacterial form, substrate transport function as well as on HeLa cells and guinea pig's corneal invasion force. The tatABC gene deletion strain had apparent changes in bacterial form, loss of substrate transporter function, and significant weakened bacterial invasion force (the number of the deletion strain invading into HeLa cells was decreased significantly, and the ability of its corneal lesion capacity of the guinea pig was significantly weakened), while the complementary strain was similar to the wild strain in the above respects. EIEC's Tat protein transport system is closely related with the biological characteristics of EIEC.

  19. Water-mediated protein-fluorophore interactions modulate the affinity of an ABC-ATPase/TNP-ADP complex.

    PubMed

    Oswald, Christine; Jenewein, Stefan; Smits, Sander H J; Holland, I Barry; Schmitt, Lutz

    2008-04-01

    TNP-modified nucleotides have been used extensively to study protein-nucleotide interactions. In the case of ABC-ATPases, application of these powerful tools has been greatly restricted due to the significantly higher affinity of the TNP-nucleotide for the corresponding ABC-ATPase in comparison to the non-modified nucleotides. To understand the molecular changes occurring upon binding of the TNP-nucleotide to an ABC-ATPase, we have determined the crystal structure of the TNP-ADP/HlyB-NBD complex at 1.6A resolution. Despite the higher affinity of TNP-ADP, no direct fluorophore-protein interactions were observed. Unexpectedly, only water-mediated interactions were detected between the TNP moiety and Tyr(477), that is engaged in pi-pi stacking with the adenine ring, as well as with two serine residues (Ser(504) and Ser(509)) of the Walker A motif. Interestingly, the side chains of these two serine residues adopt novel conformations that are not observed in the corresponding ADP structure. However, in the crystal structure of the S504A mutant, which binds TNP-ADP with similar affinity to the wild type enzyme, a novel TNP-water interaction compensates for the missing serine side chain. Since this water molecule is not present in the wild type enzyme, these results suggest that only water-mediated interactions provide a structural explanation for the increased affinity of TNP-nucleotides towards ABC-ATPases. However, our results also imply that in silico approaches such as docking or modeling cannot directly be applied to generate 'affinity-adopted' ADP- or ATP-analogs for ABC-ATPases.

  20. Trichothecene resistance in wheat: Development of molecular markers for PDR-type ABC transporter genes.

    PubMed

    Mitterbauer, R; Heinrich, M; Rauscher, R; Lemmens, M; Bürstmayr, H; Adam, G

    2003-03-01

    Infection withFusarium graminearum andF. culmorum not only causes severe yield and quality losses, the most relevant concern is the contamination of cereal foods and feeds with trichothecenes (e.g. deoxynivalenol, DON). The ability to synthesize trichothecenes has been shown to be a virulence factor ofF. graminearum on wheat and, on the other hand, toxin resistance is most likely an important component of field resistance. Our hypothesis is that pleiotropic drug resistance mediated by PDR-type ABC transporter proteins (acting as membrane located drug efflux pumps) is a relevant mechanism of DON resistance not only in yeast but also in wheat. Goal of this project is the development of molecular markers for this gene family for use in marker-assisted plant breeding programs. The technical difficulties caused by the large size of the PDR-family are discussed.

  1. The ABC transporter ABCG29 is involved in H2O2 tolerance and biocontrol traits in the fungus Clonostachys rosea.

    PubMed

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

    2016-04-01

    For successful biocontrol interactions, biological control organisms must tolerate toxic metabolites produced by themselves or plant pathogens during mycoparasitic/antagonistic interactions, by host plant during colonization of the plant, and xenobiotics present in the environment. ATP-binding cassette (ABC) transporters can play a significant role in tolerance of toxic compounds by mediating active transport across the cellular membrane. This paper reports on functional characterization of an ABC transporter ABCG29 in the biocontrol fungus Clonostachys rosea strain IK726. Gene expression analysis showed induced expression of abcG29 during exposure to the Fusarium spp. mycotoxin zearalenone (ZEA) and the fungicides Cantus, Chipco Green and Apron. Expression of abcG29 in C. rosea was significantly higher during C. rosea-C. rosea (Cr-Cr) interaction or in exposure to C. rosea culture filtrate for 2 h, compared to interaction with Fusarium graminearum or 2 h exposure to F. graminearum culture filtrate. In contrast with gene expression data, ΔabcG29 strains did not display reduced tolerance towards ZEA, fungicides or chemical agents known for inducing oxidative, cell wall or osmotic stress, compared to C. rosea WT. The exception was a significant reduction in tolerance to H2O2 (10 mM) in ΔabcG29 strains when conidia were used as an inoculum. The antagonistic ability of ΔabcG29 strains towards F. graminearum, Fusarium oxysporum or Botrytis cinerea in dual plate assays were not different compared with WT. However, in biocontrol assays ΔabcG29 strains displayed reduced ability to protect Arabidopsis thaliana leaves from B. cinerea, and barley seedling from F. graminearum as measured by an A. thaliana detached leaf assay and a barley foot rot disease assay, respectively. These data show that the ABCG29 is dispensable for ZEA and fungicides tolerance, and antagonism but not H2O2 tolerance and biocontrol effects in C. rosea.

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

    PubMed Central

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

    2016-01-01

    breast cancer-related protein, the multidrug resistance proteins 1 through 5 and the cholesterol transporters ABCA1 and ABCG1. CONCLUSIONS The ABC transporters have various roles across multiple reproductive tissues. Knowledge of efflux direction, tissue distribution, substrate specificity and regulation of the ABC transporters in the placenta and other reproductive tissues is rapidly expanding. This will allow better understanding of the disposition of specific substrates within reproductive tissues, and facilitate development of novel treatments for reproductive disorders as well as improved approaches to protecting the developing fetus. PMID:26545808

  3. Iowa ABC connections.

    DOT National Transportation Integrated Search

    2015-06-01

    For several years the Iowa Department of Transportation (DOT), Iowa State University, the Federal Highway Administration, : and several Iowa counties have been working to develop accelerated bridge construction (ABC) concepts, details, and processes....

  4. ABC transporters and cytochromes P450 in the human central nervous system: influence on brain pharmacokinetics and contribution to neurodegenerative disorders.

    PubMed

    Dutheil, Fabien; Jacob, Aude; Dauchy, Sandrine; Beaune, Philippe; Scherrmann, Jean-Michel; Declèves, Xavier; Loriot, Marie-Anne

    2010-10-01

    The identification of xenobiotic metabolizing enzymes (i.e., CYP) and transporters (i.e., ABC transporters) (XMET) in the human brain, including the BBB, raises the question whether these transporters and enzymes have specific functions in brain physiology, neuropharmacology and toxicology. Relevant literature was identified using PubMed search articles published up to March 2010. Search terms included 'ABC transporters and P450 or CYP', 'drug metabolism, effect and toxicity' and 'neurodegenerative disease (Alzheimer and Parkinson diseases)' restricted to the field of 'brain or human brain'. This review aims to provide a better understanding of XMET functions in the human brain and show their pharmacological importance for improving drug delivery and efficacy and also for managing their side effects. Finally, the impact of brain XMET activity during neurodegenerative processes is discussed, giving an opportunity to identify new markers of human brain diseases. During the last 2 decades, much evidence concerning the specific distribution patterns of XMET, their induction by xenobiotics and endobiotics and their genetic variations have made cerebral ABC transporters and CYP enzymes key elements in the way individual patients respond to centrally acting drugs.

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

    PubMed

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

    2015-07-17

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

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

    PubMed

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

    2014-10-01

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

  7. Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter

    USDA-ARS?s Scientific Manuscript database

    The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against multiple pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whet...

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

    PubMed Central

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

    2015-01-01

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

  9. ABCE1 Is a Highly Conserved RNA Silencing Suppressor

    PubMed Central

    Kärblane, Kairi; Gerassimenko, Jelena; Nigul, Lenne; Piirsoo, Alla; Smialowska, Agata; Vinkel, Kadri; Kylsten, Per; Ekwall, Karl; Swoboda, Peter; Truve, Erkki; Sarmiento, Cecilia

    2015-01-01

    ATP-binding cassette sub-family E member 1 (ABCE1) is a highly conserved protein among eukaryotes and archaea. Recent studies have identified ABCE1 as a ribosome-recycling factor important for translation termination in mammalian cells, yeast and also archaea. Here we report another conserved function of ABCE1. We have previously described AtRLI2, the homolog of ABCE1 in the plant Arabidopsis thaliana, as an endogenous suppressor of RNA silencing. In this study we show that this function is conserved: human ABCE1 is able to suppress RNA silencing in Nicotiana benthamiana plants, in mammalian HEK293 cells and in the worm Caenorhabditis elegans. Using co-immunoprecipitation and mass spectrometry, we found a number of potential ABCE1-interacting proteins that might support its function as an endogenous suppressor of RNA interference. The interactor candidates are associated with epigenetic regulation, transcription, RNA processing and mRNA surveillance. In addition, one of the identified proteins is translin, which together with its binding partner TRAX supports RNA interference. PMID:25659154

  10. Detergent Screening and Purification of the Human Liver ABC Transporters BSEP (ABCB11) and MDR3 (ABCB4) Expressed in the Yeast Pichia pastoris

    PubMed Central

    Stindt, Jan; Smits, Sander H. J.; Schmitt, Lutz

    2013-01-01

    The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters. PMID:23593265

  11. Computer simulations of transport through membranes: passive diffusion, pores, channels and transporters.

    PubMed

    Tieleman, D Peter

    2006-10-01

    A key function of biological membranes is to provide mechanisms for the controlled transport of ions, nutrients, metabolites, peptides and proteins between a cell and its environment. We are using computer simulations to study several processes involved in transport. In model membranes, the distribution of small molecules can be accurately calculated; we are making progress towards understanding the factors that determine the partitioning behaviour in the inhomogeneous lipid environment, with implications for drug distribution, membrane protein folding and the energetics of voltage gating. Lipid bilayers can be simulated at a scale that is sufficiently large to study significant defects, such as those caused by electroporation. Computer simulations of complex membrane proteins, such as potassium channels and ATP-binding cassette (ABC) transporters, can give detailed information about the atomistic dynamics that form the basis of ion transport, selectivity, conformational change and the molecular mechanism of ATP-driven transport. This is illustrated in the present review with recent simulation studies of the voltage-gated potassium channel KvAP and the ABC transporter BtuCD.

  12. NtPDR3, an iron-deficiency inducible ABC transporter in Nicotiana tabacum.

    PubMed

    Ducos, Eric; Fraysse, Staffan; Boutry, Marc

    2005-12-19

    In plants, the ABC transporter PDR (pleiotropic drug resistance) subfamily is composed of approximately 15 genes, few of which have been analyzed. We have identified NtPDR3, a Nicotiana tabacum PDR gene belonging to a cluster for which no functional data was previously available. NtPDR3 was found to be induced in suspension cells treated with methyl jasmonate, salicylic acid, 1-naphthalene acetic acid, or cembrene, a macrocyclic diterpene. In agreement with the identification of a putative iron deficiency element in the NtPDR3 transcription promoter region, we found that iron deficiency in the culture medium induced NtPDR3 expression, thus suggesting a new function of the PDR transporter family.

  13. The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

    PubMed Central

    Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

    2017-01-01

    Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

  14. The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

    PubMed

    Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

    2010-01-01

    We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

  15. Overexpression of an ABC transporter and mutations of GyrA, GyrB, and ParC in contributing to high-level ciprofloxacin resistance in Streptococcus suis type 2.

    PubMed

    Yao, Jie; Shang, Kexin; Huang, Jinhu; Ran, Wei; Kashif, Jam; Wang, Liping

    2014-04-01

    Streptococcus suis is a pathogen of zoonotic diseases. Moreover, the emergence of fluoro-quinolones (FQs) resistance in this pathogen has severe consequences for pigs and human health. In this study, the molecular mechanism of FQs resistance in S. suis type 2 (SS2) sensitive strains isolated from pigs was assessed after in vitro induction of resistance against the most frequently used FQs: ciprofloxacin, norfloxacin, and enrofloxacin. Proteome analysis, sequencing and real-time RT-PCR results strongly established an overexpression of an ABC transporter protein (other than SatAB) and topoisomerase mutations in GyrA (Ser81Arg), GyrB (Glu354Lys), and ParC (Ser79Phe) in contributing to high level ciprofloxacin resistance in SS2. Due to the overexpression of the ABC transporter, intracellular ciprofloxacin concentrations were significantly lower in the resistant strains than those of sensitive strains after 20, 35, and 60 min exposures to ciprofloxacin (p < 0.05). It was concluded that improper use of FQs is one of the main causes of the emergence of this zoonotic pathogen as a multiresistant organism against commonly used antibiotics. The existence of an efflux-like protein is an incentive to find new drug targets to avoid the spread of FQs-resistant S. suis isolates in pigs and the human population.

  16. NFκBP65 transcription factor modulates resistance to doxorubicin through ABC transporters in breast cancer.

    PubMed

    Velaei, Kobra; Samadi, Nasser; Soltani, Sina; Barazvan, Balal; Soleimani Rad, Jafar

    2017-07-01

    Shedding light on chemoresistance biology of breast cancer could contribute to enhance the clinical outcome. Intrinsic or acquired resistance to chemotherapy is a major problem in breast cancer treatment. The NFκB pathway by siRNAP65 and JSH-23 as a translocational inhibitor of NFκBP65 in the doxorubicin-resistant MCF-7 (MCF-7/Dox) and MCF-7 cells was blocked. Then, the ABC transporter expression and function were assessed by real-time qRT-PCR and flow cytometry, respectively. Induction of apoptosis was evaluated after inhibition of the NFΚB pathway as well. Our study underlined the upregulation of NFκBP65 and anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax in the MCF-7/Dox cells compared with control MCF-7 cells. Here, we showed that interplay between nuclear factor kappa B P65 (NFkBP65) as a transcriptional regulator and ABC transporters in the MCF-7/Dox cancer cells. We found that inhibition of the elevated expression of NFκBP65 in the resistant breast cancer, whether translocational inhibition or silencing by siRNA, decreased the expression and function of MDR1 and MRP1 efflux pumps. Furthermore, the blockade of NFκBP65 promoted apoptosis via modulating Bcl-2 and BAX expression. After inhibition of the NFκBP65 signaling pathway, elevated baseline expression of survival Bcl-2 gene in the resistant breast cells significantly decreased. Suppression of the NFκB pathway has a profound dual impact on promoting the intrinsic apoptotic pathway and reducing ABC transporter function and expression, which are some of the chemoresistance features. It was speculated that the NFκB pathway directly acts on doxorubicin-induced MDR1 and MRP1 expression in MCF-7/Dox cells.

  17. Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump

    PubMed Central

    Llabrés, Salomé; Neuberger, Arthur; Blaza, James N.; Bai, Xiao-chen; Okada, Ui; Murakami, Satoshi; van Veen, Hendrik W.; Zachariae, Ulrich; Scheres, Sjors H.W.; Luisi, Ben F.

    2017-01-01

    The MacA-MacB-TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain (TMD) with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism. PMID:28504659

  18. TaABC1, a member of the activity of bc1 complex protein kinase family from common wheat, confers enhanced tolerance to abiotic stresses in Arabidopsis

    PubMed Central

    Wang, Caixiang; Jing, Ruilian; Mao, Xinguo; Chang, Xiaoping; Li, Ang

    2011-01-01

    Abiotic stresses such as drought, salinity, and low temperature have drastic effects on plant growth and development. However, the molecular mechanisms regulating biochemical and physiological changes in response to stresses are not well understood. Protein kinases are major signal transduction factors among the reported molecular mechanisms mediating acclimation to environmental changes. Protein kinase ABC1 (activity of bc1 complex) is involved in regulating coenzyme Q biosynthesis in mitochondria in yeast (Saccharomyces cersvisiae), and in balancing oxidative stress in chloroplasts in Arabidopsis thaliana. In the current study, TaABC1 (Triticum aestivum L. activity of bc1 complex) protein kinase was localized to the cell membrane, cytoplasm, and nucleus. The effects of overexpressing TaABC1 in transgenic Arabidopsis plants on responses to drought, salt, and cold stress were further investigated. Transgenic Arabidopsis overexpressing the TaABC1 protein showed lower water loss and higher osmotic potential, photochemistry efficiency, and chlorophyll content, while cell membrane stability and controlled reactive oxygen species homeostasis were maintained. In addition, overexpression of TaABC1 increased the expression of stress-responsive genes, such as DREB1A, DREB2A, RD29A, ABF3, KIN1, CBF1, LEA, and P5CS, detected by real-time PCR analysis. The results suggest that TaABC1 overexpression enhances drought, salt, and cold stress tolerance in Arabidopsis, and imply that TaABC1 may act as a regulatory factor involved in a multiple stress response pathways. PMID:21115661

  19. The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koropatkin, Nicole M.; Koppenaal, David W.; Pakrasi, Himadri B.

    2007-01-26

    Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO₂ over O₂, Cyanobacteria have developed highly efficient CO₂concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here we describe the structure of the bicarbonate binding protein, CmpA, in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein, NrtA. CmpA binds carbonic acid at the entrance to themore » ligand-binding pocket whereas bicarbonate binds in nearly an identical location compared to nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca²⁺ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal is highly cooperative and suggests that CmpA co-transports bicarbonate and calcium.« less

  20. Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design

    NASA Astrophysics Data System (ADS)

    Ishikawa, Toshihisa; Tamura, Ai; Saito, Hikaru; Wakabayashi, Kanako; Nakagawa, Hiroshi

    2005-10-01

    In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.

  1. ABCE1 is essential for S phase progression in human cells

    PubMed Central

    Toompuu, Marina; Kärblane, Kairi; Pata, Pille; Truve, Erkki; Sarmiento, Cecilia

    2016-01-01

    ABSTRACT ABCE1 is a highly conserved protein universally present in eukaryotes and archaea, which is crucial for the viability of different organisms. First identified as RNase L inhibitor, ABCE1 is currently recognized as an essential translation factor involved in several stages of eukaryotic translation and ribosome biogenesis. The nature of vital functions of ABCE1, however, remains unexplained. Here, we study the role of ABCE1 in human cell proliferation and its possible connection to translation. We show that ABCE1 depletion by siRNA results in a decreased rate of cell growth due to accumulation of cells in S phase, which is accompanied by inefficient DNA synthesis and reduced histone mRNA and protein levels. We infer that in addition to the role in general translation, ABCE1 is involved in histone biosynthesis and DNA replication and therefore is essential for normal S phase progression. In addition, we analyze whether ABCE1 is implicated in transcript-specific translation via its association with the eIF3 complex subunits known to control the synthesis of cell proliferation-related proteins. The expression levels of a few such targets regulated by eIF3A, however, were not consistently affected by ABCE1 depletion. PMID:26985706

  2. Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.

    PubMed

    Howlett, Robert M; Hughes, Bethan M; Hitchcock, Andrew; Kelly, David J

    2012-06-01

    Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

  3. A Neisseria meningitidis fbpABC mutant is incapable of using nonheme iron for growth.

    PubMed

    Khun, H H; Kirby, S D; Lee, B C

    1998-05-01

    The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway.

  4. A Neisseria meningitidis fbpABC Mutant Is Incapable of Using Nonheme Iron for Growth

    PubMed Central

    Khun, Heng H.; Kirby, Shane D.; Lee, B. Craig

    1998-01-01

    The neisserial fbpABC locus has been proposed to act as an iron-specific ABC transporter system. To confirm this assigned function, we constructed an fbpABC mutant in Neisseria meningitidis by insertional inactivation of fbpABC with a selectable antibiotic marker. The mutant was unable to use iron supplied from human transferrin, human lactoferrin, or iron chelates. However, the use of iron from heme and human hemoglobin was unimpaired. These results support the obligatory participation of fbpABC in neisserial periplasmic iron transport and do not indicate a role for this genetic locus in the heme iron pathway. PMID:9573125

  5. The structure of the human ABC transporter ABCG2 reveals a novel mechanism for drug extrusion.

    PubMed

    Khunweeraphong, Narakorn; Stockner, Thomas; Kuchler, Karl

    2017-10-23

    The human ABC transporter ABCG2 (Breast Cancer Resistance Protein, BCRP) is implicated in anticancer resistance, in detoxification across barriers and linked to gout. Here, we generate a novel atomic model of ABCG2 using the crystal structure of ABCG5/G8. Extensive mutagenesis verifies the structure, disclosing hitherto unrecognized essential residues and domains in the homodimeric ABCG2 transporter. The elbow helix, the first intracellular loop (ICL1) and the nucleotide-binding domain (NBD) constitute pivotal elements of the architecture building the transmission interface that borders a central cavity which acts as a drug trap. The transmission interface is stabilized by salt-bridge interactions between the elbow helix and ICL1, as well as within ICL1, which is essential to control the conformational switch of ABCG2 to the outward-open drug-releasing conformation. Importantly, we propose that ICL1 operates like a molecular spring that holds the NBD dimer close to the membrane, thereby enabling efficient coupling of ATP hydrolysis during the catalytic cycle. These novel mechanistic data open new opportunities to therapeutically target ABCG2 in the context of related diseases.

  6. Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach.

    PubMed

    Alvarez, Angel H; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

    2015-10-01

    Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD.

  7. Identification of immunodominant proteins from Mannheimia haemolytica and Histophilus somni by an immunoproteomic approach

    PubMed Central

    Alvarez, Angel H.; Gutiérrez-Ortega, Abel; Hernández-Gutiérrez, Rodolfo

    2015-01-01

    Mannheimia haemolytica and Histophilus somni are frequently isolated from diseased cattle with bovine respiratory disease (BRD). They compromise animal lung function and the immune responses generated are not sufficient to limit infection. Identification of specific immunogenic antigens for vaccine development represents a great challenge. Immunogenic proteins were identified by immunoproteomic approach with sera from cattle immunized with a commercial cellular vaccine of M. haemolytica and H. somni. Proteins of M. haemolytica were identified as solute ABC transporter, iron-binding protein, and hypothetical protein of capsular biosynthesis. Histophilus somni proteins correspond to porin, amino acid ABC transporter, hypothetical outer membrane protein, cysteine synthase, and outer membrane protein P6. Although these antigens share strong similarities with other proteins from animal pathogens, the ABC system proteins have been associated with virulence and these proteins could be considered as potential vaccine candidates for BRD. PMID:26424916

  8. A Mutation within the Extended X Loop Abolished Substrate-induced ATPase Activity of the Human Liver ATP-binding Cassette (ABC) Transporter MDR3*

    PubMed Central

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-01-01

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. PMID:25533467

  9. Trafficking and processing of bacterial proteins by mammalian cells: Insights from chondroitinase ABC.

    PubMed

    Muir, Elizabeth; Raza, Mansoor; Ellis, Clare; Burnside, Emily; Love, Fiona; Heller, Simon; Elliot, Matthew; Daniell, Esther; Dasgupta, Debayan; Alves, Nuno; Day, Priscilla; Fawcett, James; Keynes, Roger

    2017-01-01

    There is very little reported in the literature about the relationship between modifications of bacterial proteins and their secretion by mammalian cells that synthesize them. We previously reported that the secretion of the bacterial enzyme Chondroitinase ABC by mammalian cells requires the strategic removal of at least three N-glycosylation sites. The aim of this study was to determine if it is possible to enhance the efficacy of the enzyme as a treatment for spinal cord injury by increasing the quantity of enzyme secreted or by altering its cellular location. To determine if the efficiency of enzyme secretion could be further increased, cells were transfected with constructs encoding the gene for chondroitinase ABC modified for expression by mammalian cells; these contained additional modifications of strategic N-glycosylation sites or alternative signal sequences to direct secretion of the enzyme from the cells. We show that while removal of certain specific N-glycosylation sites enhances enzyme secretion, N-glycosylation of at least two other sites, N-856 and N-773, is essential for both production and secretion of active enzyme. Furthermore, we find that the signal sequence directing secretion also influences the quantity of enzyme secreted, and that this varies widely amongst the cell types tested. Last, we find that replacing the 3'UTR on the cDNA encoding Chondroitinase ABC with that of β-actin is sufficient to target the enzyme to the neuronal growth cone when transfected into neurons. This also enhances neurite outgrowth on an inhibitory substrate. Some intracellular trafficking pathways are adversely affected by cryptic signals present in the bacterial gene sequence, whilst unexpectedly others are required for efficient secretion of the enzyme. Furthermore, targeting chondroitinase to the neuronal growth cone promotes its ability to increase neurite outgrowth on an inhibitory substrate. These findings are timely in view of the renewed prospects for

  10. 75 FR 49549 - ABC & D Recycling, Inc.-Lease and Operation Exemption-a Line of Railroad in Ware, MA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-13

    ... DEPARTMENT OF TRANSPORTATION Surface Transportation Board [Docket No. FD 35397] ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, MA ABC & D Recycling, Inc. (ABC & D..., ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, Massachusetts (STB...

  11. Investigating the dynamic nature of the ABC transporters: ABCB1 and MsbA as examples for the potential synergies of MD theory and EPR applications.

    PubMed

    Stockner, Thomas; Mullen, Anna; MacMillan, Fraser

    2015-10-01

    ABC transporters are primary active transporters found in all kingdoms of life. Human multidrug resistance transporter ABCB1, or P-glycoprotein, has an extremely broad substrate spectrum and confers resistance against chemotherapy drug treatment in cancer cells. The bacterial ABC transporter MsbA is a lipid A flippase and a homolog to the human ABCB1 transporter, with which it partially shares its substrate spectrum. Crystal structures of MsbA and ABCB1 have been solved in multiple conformations, providing a glimpse into the possible conformational changes the transporter could be going through during the transport cycle. Crystal structures are inherently static, while a dynamic picture of the transporter in motion is needed for a complete understanding of transporter function. Molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy can provide structural information on ABC transporters, but the strength of these two methods lies in the potential to characterise the dynamic regime of these transporters. Information from the two methods is quite complementary. MD simulations provide an all atom dynamic picture of the time evolution of the molecular system, though with a narrow time window. EPR spectroscopy can probe structural, environmental and dynamic properties of the transporter in several time regimes, but only through the attachment sites of an exogenous spin label. In this review the synergistic effects that can be achieved by combining the two methods are highlighted, and a brief methodological background is also presented. © 2015 Authors; published by Portland Press Limited.

  12. Molecular identification of an ABC transporter complex for manganese: analysis of a cyanobacterial mutant strain impaired in the photosynthetic oxygen evolution process.

    PubMed Central

    Bartsevich, V V; Pakrasi, H B

    1995-01-01

    During photosynthesis, the photosystem II (PSII) pigment-protein complex catalyzes oxygen evolution, a reaction in which a four-manganese ensemble plays a crucial role. Using a newly developed selection scheme, we have isolated BP13, a random photosynthesis-deficient mutant strain of the cyanobacterium, Synechocystis 6803. This mutant grew slowly under photoautotrophic conditions, and had a low oxygen evolution activity. Biochemical analysis revealed that the lesion in this mutant strain had specifically affected the Mn ensemble in PSII. Interestingly, incubation of BP13 cells with micromolar levels of added Mn induced rapid recovery of oxygen evolution activity. The mutant could be complemented with a fragment of wild-type chromosomal DNA containing three closely linked genes, mntA, mntB and mntC. These gene products showed significant sequence similarities with polypeptide components of bacterial permeases that are members of the 'ABC (ATP binding cassette) superfamily' of transporter proteins. We determined that in the BP13 strain, a single nucleotide change had resulted in the replacement of an alanine by an aspartic acid residue in MntA, a soluble protein containing ATP binding motifs. These results suggest that the mntCAB gene cluster encodes polypeptide components of a Mn transporter, the first such protein complex identified in any organism. PMID:7743991

  13. Polymorphisms in ABC Transporter Genes and Concentrations of Mercury in Newborns – Evidence from Two Mediterranean Birth Cohorts

    PubMed Central

    Llop, Sabrina; Engström, Karin; Ballester, Ferran; Franforte, Elisa; Alhamdow, Ayman; Pisa, Federica; Tratnik, Janja Snoj; Mazej, Datja; Murcia, Mario; Rebagliato, Marisa; Bustamante, Mariona; Sunyer, Jordi; Sofianou-Katsoulis, Αikaterini; Prasouli, Alexia; Antonopoulou, Eleni; Antoniadou, Ioanna; Nakou, Sheena; Barbone, Fabio; Horvat, Milena; Broberg, Karin

    2014-01-01

    Background The genetic background may influence methylmercury (MeHg) metabolism and neurotoxicity. ATP binding cassette (ABC) transporters actively transport various xenobiotics across biological membranes. Objective To investigate the role of ABC polymorphisms as modifiers of prenatal exposure to MeHg. Methods The study population consisted of participants (n = 1651) in two birth cohorts, one in Italy and Greece (PHIME) and the other in Spain (INMA). Women were recruited during pregnancy in Italy and Spain, and during the perinatal period in Greece. Total mercury concentrations were measured in cord blood samples by atomic absorption spectrometry. Maternal fish intake during pregnancy was determined from questionnaires. Polymorphisms (n = 5) in the ABC genes ABCA1, ABCB1, ABCC1 and ABCC2 were analysed in both cohorts. Results ABCB1 rs2032582, ABCC1 rs11075290, and ABCC2 rs2273697 modified the associations between maternal fish intake and cord blood mercury concentrations. The overall interaction coefficient between rs2032582 and log2-transformed fish intake was negative for carriers of GT (β = −0.29, 95%CI −0.47, −0.12) and TT (β = −0.49, 95%CI −0.71, −0.26) versus GG, meaning that for a doubling in fish intake of the mothers, children with the rs2032582 GG genotype accumulated 35% more mercury than children with TT. For rs11075290, the interaction coefficient was negative for carriers of TC (β = −0.12, 95%CI −0.33, 0.09), and TT (β = −0.28, 95%CI −0.51, −0.06) versus CC. For rs2273697, the interaction coefficient was positive when combining GA+AA (β = 0.16, 95%CI 0.01, 0.32) versus GG. Conclusion The ABC transporters appear to play a role in accumulation of MeHg during early development. PMID:24831289

  14. Caenorhabditis elegans ABCRNAi transporters interact genetically with rde-2 and mut-7.

    PubMed

    Sundaram, Prema; Han, Wang; Cohen, Nancy; Echalier, Benjamin; Albin, John; Timmons, Lisa

    2008-02-01

    RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABC(RNAi) mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABC(RNAi) gene class. Genetic complementation tests reveal functions for ABC(RNAi) transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABC(RNAi) proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABC(RNAi) mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABC(RNAi) gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABC(RNAi) transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity.

  15. Multiple ABC transporters are involved in the acquisition of petrobactin in Bacillus anthracis

    PubMed Central

    Dixon, Shandee D.; Janes, Brian K.; Bourgis, Alexandra; Carlson, Paul E.; Hanna, Philip C.

    2012-01-01

    Summary In Bacillus anthracis the siderophore petrobactin is vital for iron acquisition and virulence. The petrobactin-binding receptor FpuA is required for these processes. Here additional components of petrobactin reacquisition are described. To identify these proteins, mutants of candidate permease and ATPase genes were generated allowing for characterization of multiple petrobactin ATP-binding cassette (ABC)-import systems. Either of two distinct permeases, FpuB or FatCD, are required for iron acquisition and play redundant roles in petrobactin transport. A mutant strain lacking both permeases, ΔfpuBΔfatCD, was incapable of using petrobactin as an iron source and exhibited attenuated virulence in a murine model of inhalational anthrax infection. ATPase mutants were generated in either of the permease mutant backgrounds to identify the ATPase(s) interacting with each individual permease channel. Mutants lacking the FpuB permease and FatE ATPase (ΔfpuBΔfatE) and a mutant lacking the distinct ATPases FpuC and FpuD generated in the ΔfatCD background (ΔfatCDΔfpuCΔfpuD) displayed phenotypic characteristics of a mutant deficient in petrobactin import. A mutant lacking all three of the identified ATPases (ΔfatEΔfpuCΔfpuD) exhibited the same growth defect in iron-depleted conditions. Taken together, these results provide the first description of the permease and ATPase proteins required for the import of petrobactin in B. anthracis. PMID:22429808

  16. Phosphorylation is required for the pathogen defense function of the Arabidopsis PEN3 ABC transporter.

    PubMed

    Underwood, William; Somerville, Shauna C

    2017-10-03

    The Arabidopsis PEN3 ABC transporter accumulates at sites of pathogen detection, where it is involved in defense against a number of pathogens. Perception of PAMPs by pattern recognition receptors initiates recruitment of PEN3 and also leads to PEN3 phosphorylation at multiple amino acid residues. Whether PAMP-induced phosphorylation of PEN3 is important for its defense function or focal recruitment has not been addressed. In this study, we evaluated the role of PEN3 phosphorylation in modulating the localization and defense function of the transporter. We report that PEN3 phosphorylation is critical for its function in defense, but dispensable for recruitment to powdery mildew penetration sites. These results indicate that PAMP-induced phosphorylation is likely to regulate the transport activity of PEN3.

  17. A mutation within the extended X loop abolished substrate-induced ATPase activity of the human liver ATP-binding cassette (ABC) transporter MDR3.

    PubMed

    Kluth, Marianne; Stindt, Jan; Dröge, Carola; Linnemann, Doris; Kubitz, Ralf; Schmitt, Lutz

    2015-02-20

    The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells

    PubMed Central

    Yeh, Johannes T.-H.; Nam, Kwangho; Yeh, Joshua T.-H.; Perrimon, Norbert

    2017-01-01

    The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter. PMID:28176814

  19. Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

    PubMed

    Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

    2013-01-01

    Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation

  20. 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...

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

    PubMed

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

    2016-03-01

    Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development. © 2015 John Wiley & Sons Ltd.

  2. Characterization and regulation of the resistance-nodulation-cell division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora.

    PubMed

    Pletzer, Daniel; Weingart, Helge

    2014-07-11

    The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the efflux pumps are involved in

  3. Characterization and regulation of the Resistance-Nodulation-Cell Division-type multidrug efflux pumps MdtABC and MdtUVW from the fire blight pathogen Erwinia amylovora

    PubMed Central

    2014-01-01

    Background The Gram-negative bacterium Erwinia amylovora is the causal agent of the devastating disease fire blight in rosaceous plants such as apple, pear, quince, raspberry, and cotoneaster. In order to survive and multiply in a host, microbes must be able to circumvent the toxic effects of antimicrobial plant compounds, such as flavonoids and tannins. E. amylovora uses multidrug efflux transporters that recognize and actively export toxic compounds out of the cells. Here, two heterotrimeric resistance-nodulation-cell division (RND)-type multidrug efflux pumps, MdtABC and MdtUVW, from E. amylovora were identified. These RND systems are unusual in that they contain two different RND proteins forming a functional pump. Results To find the substrate specificities of the two efflux systems, we overexpressed the transporters in a hypersensitive mutant lacking the major RND pump AcrB. Both transporters mediated resistance to several flavonoids, fusidic acid and novobiocin. Additionally, MdtABC mediated resistance towards josamycin, bile salts and silver nitrate, and MdtUVW towards clotrimazole. The ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock was reduced. Quantitative RT-PCR analyses revealed that the expression of the transporter genes was induced during infection of apple rootstock. The polyphenolic plant compound tannin, as well as the heavy metal salt tungstate was found to induce the expression of mdtABC. Finally, the expression of the mdtABC genes was shown to be regulated by BaeR, the response regulator of the two-component system BaeSR, a cell envelope stress response system that controls the adaptive responses to changes in the environment. Conclusions The expression of MdtABC and MdtUVW is induced during growth of E. amylovora in planta. We identified the plant polyphenol tannin as inducer of mdtABC expression. The reduced ability of the mdtABC- and mdtUVW-deficient mutants to multiply in apple rootstock suggests that the

  4. 75 FR 11991 - ABC & D Recycling, Inc.-Lease and Operation Exemption-a Line of Railroad in Ware, MA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... DEPARTMENT OF TRANSPORTATION Surface Transportation Board [STB Finance Docket No. 35356] ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, MA ABC & D Recycling, Inc. (ABC & D), a noncarrier, has filed a verified notice of exemption under 49 CFR 1150.31 to lease from O...

  5. Chemiluminescence Immunoassay for the Detection of Antibodies against the 2C and 3ABC Nonstructural Proteins Induced by Infecting Pigs with Foot-and-Mouth Disease Virus.

    PubMed

    Liu, Zezhong; Shao, Junjun; Zhao, Furong; Zhou, Guangqing; Gao, Shandian; Liu, Wei; Lv, Jianliang; Li, Xiumei; Li, Yangfan; Chang, Huiyun; Zhang, Yongguang

    2017-08-01

    The potential diagnostic value of chemiluminescence immunoassays (CLIAs) has been accepted in recent years, although their use for foot-and-mouth disease (FMD) diagnostics has not been reported. Full-length 3ABC and 2C proteins were expressed in bacteria and purified by affinity chromatography to develop a rapid and accurate approach to distinguish pigs infected with foot-and-mouth disease virus (FMDV) from vaccinated pigs. The recombinant proteins were then used as antigens to develop two CLIAs for the detection of antibodies against nonstructural viral proteins. The diagnostic performance of the two assays was compared by analyzing serum from pigs (naive pigs, n = 63; vaccinated, uninfected pigs, n = 532; naive, infected pigs, n = 117) with a known infection status. The 3ABC-2C CLIA had a higher accuracy rate, with a diagnostic sensitivity of 100% and a diagnostic specificity of 96.5%, than the 3ABC CLIA, which had a diagnostic sensitivity of 95.7% and a diagnostic specificity of 96.0%. The results of the 3ABC-2C CLIA also had a high rate of concordance with those of two commercial FMDV enzyme-linked immunosorbent assay (ELISA) kits used to assess serum collected from 962 pigs in the field (96.2% and 97.8%, respectively). The 3ABC-2C CLIA detected infection in serum samples from infected pigs earlier than the commercial ELISA kits. In addition, the 3ABC-2C CLIA produced results within 15 min. On the basis of these findings, the 3ABC-2C CLIA could serve as the foundation for the development of penside FMD diagnostics and offers an alternative method to detect FMDV infections. Copyright © 2017 American Society for Microbiology.

  6. A Novel Class of Modular Transporters for Vitamins in Prokaryotes ▿ †

    PubMed Central

    Rodionov, Dmitry A.; Hebbeln, Peter; Eudes, Aymerick; ter Beek, Josy; Rodionova, Irina A.; Erkens, Guus B.; Slotboom, Dirk J.; Gelfand, Mikhail S.; Osterman, Andrei L.; Hanson, Andrew D.; Eitinger, Thomas

    2009-01-01

    The specific and tightly controlled transport of numerous nutrients and metabolites across cellular membranes is crucial to all forms of life. However, many of the transporter proteins involved have yet to be identified, including the vitamin transporters in various human pathogens, whose growth depends strictly on vitamin uptake. Comparative analysis of the ever-growing collection of microbial genomes coupled with experimental validation enables the discovery of such transporters. Here, we used this approach to discover an abundant class of vitamin transporters in prokaryotes with an unprecedented architecture. These transporters have energy-coupling modules comprised of a conserved transmembrane protein and two nucleotide binding proteins similar to those of ATP binding cassette (ABC) transporters, but unlike ABC transporters, they use small integral membrane proteins to capture specific substrates. We identified 21 families of these substrate capture proteins, each with a different specificity predicted by genome context analyses. Roughly half of the substrate capture proteins (335 cases) have a dedicated energizing module, but in 459 cases distributed among almost 100 gram-positive bacteria, including numerous human pathogens, different and unrelated substrate capture proteins share the same energy-coupling module. The shared use of energy-coupling modules was experimentally confirmed for folate, thiamine, and riboflavin transporters. We propose the name energy-coupling factor transporters for the new class of membrane transporters. PMID:18931129

  7. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter

    PubMed Central

    Benedet, Mattia; Falchi, Federica A.; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra

    2016-01-01

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptFSupC). In complementation tests, lptFSupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine. PMID:27529623

  8. The Lack of the Essential LptC Protein in the Trans-Envelope Lipopolysaccharide Transport Machine Is Circumvented by Suppressor Mutations in LptF, an Inner Membrane Component of the Escherichia coli Transporter.

    PubMed

    Benedet, Mattia; Falchi, Federica A; Puccio, Simone; Di Benedetto, Cristiano; Peano, Clelia; Polissi, Alessandra; Dehò, Gianni

    2016-01-01

    The lipopolysaccharide (LPS) transport (Lpt) system is responsible for transferring LPS from the periplasmic surface of the inner membrane (IM) to the outer leaflet of the outer membrane (OM), where it plays a crucial role in OM selective permeability. In E. coli seven essential proteins are assembled in an Lpt trans-envelope complex, which is conserved in γ-Proteobacteria. LptBFG constitute the IM ABC transporter, LptDE form the OM translocon for final LPS delivery, whereas LptC, an IM-anchored protein with a periplasmic domain, interacts with the IM ABC transporter, the periplasmic protein LptA, and LPS. Although essential, LptC can tolerate several mutations and its role in LPS transport is unclear. To get insights into the functional role of LptC in the Lpt machine we searched for viable mutants lacking LptC by applying a strong double selection for lptC deletion mutants. Genome sequencing of viable ΔlptC mutants revealed single amino acid substitutions at a unique position in the predicted large periplasmic domain of the IM component LptF (LptFSupC). In complementation tests, lptFSupC mutants suppress lethality of both ΔlptC and lptC conditional expression mutants. Our data show that mutations in a specific residue of the predicted LptF periplasmic domain can compensate the lack of the essential protein LptC, implicate such LptF domain in the formation of the periplasmic bridge between the IM and OM complexes, and suggest that LptC may have evolved to improve the performance of an ancestral six-component Lpt machine.

  9. Alzheimer's disease: neuroprogesterone, epoxycholesterol, and ABC transporters as determinants of neurodesmosterol tissue levels and its role in amyloid protein processing.

    PubMed

    Javitt, Norman B

    2013-01-01

    Evidence is emerging that during the development of Alzheimer's disease (AD), changes in the synthesis and metabolism of cholesterol and progesterone are occurring that may or may not affect the progression of the disease. The concept arose from the recognition that dehydrocholesterol 24-reductase (DHCR24/Seladin-1), one of the nine enzymes in the endoplasmic reticulum that determines the transformation of lanosterol to cholesterol, is selectively reduced in late AD. As a consequence, the tissue level of desmosterol increases, affecting the expression of ABC transporters and the structure of lipid rafts, both determinants of amyloid-β processing. However, the former effect is considered beneficial and the latter detrimental to processing. Other determinants of desmosterol tissue levels are 24,25 epoxycholesterol and the ABCG1 and ABCG4 transporters. Progesterone and its metabolites are determinants of tissue levels of desmosterol and several other sterol intermediates in cholesterol synthesis. Animal models indicate marked elevations in the tissue levels of these sterols at early time frames in the progression of neurodegenerative diseases. The low level of neuroprogesterone and metabolites in AD are consonant with the low level of desmosterol and may have a role in amyloid-β processing. The sparse data that has accumulated appears to be a sufficient basis for proposing a systematic evaluation of the biologic roles of sterol intermediates in the slowly progressive neurodegeneration characteristic of AD.

  10. The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics.

    PubMed

    Pletzer, Daniel; Braun, Yvonne; Dubiley, Svetlana; Lafon, Corinne; Köhler, Thilo; Page, Malcolm G P; Mourez, Michael; Severinov, Konstantin; Weingart, Helge

    2015-07-01

    Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake

  11. Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens

    PubMed Central

    Wilms, Ina; Möller, Philip; Stock, Anna-Maria; Gurski, Rosemarie; Lai, Erh-Min

    2012-01-01

    The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of an hfq mutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineer Agrobacterium tumefaciens binds to the previously described sRNA AbcR1 and its target mRNA atu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in an hfq mutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility of A. tumefaciens. The hfq mutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation by A. tumefaciens. PMID:22821981

  12. Regulation of Expression of abcA and Its Response to Environmental Conditions

    PubMed Central

    Villet, Regis A.; Truong-Bolduc, Que Chi; Wang, Yin; Estabrooks, Zoe; Medeiros, Heidi

    2014-01-01

    The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic β-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA. PMID:24509312

  13. Emission of volatile organic compounds from petunia flowers is facilitated by an ABC transporter.

    PubMed

    Adebesin, Funmilayo; Widhalm, Joshua R; Boachon, Benoît; Lefèvre, François; Pierman, Baptiste; Lynch, Joseph H; Alam, Iftekhar; Junqueira, Bruna; Benke, Ryan; Ray, Shaunak; Porter, Justin A; Yanagisawa, Makoto; Wetzstein, Hazel Y; Morgan, John A; Boutry, Marc; Schuurink, Robert C; Dudareva, Natalia

    2017-06-30

    Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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

    PubMed Central

    Cole, Susan P. C.

    2014-01-01

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

  15. Comparative genomics of transport proteins in developmental bacteria: Myxococcus xanthus and Streptomyces coelicolor

    PubMed Central

    2013-01-01

    Background Two of the largest fully sequenced prokaryotic genomes are those of the actinobacterium, Streptomyces coelicolor (Sco), and the δ-proteobacterium, Myxococcus xanthus (Mxa), both differentiating, sporulating, antibiotic producing, soil microbes. Although the genomes of Sco and Mxa are the same size (~9 Mbp), Sco has 10% more genes that are on average 10% smaller than those in Mxa. Results Surprisingly, Sco has 93% more identifiable transport proteins than Mxa. This is because Sco has amplified several specific types of its transport protein genes, while Mxa has done so to a much lesser extent. Amplification is substrate- and family-specific. For example, Sco but not Mxa has amplified its voltage-gated ion channels but not its aquaporins and mechano-sensitive channels. Sco but not Mxa has also amplified drug efflux pumps of the DHA2 Family of the Major Facilitator Superfamily (MFS) (49 versus 6), amino acid transporters of the APC Family (17 versus 2), ABC-type sugar transport proteins (85 versus 6), and organic anion transporters of several families. Sco has not amplified most other types of transporters. Mxa has selectively amplified one family of macrolid exporters relative to Sco (16 versus 1), consistent with the observation that Mxa makes more macrolids than does Sco. Conclusions Except for electron transport carriers, there is a poor correlation between the types of transporters found in these two organisms, suggesting that their solutions to differentiative and metabolic needs evolved independently. A number of unexpected and surprising observations are presented, and predictions are made regarding the physiological functions of recognizable transporters as well as the existence of yet to be discovered transport systems in these two important model organisms and their relatives. The results provide insight into the evolutionary processes by which two dissimilar prokaryotes evolved complexity, particularly through selective chromosomal gene

  16. The Yersinia pestis Siderophore, Yersiniabactin, and the ZnuABC system both contribute to Zinc acquisition and the development of lethal septicemic plague in mice

    PubMed Central

    Bobrov, Alexander G.; Kirillina, Olga; Fetherston, Jacqueline D.; Miller, M. Clarke; Burlison, Joseph A.; Perry, Robert D.

    2014-01-01

    Summary Bacterial pathogens must overcome host sequestration of zinc (Zn2+), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn2+ by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn2+-deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn2+ acquisition. Studies with the Zn2+-dependent transcriptional reporter znuA∷lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn2+. However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, that are required for Fe3+ acquisition by Ybt, are not needed for Ybt-dependent Zn2+ uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn2+ uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicemic plague mouse model. PMID:24979062

  17. The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice.

    PubMed

    Bobrov, Alexander G; Kirillina, Olga; Fetherston, Jacqueline D; Miller, M Clarke; Burlison, Joseph A; Perry, Robert D

    2014-08-01

    Bacterial pathogens must overcome host sequestration of zinc (Zn(2+) ), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn(2+) by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn(2+) -deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn(2+) acquisition. Studies with the Zn(2+) -dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn(2+) . However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe(3+) acquisition by Ybt, are not needed for Ybt-dependent Zn(2+) uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn(2+) uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model. © 2014 John Wiley & Sons Ltd.

  18. Substrate Binding Protein DppA1 of ABC Transporter DppBCDF Increases Biofilm Formation in Pseudomonas aeruginosa by Inhibiting Pf5 Prophage Lysis

    PubMed Central

    Lee, Yunho; Song, Sooyeon; Sheng, Lili; Zhu, Lei; Kim, Jun-Seob; Wood, Thomas K.

    2018-01-01

    Filamentous phage impact biofilm development, stress tolerance, virulence, biofilm dispersal, and colony variants. Previously, we identified 137 Pseudomonas aeruginosa PA14 mutants with more than threefold enhanced and 88 mutants with more than 10-fold reduced biofilm formation by screening 5850 transposon mutants (PLoS Pathogens 5: e1000483, 2009). Here, we characterized the function of one of these 225 mutations, dppA1 (PA14_58350), in regard to biofilm formation. DppA1 is a substrate-binding protein (SBP) involved in peptide utilization via the DppBCDF ABC transporter system. We show that compared to the wild-type strain, inactivating dppA1 led to 68-fold less biofilm formation in a static model and abolished biofilm formation in flow cells. Moreover, the dppA1 mutant had a delay in swarming and produced 20-fold less small-colony variants, and both biofilm formation and swarming were complemented by producing DppA1. A whole-transcriptome analysis showed that only 10 bacteriophage Pf5 genes were significantly induced in the biofilm cells of the dppA1 mutant compared to the wild-type strain, and inactivation of dppA1 resulted in a 600-fold increase in Pf5 excision and a million-fold increase in phage production. As expected, inactivating Pf5 genes PA0720 and PA0723 increased biofilm formation substantially. Inactivation of DppA1 also reduced growth (due to cell lysis). Hence, DppA1 increases biofilm formation by repressing Pf5 prophage. PMID:29416528

  19. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    PubMed

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-04

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

  20. An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

    PubMed Central

    Bearden, Scott W.; Staggs, Teanna M.; Perry, Robert D.

    1998-01-01

    The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The ∼21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media. PMID:9495751

  1. An ABC transporter system of Yersinia pestis allows utilization of chelated iron by Escherichia coli SAB11.

    PubMed

    Bearden, S W; Staggs, T M; Perry, R D

    1998-03-01

    The acquisition of iron is an essential component in the pathogenesis of Yersinia pestis, the agent of bubonic and pneumonic plague. A cosmid library derived from the genomic DNA of Y. pestis KIM6+ was used for transduction of an Escherichia coli mutant (SAB11) defective in the biosynthesis of the siderophore enterobactin. Recombinant plasmids which had a common 13-kb BamHI fragment were isolated from SAB11 transductants in which growth but not enterobactin synthesis was restored on media containing the iron chelator EDDA [ethylenediamine-di(o-hydroxyphenyl acetic acid)]. Subcloning and transposon mutagenesis revealed a 5.6-kb region, designated yfe, essential for SAB11 growth stimulation. In vitro transcription-translation analysis identified polypeptides of 18, 29.5, 32, and 33 kDa encoded by the yfe locus. Sequence analysis shows this locus to be comprised of five genes in two separate operons which have potential Fur-binding sequences in both promoters. A putative polycistronic operon, yfeABCD, is Fur regulated and responds to iron and manganese. A functional Fur protein is required for the observed manganese repression of this operon. This operon encodes polypeptides which have strong similarity to the ATP-binding cassette (ABC) family of transporters and include a periplasmic binding protein (YfeA), an ATP-binding protein (YfeB), and two integral membrane proteins (YfeC and -D), which likely function in the acquisition of inorganic iron and possibly other ions. The approximately 21-kDa protein encoded by the separately transcribed yfeE gene may be located in the cell envelope, since a yfeE::TnphoA fusion is PhoA+. Mutations in this gene abrogate growth of SAB11 on iron-chelated media.

  2. Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

    PubMed Central

    Baglieri, Jacopo; Beck, Daniel; Vasisht, Nishi; Smith, Corinne J.; Robinson, Colin

    2012-01-01

    The twin-arginine translocation (Tat) system transports folded proteins across bacterial and plant thylakoid membranes. Most current models for the translocation mechanism propose the coalescence of a substrate-binding TatABC complex with a separate TatA complex. In Escherichia coli, TatA complexes are widely believed to form the translocation pore, and the size variation of TatA has been linked to the transport of differently sized substrates. Here, we show that the TatA paralog TatE can substitute for TatA and support translocation of Tat substrates including AmiA, AmiC, and TorA. However, TatE is found as much smaller, discrete complexes. Gel filtration and blue native electrophoresis suggest sizes between ∼50 and 110 kDa, and single-particle processing of electron micrographs gives size estimates of 70–90 kDa. Three-dimensional models of the two principal TatE complexes show estimated diameters of 6–8 nm and potential clefts or channels of up to 2.5 nm diameter. The ability of TatE to support translocation of the 90-kDa TorA protein suggests alternative translocation models in which single TatA/E complexes do not contribute the bulk of the translocation channel. The homogeneity of both the TatABC and the TatE complexes further suggests that a discrete Tat translocase can translocate a variety of substrates, presumably through the use of a flexible channel. The presence and possible significance of double- or triple-ring TatE forms is discussed. PMID:22190680

  3. Functional significance of the E loop, a novel motif conserved in the lantibiotic immunity ATP-binding cassette transport systems.

    PubMed

    Okuda, Ken-ichi; Yanagihara, Sae; Sugayama, Tomomichi; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

    2010-06-01

    Lantibiotics are peptide-derived antibacterial substances produced by some Gram-positive bacteria and characterized by the presence of unusual amino acids, like lanthionines and dehydrated amino acids. Because lantibiotic producers may be attacked by self-produced lantibiotics, they express immunity proteins on the cytoplasmic membrane. An ATP-binding cassette (ABC) transport system mediated by the LanFEG protein complex is a major system in lantibiotic immunity. Multiple-sequence alignment analysis revealed that LanF proteins contain the E loop, a variant of the Q loop, which is a well-conserved motif in the nucleotide-binding domains (NBDs) of general ABC transporters. To elucidate E loop function, we introduced a mutation in the NukF protein, which is involved in the nukacin-ISK-1 immunity system. Amino acid replacement of glutamic acid in the E loop with glutamine (E85Q) resulted in slight decreases in the immunity level and transport activity. Additionally, the E85A mutation severely impaired the immunity level and transport activity. On the other hand, ATPase activities of purified E85Q and E85A mutants were almost similar to that of the wild type. These results suggested that the E loop found in ABC transporters involved in lantibiotic immunity plays a significant role in the function of these transporters, especially in the structural change of transmembrane domains.

  4. Separating the roles of acropetal and basipetal auxin transport on gravitropism with mutations in two Arabidopsis multidrug resistance-like ABC transporter genes.

    PubMed

    Lewis, Daniel R; Miller, Nathan D; Splitt, Bessie L; Wu, Guosheng; Spalding, Edgar P

    2007-06-01

    Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90 degrees reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation.

  5. Structural basis for allosteric cross-talk between the asymmetric nucleotide binding sites of a heterodimeric ABC exporter.

    PubMed

    Hohl, Michael; Hürlimann, Lea M; Böhm, Simon; Schöppe, Jendrik; Grütter, Markus G; Bordignon, Enrica; Seeger, Markus A

    2014-07-29

    ATP binding cassette (ABC) transporters mediate vital transport processes in every living cell. ATP hydrolysis, which fuels transport, displays positive cooperativity in numerous ABC transporters. In particular, heterodimeric ABC exporters exhibit pronounced allosteric coupling between a catalytically impaired degenerate site, where nucleotides bind tightly, and a consensus site, at which ATP is hydrolyzed in every transport cycle. Whereas the functional phenomenon of cooperativity is well described, its structural basis remains poorly understood. Here, we present the apo structure of the heterodimeric ABC exporter TM287/288 and compare it to the previously solved structure with adenosine 5'-(β,γ-imido)triphosphate (AMP-PNP) bound at the degenerate site. In contrast to other ABC exporter structures, the nucleotide binding domains (NBDs) of TM287/288 remain in molecular contact even in the absence of nucleotides, and the arrangement of the transmembrane domains (TMDs) is not influenced by AMP-PNP binding, a notion confirmed by double electron-electron resonance (DEER) measurements. Nucleotide binding at the degenerate site results in structural rearrangements, which are transmitted to the consensus site via two D-loops located at the NBD interface. These loops owe their name from a highly conserved aspartate and are directly connected to the catalytically important Walker B motif. The D-loop at the degenerate site ties the NBDs together even in the absence of nucleotides and substitution of its aspartate by alanine is well-tolerated. By contrast, the D-loop of the consensus site is flexible and the aspartate to alanine mutation and conformational restriction by cross-linking strongly reduces ATP hydrolysis and substrate transport.

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

    PubMed Central

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

    2013-01-01

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

  7. Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

    PubMed

    Tanaka, Shiho; Endo, Haruka; Adegawa, Satomi; Iizuka, Ami; Imamura, Kazuhiro; Kikuta, Shingo; Sato, Ryoichi

    2017-12-01

    Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770 DYWL 773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770 DYWL 773 of ECL 4 in the ABCC2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Transport capabilities of environmental Pseudomonads for sulfur compounds

    DOE PAGES

    Zerbs, Sarah; Korajczyk, Peter J.; Noirot, Philippe H.; ...

    2017-01-27

    Sulfur is an essential element in plant rhizospheres and microbial activity plays a key role in increasing the biological availability of sulfur in soil environments. To better understand the mechanisms facilitating the exchange of sulfur-containing molecules in soil, we profiled the binding specificities of eight previously uncharacterized ABC transporter solute-binding proteins from plant-associated Pseudomonads. A high-throughput screening procedure indicated eighteen significant organosulfur binding ligands, with at least one high-quality screening hit for each protein target. Calorimetric and spectroscopic methods were used to validate the best ligand assignments and catalog the thermodynamic properties of the protein-ligand interactions. Two novel high-affinity ligandmore » binding activities were identified and quantified in this set of solute binding proteins. Bacteria were cultured in minimal media with screening library components supplied as the sole sulfur sources, demonstrating that these organosulfur compounds can be metabolized and confirming the relevance of ligand assignments. These results expand the set of experimentally validated ligands amenable to transport by this ABC transporter family and demonstrate the complex range of protein-ligand interactions that can be accomplished by solute-binding proteins. As a result, characterizing new nutrient import pathways provides insight into Pseudomonad metabolic capabilities which can be used to further interrogate bacterial survival and participation in soil and rhizosphere communities.« less

  9. Expression, purification and thermostability of MBP-chondroitinase ABC I from Proteus vulgaris.

    PubMed

    Chen, Zhenya; Li, Ye; Yuan, Qipeng

    2015-01-01

    Chondroitinase ABC I (ChSase ABC I) which can degrade chondroitin sulfate (CS) and other glycosaminoglycan to oligosaccharide or unsaturated disaccharide, was fusionally expressed with maltose-binding protein (MBP) in Escherichia coli BL21(DE3) (E. coli BL21(DE3)) and purified for the first time in this study. The result showed that the productivity of recombinant MBP-ChSase ABC I was 3180 IU/(L fermentation liquor) with CS A as substrate, and the productivity might be the highest level when compared to the reported ones. The specific activity of recombinant MBP-ChSase ABC I was 76 IU/(mg protein) after purification. The Vmax, Km and kcat were 18.7 ± 0.3 μmol/Ls, 73.1 ± 4.1 μmol/L and 586.7 ± 10.8 s(-1), respectively. Enzyme activity of the purified enzyme remained about 78% after 210 min when the enzyme incubated at 30 °C. This study introduces a rapid method for highly expressing ChSase ABC I, and the method could be adopted in the process of industrial production. Furthermore the investigation of thermostability might lead to an important guide in clinical treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Genetic polymorphisms of ATP-binding cassette (ABC) proteins, overall survival and drug toxicity in patients with Acute Myeloid Leukemia

    PubMed Central

    Hampras, Shalaka S; Sucheston, Lara; Weiss, Joli; Baer, Maria R; Zirpoli, Gary; Singh, Prashant K; Wetzler, Meir; Chennamaneni, Raj; Blanco, Javier G; Ford, LaurieAnn; Moysich, Kirsten B

    2010-01-01

    The overall survival of patients with acute myeloid leukemia (AML) remains poor due to both intrinsic and acquired chemotherapy resistance. Over expression of ATP binding cassette (ABC) proteins in AML cells has been suggested as a putative mechanism of drug resistance. Genetic variation among individuals affecting the expression or function of these proteins may contribute to inter-individual variation in treatment outcomes. DNA from pre-treatment bone marrow or blood samples from 261 patients age 20-85 years, who received cytarabine and anthracycline-based therapy at Roswell Park Cancer Institute between 1994 and 2006, was genotyped for eight non-synonymous single nucleotide polymorphisms in the ABCB1, ABCC1 and ABCG2 drug transporter genes. Heterozygous (AG) or homozygous (AA) variant genotypes for rs2231137 (G34A) in the ABCG2 (BRCP) gene, compared to the wild type (GG) genotype were associated with both significantly improved survival (HR=0.44, 95%CI=0.25-0.79), and increased odds for toxicity (OR=8.41, 95%CI= 1.10-64.28). Thus genetic polymorphisms in the ABCG2 (BRCP) gene may contribute to differential survival outcomes and toxicities in AML patients via a mechanism of decreased drug efflux in both, AML cells and normal progenitors. PMID:21311724

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

    PubMed

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

    2016-01-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Zhaojing; Xu, Yonghong; Meng, Xiangning

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

  13. Characterization of a lactose-responsive promoter of ATP-binding cassette (ABC) transporter gene from Lactobacillus acidophilus 05-172.

    PubMed

    Zeng, Zhu; Zuo, Fanglei; Yu, Rui; Zhang, Bo; Ma, Huiqin; Chen, Shangwu

    2017-09-01

    A novel lactose-responsive promoter of the ATP-binding cassette (ABC) transporter gene Lba1680 of Lactobacillus acidophilus strain 05-172 isolated from a traditionally fermented dairy product koumiss was characterized. In L. acidophilus 05-172, expression of Lba1680 was induced by lactose, with lactose-induced transcription of Lba1680 being 6.1-fold higher than that induced by glucose. This is in contrast to L. acidophilus NCFM, a strain isolated from human feces, in which expression of Lba1680 and Lba1679 is induced by glucose. Both gene expression and enzyme activity assays in L. paracasei transformed with a vector containing the inducible Lba1680 promoter (PLba1680) of strain 05-172 and a heme-dependent catalase gene as reporter confirmed that PLba1680 is specifically induced by lactose. Its regulatory expression could not be repressed by glucose, and was independent of cAMP receptor protein. This lactose-responsive promoter might be used in the expression of functional genes in L. paracasei incorporated into a lactose-rich environment, such as dairy products. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. MntABC and MntH Contribute to Systemic Staphylococcus aureus Infection by Competing with Calprotectin for Nutrient Manganese

    PubMed Central

    Kehl-Fie, Thomas E.; Zhang, Yaofang; Moore, Jessica L.; Farrand, Allison J.; Hood, M. Indriati; Rathi, Subodh; Chazin, Walter J.; Caprioli, Richard M.

    2013-01-01

    During infection, vertebrates limit access to manganese and zinc, starving invading pathogens, such as Staphylococcus aureus, of these essential metals in a process termed “nutritional immunity.” The manganese and zinc binding protein calprotectin is a key component of the nutrient-withholding response, and mice lacking this protein do not sequester manganese from S. aureus liver abscesses. One potential mechanism utilized by S. aureus to minimize host-imposed manganese and zinc starvation is the expression of the metal transporters MntABC and MntH. We performed transcriptional analyses of both mntA and mntH, which revealed increased expression of both systems in response to calprotectin treatment. MntABC and MntH compete with calprotectin for manganese, which enables S. aureus growth and retention of manganese-dependent superoxide dismutase activity. Loss of MntABC and MntH results in reduced staphylococcal burdens in the livers of wild-type but not calprotectin-deficient mice, suggesting that these systems promote manganese acquisition during infection. During the course of these studies, we observed that metal content and the importance of calprotectin varies between murine organs, and infection leads to profound changes in the anatomical distribution of manganese and zinc. In total, these studies provide insight into the mechanisms utilized by bacteria to evade host-imposed nutrient metal starvation and the critical importance of restricting manganese availability during infection. PMID:23817615

  15. Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

    PubMed

    Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

    2016-09-01

    ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

  16. Multidrug resistance-associated protein 4 is a bile transporter of Clonorchis sinensis simulated by in silico docking.

    PubMed

    Dai, Fuhong; Yoo, Won Gi; Lee, Ji-Yun; Lu, Yanyan; Pak, Jhang Ho; Sohn, Woon-Mok; Hong, Sung-Jong

    2017-11-21

    Multidrug resistance-associated protein 4 (MRP4) is a member of the C subfamily of the ABC family of ATP-binding cassette (ABC) transporters. MRP4 regulates ATP-dependent efflux of various organic anionic substrates and bile acids out of cells. Since Clonorchis sinensis lives in host's bile duct, accumulation of bile juice can be toxic to the worm's tissues and cells. Therefore, C. sinensis needs bile transporters to reduce accumulation of bile acids within its body. We cloned MRP4 (CsMRP4) from C. sinensis and obtained a cDNA encoding an open reading frame of 1469 amino acids. Phylogenetic analysis revealed that CsMRP4 belonged to the MRP/SUR/CFTR subfamily. A tertiary structure of CsMRP4 was generated by homology modeling based on multiple structures of MRP1 and P-glycoprotein. CsMRP4 had two membrane-spanning domains (MSD1 & 2) and two nucleotide-binding domains (NBD1 & 2) as common structural folds. Docking simulation with nine bile acids showed that CsMRP4 transports bile acids through the inner cavity. Moreover, it was found that CsMRP4 mRNA was more abundant in the metacercariae than in the adults. Mouse immune serum, generated against the CsMRP4-NBD1 (24.9 kDa) fragment, localized CsMRP4 mainly in mesenchymal tissues and oral and ventral suckers of the metacercariae and the adults. Our findings shed new light on MRPs and their homologs and provide a platform for further structural and functional investigations on the bile transporters and parasites' survival.

  17. Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis.

    PubMed

    Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S; Bilalis, Dimitrios

    2016-04-20

    Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha(-1)) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance.

  18. Environmental Conditions Influence Induction of Key ABC-Transporter Genes Affecting Glyphosate Resistance Mechanism in Conyza canadensis

    PubMed Central

    Tani, Eleni; Chachalis, Demosthenis; Travlos, Ilias S.; Bilalis, Dimitrios

    2016-01-01

    Conyza canadensis has been reported to be the most frequent weed species that evolved resistance to glyphosate in various parts of the world. The objective of the present study was to investigate the effect of environmental conditions (temperature and light) on the expression levels of the EPSPS gene and two major ABC-transporter genes (M10 and M11) on glyphosate susceptible (GS) and glyphosate resistant (GR) horseweed populations, collected from several regions across Greece. Real-time PCR was conducted to determine the expression level of the aforementioned genes when glyphosate was applied at normal (1×; 533 g·a.e.·ha−1) and high rates (4×, 8×), measured at an early one day after treatment (DAT) and a later stage (four DAT) of expression. Plants were exposed to light or dark conditions, at three temperature regimes (8, 25, 35 °C). GR plants were made sensitive when exposed to 8 °C with light; those sensitized plants behaved biochemically (shikimate accumulation) and molecularly (expression of EPSPS and ABC-genes) like the GS plants. Results from the current study show the direct link between the environmental conditions and the induction level of the above key genes that likely affect the efficiency of the proposed mechanism of glyphosate resistance. PMID:27104532

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

    PubMed

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

    2014-12-01

    Sterol uptake in fungi is a multistep process that involves interaction between external sterols and the cell wall, incorporation of sterol molecules into the plasma membrane, and subsequent integration into intracellular membranes for turnover. ATP-binding cassette (ABC) transporters have been implicated in sterol uptake, but key features of their activity remain to be elucidated. Here, we apply fluorescent cholesterol (NBD-cholesterol) to monitor sterol uptake under anaerobic and aerobic conditions in two fungal species, Candida glabrata (Cg) and Saccharomyces cerevisiae (Sc). We found that in both fungal species, ABC transporter-dependent uptake of cholesterol under anaerobic conditions and in mutants lacking HEM1 gene is promoted in the presence of the serum protein albumin that is able to bind the sterol molecule. Furthermore, the C. glabrata ABC transporter CgAus1p expressed in S. cerevisiae requires the presence of serum or albumin for efficient cholesterol uptake. These results suggest that albumin can serve as sterol donor in ABC transporter-dependent sterol uptake, a process potentially important for growth of C. glabrata inside infected humans. © 2014 The Authors. FEMS Yeast Research published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

  20. Interaction of Food Additives with Intestinal Efflux Transporters.

    PubMed

    Sjöstedt, Noora; Deng, Feng; Rauvala, Oskari; Tepponen, Tuomas; Kidron, Heidi

    2017-11-06

    Breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2) and P-glycoprotein (P-gp) are ABC transporters that are expressed in the intestine, where they are involved in the efflux of many drugs from enterocytes back into the intestinal lumen. The inhibition of BCRP, MRP2, and P-gp can result in enhanced absorption and exposure of substrate drugs. Food additives are widely used by the food industry to improve the stability, flavor, and consistency of food products. Although they are considered safe for consumption, their interactions with intestinal transporters are poorly characterized. Therefore, in this study, selected food additives, including preservatives, colorants, and sweeteners, were studied in vitro for their inhibitory effects on intestinal ABC transporters. Among the studied compounds, several colorants were able to inhibit BCRP and MRP2, whereas P-gp was fairly insensitive to inhibition. Additionally, one sweetener was identified as a potent inhibitor of BCRP. Dose-response studies revealed that the IC 50 values of the inhibitors were lower than the estimated intestinal concentrations after the consumption of beverages containing food colorants. This suggests that there is potential for previously unrecognized transporter-mediated food additive-drug interactions.

  1. Vibrio cholerae NspS, a homologue of ABC-type periplasmic solute binding proteins, facilitates transduction of polyamine signals independent of their transport

    PubMed Central

    Cockerell, Steven R.; Rutkovsky, Alex C.; Zayner, Josiah P.; Cooper, Rebecca E.; Porter, Lindsay R.; Pendergraft, Sam S.; Parker, Zach M.; McGinnis, Marcus W.

    2014-01-01

    The polyamines norspermidine and spermidine are among the environmental signals that regulate Vibrio cholerae biofilm formation. The effects of these polyamines are mediated by NspS, a member of the bacterial periplasmic solute binding protein superfamily. Almost all members of this superfamily characterized to date are components of ATP-binding cassette-type transporters involved in nutrient uptake. Consequently, in the current annotation of the V. cholerae genome, NspS has been assigned a function in transport. The objective of this study was to further characterize NspS and investigate its potential role in transport. Our results support a role for NspS in signal transduction in response to norspermidine and spermidine, but not their transport. In addition, we provide evidence that these polyamine signals are processed by c-di-GMP signalling networks in the cell. Furthermore, we present comparative genomics analyses which reveal the presence of NspS-like proteins in a variety of bacteria, suggesting that periplasmic ligand binding proteins may be widely utilized for sensory transduction. PMID:24530989

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

    PubMed

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

    2015-11-01

    Abacavir is as a frequent part of combination antiretroviral therapy used in pregnant women. The aim of this study was to investigate, using in vitro, in situ and ex vivo experimental approaches, whether the transplacental pharmacokinetics of abacavir is affected by ATP-binding cassette (ABC) efflux transporters functionally expressed in the placenta: P-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2), multidrug resistance-associated protein 2 (ABCC2) and multidrug resistance-associated protein 5 (ABCC5). In vitro transport assays revealed that abacavir is a substrate of human ABCB1 and ABCG2 transporters but not of ABCC2 or ABCC5. In addition, in situ experiments using dually perfused rat term placenta confirmed interactions of abacavir with placental Abcb1/Abcg2. In contrast, uptake studies in human placental villous fragments did not reveal any interaction of abacavir with efflux transporters suggesting a large contribution of passive diffusion and/or influx mechanisms to net transplacental abacavir transfer. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Forkhead-associated (FHA) Domain Containing ABC Transporter Rv1747 Is Positively Regulated by Ser/Thr Phosphorylation in Mycobacterium tuberculosis*

    PubMed Central

    Spivey, Vicky L.; Molle, Virginie; Whalan, Rachael H.; Rodgers, Angela; Leiba, Jade; Stach, Lasse; Walker, K. Barry; Smerdon, Stephen J.; Buxton, Roger S.

    2011-01-01

    One major signaling method employed by Mycobacterium tuberculosis, the causative agent of tuberculosis, is through reversible phosphorylation of proteins mediated by protein kinases and phosphatases. This study concerns one of these enzymes, the serine/threonine protein kinase PknF, that is encoded in an operon with Rv1747, an ABC transporter that is necessary for growth of M. tuberculosis in vivo and contains two forkhead-associated (FHA) domains. FHA domains are phosphopeptide recognition motifs that specifically recognize phosphothreonine-containing epitopes. Experiments to determine how PknF regulates the function of Rv1747 demonstrated that phosphorylation occurs on two specific threonine residues, Thr-150 and Thr-208. To determine the in vivo consequences of phosphorylation, infection experiments were performed in bone marrow-derived macrophages and in mice using threonine-to-alanine mutants of Rv1747 that prevent specific phosphorylation and revealed that phosphorylation positively modulates Rv1747 function in vivo. The role of the FHA domains in this regulation was further demonstrated by isothermal titration calorimetry, using peptides containing both phosphothreonine residues. FHA-1 domain mutation resulted in attenuation in macrophages highlighting the critical role of this domain in Rv1747 function. A mutant deleted for pknF did not, however, have a growth phenotype in an infection, suggesting that other kinases can fulfill its role when it is absent. This study provides the first information on the molecular mechanism(s) regulating Rv1747 through PknF-dependent phosphorylation but also indicates that phosphorylation activates Rv1747, which may have important consequences in regulating growth of M. tuberculosis. PMID:21622570

  4. Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

    PubMed

    Murota, Yoshitaka; Tabu, Kouichi; Taga, Tetsuya

    2016-11-04

    Elucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism. The metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively. SP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of

  5. Purification and characterisation of the yeast plasma membrane ATP binding cassette transporter Pdr11p

    PubMed Central

    Laub, Katrine Rude; Marek, Magdalena; Stanchev, Lyubomir Dimitrov; Herrera, Sara Abad; Kanashova, Tamara; Bourmaud, Adèle; Dittmar, Gunnar

    2017-01-01

    The ATP binding cassette (ABC) transporters Pdr11p and its paralog Aus1p are expressed under anaerobic growth conditions at the plasma membrane of the yeast Saccharomyces cerevisiae and are required for sterol uptake. However, the precise mechanism by which these ABC transporters facilitate sterol movement is unknown. In this study, an overexpression and purification procedure was developed with the aim to characterise the Pdr11p transporter. Engineering of Pdr11p variants fused at the C terminus with green fluorescent protein (Pdr11p-GFP) and containing a FLAG tag at the N terminus facilitated expression analysis and one-step purification, respectively. The detergent-solubilised and purified protein displayed a stable ATPase activity with a broad pH optimum near 7.4. Mutagenesis of the conserved lysine to methionine (K788M) in the Walker A motif abolished ATP hydrolysis. Remarkably, and in contrast to Aus1p, ATPase activity of Pdr11p was insensitive to orthovanadate and not specifically stimulated by phosphatidylserine upon reconstitution into liposomes. Our results highlight distinct differences between Pdr11p and Aus1p and create an experimental basis for further biochemical studies of both ABC transporters to elucidate their function. PMID:28922409

  6. Overexpression of the ABC transporter AvtAB increases avermectin production in Streptomyces avermitilis.

    PubMed

    Qiu, Jingfan; Zhuo, Ying; Zhu, Dongqing; Zhou, Xiufen; Zhang, Lixin; Bai, Linquan; Deng, Zixin

    2011-10-01

    Avermectins are 16-membered macrocyclic polyketides with potent antiparasitic activities, produced by Streptomyces avermitilis. Upstream of the avermectin biosynthetic gene cluster, there is the avtAB operon encoding the ABC transporter AvtAB, which is highly homologous to the mammalian multidrug efflux pump P-glycoprotein (Pgp). Inactivation of avtAB had no effect, but increasing the concentration of avtAB mRNA 30-500-fold, using a multi-copy plasmid in S. avermitilis, enhanced avermectin production about two-fold both in the wild-type and in a high-yield producer strain on agar plates. In liquid industrial fermentation medium, the overall productivity of avermectin B1a in the engineered high-yield producer was improved for about 50%, from 3.3 to 4.8 g/l. In liquid YMG medium, moreover, the ratio of intracellular to extracellular accumulation of avermectin B1a was dropped from 6:1 to 4.5:1 in response to multiple copies of avtAB. Additionally, the overexpression of avtAB did not cause any increased expression of the avermectin biosynthetic genes through RT-PCR analysis. We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production inside the cell. This strategy may be useful for enhancing the production of other antibiotics.

  7. Strategies for neurotrophin-3 and chondroitinase ABC release from freeze-cast chitosan-alginate nerve-guidance scaffolds.

    PubMed

    Francis, Nicola L; Hunger, Philipp M; Donius, Amalie E; Wegst, Ulrike G K; Wheatley, Margaret A

    2017-01-01

    Freeze casting, or controlled unidirectional solidification, can be used to fabricate chitosan-alginate (C-A) scaffolds with highly aligned porosity that are suitable for use as nerve-guidance channels. To augment the guidance of growth across a spinal cord injury lesion, these scaffolds are now evaluated in vitro to assess their ability to release neurotrophin-3 (NT-3) and chondroitinase ABC (chABC) in a controlled manner. Protein-loaded microcapsules were incorporated into C-A scaffolds prior to freeze casting without affecting the original scaffold architecture. In vitro protein release was not significantly different when comparing protein loaded directly into the scaffolds with release from scaffolds containing incorporated microcapsules. NT-3 was released from the C-A scaffolds for 8 weeks in vitro, while chABC was released for up to 7 weeks. Low total percentages of protein released from the scaffolds over this time period were attributed to limitation of diffusion by the interpenetrating polymer network matrix of the scaffold walls. NT-3 and chABC released from the scaffolds retained bioactivity, as determined by a neurite outgrowth assay, and the promotion of neurite growth across an inhibitory barrier of chondroitin sulphate proteoglycans. This demonstrates the potential of these multifunctional scaffolds for enhancing axonal regeneration through growth-inhibiting glial scars via the sustained release of chABC and NT-3. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Role of cis-trans proline isomerization in the function of pathogenic enterobacterial Periplasmic Binding Proteins

    PubMed Central

    Cortes-Hernandez, Paulina

    2017-01-01

    Periplasmic Binding Proteins (PBPs) trap nutrients for their internalization into bacteria by ABC transporters. Ligand binding triggers PBP closure by bringing its two domains together like a Venus flytrap. The atomic determinants that control PBP opening and closure for nutrient capture and release are not known, although it is proposed that opening and ligand release occur while in contact with the ABC transporter for concurrent substrate translocation. In this paper we evaluated the effect of the isomerization of a conserved proline, located near the binding site, on the propensity of PBPs to open and close. ArgT/LAO from Salmonella typhimurium and HisJ from Escherichia coli were studied through molecular mechanics at two different temperatures: 300 and 323 K. Eight microseconds were simulated per protein to analyze protein opening and closure in the absence of the ABC transporter. We show that when the studied proline is in trans, closed empty LAO and HisJ can open. In contrast, with the proline in cis, opening transitions were much less frequent and characterized by smaller changes. The proline in trans also renders the open trap prone to close over a ligand. Our data suggest that the isomerization of this conserved proline modulates the PBP mechanism: the proline in trans allows the exploration of conformational space to produce trap opening and closure, while in cis it restricts PBP movement and could limit ligand release until in productive contact with the ABC transporter. This is the first time that a proline isomerization has been related to the control of a large conformational change like the PBP flytrap mechanism. PMID:29190818

  9. Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Le; Ghimire-Rijal, Sudipa; Lucas, Sarah L.

    Here, the ligand-induced conformational changes of periplasmic binding proteins (PBP) play a key role in the acquisition of metabolites in ATP binding cassette (ABC) transport systems. This conformational change allows for differential recognition of the ligand occupancy of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis in the transporter, a phenomenon in which ATP hydrolysis is not coupled to metabolite transport. In many systems, the PBP conformational change is insufficient at eliminating futile ATP hydrolysis. Here we identify an additional state of the PBP that is also allosterically regulated by the ligand. Ligand binding to the homodimericmore » apo PBP leads to a tightening of the interface alpha-helices so that the hydrogen bonding pattern shifts to that of a 3 10 helix, in-turn altering the contacts and the dynamics of the protein interface so that the monomer exists in the presence of ligand.« less

  10. Periplasmic Binding Protein Dimer Has a Second Allosteric Event Tied to Ligand Binding

    DOE PAGES

    Li, Le; Ghimire-Rijal, Sudipa; Lucas, Sarah L.; ...

    2017-09-06

    Here, the ligand-induced conformational changes of periplasmic binding proteins (PBP) play a key role in the acquisition of metabolites in ATP binding cassette (ABC) transport systems. This conformational change allows for differential recognition of the ligand occupancy of the PBP by the ABC transporter. This minimizes futile ATP hydrolysis in the transporter, a phenomenon in which ATP hydrolysis is not coupled to metabolite transport. In many systems, the PBP conformational change is insufficient at eliminating futile ATP hydrolysis. Here we identify an additional state of the PBP that is also allosterically regulated by the ligand. Ligand binding to the homodimericmore » apo PBP leads to a tightening of the interface alpha-helices so that the hydrogen bonding pattern shifts to that of a 3 10 helix, in-turn altering the contacts and the dynamics of the protein interface so that the monomer exists in the presence of ligand.« less

  11. Accelerated bridge construction (ABC) decision making and economic modeling tool.

    DOT National Transportation Integrated Search

    2011-12-01

    In this FHWA-sponsored pool funded study, a set of decision making tools, based on the Analytic Hierarchy Process (AHP) was developed. This tool set is prepared for transportation specialists and decision-makers to determine if ABC is more effective ...

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

    PubMed

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

    2015-11-01

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

  13. N-retinylidene-phosphatidylethanolamine is the preferred retinoid substrate for the photoreceptor-specific ABC transporter ABCA4 (ABCR).

    PubMed

    Beharry, Seelochan; Zhong, Ming; Molday, Robert S

    2004-12-24

    ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, approximately 0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent K(d) of 2-5 microm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5'-(beta,gamma-imido)triphosphate and guanosine 5'-(beta,gamma-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.

  14. Zinc transporters YbtX and ZnuABC are required for the virulence of Yersinia pestis in bubonic and pneumonic plague in mice.

    PubMed

    Bobrov, Alexander G; Kirillina, Olga; Fosso, Marina Y; Fetherston, Jacqueline D; Miller, M Clarke; VanCleave, Tiva T; Burlison, Joseph A; Arnold, William K; Lawrenz, Matthew B; Garneau-Tsodikova, Sylvie; Perry, Robert D

    2017-06-21

    A number of bacterial pathogens require the ZnuABC Zinc (Zn 2+ ) transporter and/or a second Zn 2+ transport system to overcome Zn 2+ sequestration by mammalian hosts. Previously we have shown that in addition to ZnuABC, Yersinia pestis possesses a second Zn 2+ transporter that involves components of the yersiniabactin (Ybt), siderophore-dependent iron transport system. Synthesis of the Ybt siderophore and YbtX, a member of the major facilitator superfamily, are both critical components of the second Zn 2+ transport system. Here we demonstrate that a ybtX znu double mutant is essentially avirulent in mouse models of bubonic and pneumonic plague while a ybtX mutant retains high virulence in both plague models. While sequestration of host Zn is a key nutritional immunity factor, excess Zn appears to have a significant antimicrobial role in controlling intracellular bacterial survival. Here, we demonstrate that ZntA, a Zn 2+ exporter, plays a role in resistance to Zn toxicity in vitro, but that a zntA zur double mutant retains high virulence in both pneumonic and bubonic plague models and survival in macrophages. We also confirm that Ybt does not directly bind Zn 2+ in vitro under the conditions tested. However, we detect a significant increase in Zn 2+ -binding ability of filtered supernatants from a Ybt + strain compared to those from a strain unable to produce the siderophore, supporting our previously published data that Ybt biosynthetic genes are involved in the production of a secreted Zn-binding molecule (zincophore). Our data suggest that Ybt or a modified Ybt participate in or promote Zn-binding activity in culture supernatants and is involved in Zn acquisition in Y. pestis.

  15. Characterization and expression profiling of ATP-binding cassette transporter genes in the diamondback moth, Plutella xylostella (L.).

    PubMed

    Qi, Weiping; Ma, Xiaoli; He, Weiyi; Chen, Wei; Zou, Mingmin; Gurr, Geoff M; Vasseur, Liette; You, Minsheng

    2016-09-27

    ATP-binding cassette (ABC) transporters are one of the major transmembrane protein families found in all organisms and play important roles in transporting a variety of compounds across intra and extra cellular membranes. In some species, ABC transporters may be involved in the detoxification of substances such as insecticides. The diamondback moth, Plutella xylostella (L.), a destructive pest of cruciferous crops worldwide, is an important species to study as it is resistant to many types of insecticides as well as biological control Bacillus thuringiensis toxins. A total of 82 ABC genes were identified from our published P. xylostella genome, and grouped into eight subfamilies (ABCA-H) based on phylogenetic analysis. Genes of subfamilies ABCA, ABCC and ABCH were found to be expanded in P. xylostella compared with those in Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens. Phylogenetic analysis indicated that many of the ABC transporters in P. xylostella are orthologous to the well-studied ABC transporter genes in the seven other species. Transcriptome- and qRT-PCR-based analysis elucidated physiological effects of ABC gene expressions of P. xylostella which were developmental stage- and tissue-specific as well as being affected by whether or not the insects were from an insecticide-resistant strain. Two ABCC and one ABCA genes were preferentially expressed in midgut of the 4th-instar larvae of a susceptible strain (Fuzhou-S) suggesting their potential roles in metabolizing plant defensive chemicals. Most of the highly expressed genes in insecticide-resistant strains were also predominantly expressed in the tissues of Malpighian tubules and midgut. This is the most comprehensive study on identification, characterization and expression profiling of ABC transporter genes in P. xylostella to date. The diversified features and expression patterns of this gene family may be associated with

  16. Expression analysis of MDR1, BCRP and MRP3 transporter proteins in different in vitro and ex vivo cornea models for drug absorption studies.

    PubMed

    Verstraelen, Jessica; Reichl, Stephan

    2013-01-30

    Ocular drug absorption studies are required for the development of new drugs or drug delivery systems for eye treatment. Such preclinical investigations on transcorneal drug absorption are performed ex vivo with the excised corneas of experimental animals or in vitro using corneal cell culture models. The data currently available on the expression of ABC transporter proteins in corneal tissue is limited or contradictory. This study describes, for the first time, the comparison of the expression of ABC transporters, in particular, MDR1, BCRP and MRP3, between human cornea cell culture models and the most commonly used ex vivo models, namely, rabbit and porcine corneas, conducted in the same laboratory. The expression levels and functionality were determined by means of PCR, western blot, immunohistochemistry and bidirectional permeation studies using specific substrates and inhibitors. The results clearly indicate species-dependent expression of the studied efflux transporters. In the rabbit cornea, the expression and activity of MDR1 transporter was confirmed, whereas human cell culture models and porcine corneas did not show MDR1 expression. However, human cornea models possessed MRP3 and BCRP expression, whereas no functional expression was found in rabbit and porcine corneas. Therefore, the translation of transcorneal permeation data from animal experiments to humans should be performed with caution. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Transporter taxonomy - a comparison of different transport protein classification schemes.

    PubMed

    Viereck, Michael; Gaulton, Anna; Digles, Daniela; Ecker, Gerhard F

    2014-06-01

    Currently, there are more than 800 well characterized human membrane transport proteins (including channels and transporters) and there are estimates that about 10% (approx. 2000) of all human genes are related to transport. Membrane transport proteins are of interest as potential drug targets, for drug delivery, and as a cause of side effects and drug–drug interactions. In light of the development of Open PHACTS, which provides an open pharmacological space, we analyzed selected membrane transport protein classification schemes (Transporter Classification Database, ChEMBL, IUPHAR/BPS Guide to Pharmacology, and Gene Ontology) for their ability to serve as a basis for pharmacology driven protein classification. A comparison of these membrane transport protein classification schemes by using a set of clinically relevant transporters as use-case reveals the strengths and weaknesses of the different taxonomy approaches.

  18. The deca-GX3 proteins Yae1-Lto1 function as adaptors recruiting the ABC protein Rli1 for iron-sulfur cluster insertion

    PubMed Central

    Paul, Viktoria Désirée; Mühlenhoff, Ulrich; Stümpfig, Martin; Seebacher, Jan; Kugler, Karl G; Renicke, Christian; Taxis, Christof; Gavin, Anne-Claude; Pierik, Antonio J; Lill, Roland

    2015-01-01

    Cytosolic and nuclear iron-sulfur (Fe-S) proteins are involved in many essential pathways including translation and DNA maintenance. Their maturation requires the cytosolic Fe-S protein assembly (CIA) machinery. To identify new CIA proteins we employed systematic protein interaction approaches and discovered the essential proteins Yae1 and Lto1 as binding partners of the CIA targeting complex. Depletion of Yae1 or Lto1 results in defective Fe-S maturation of the ribosome-associated ABC protein Rli1, but surprisingly no other tested targets. Yae1 and Lto1 facilitate Fe-S cluster assembly on Rli1 in a chain of binding events. Lto1 uses its conserved C-terminal tryptophan for binding the CIA targeting complex, the deca-GX3 motifs in both Yae1 and Lto1 facilitate their complex formation, and Yae1 recruits Rli1. Human YAE1D1 and the cancer-related ORAOV1 can replace their yeast counterparts demonstrating evolutionary conservation. Collectively, the Yae1-Lto1 complex functions as a target-specific adaptor that recruits apo-Rli1 to the generic CIA machinery. DOI: http://dx.doi.org/10.7554/eLife.08231.001 PMID:26182403

  19. Opioid transport by ATP-binding cassette transporters at the blood-brain barrier: implications for neuropsychopharmacology.

    PubMed

    Tournier, Nicolas; Declèves, Xavier; Saubaméa, Bruno; Scherrmann, Jean-Michel; Cisternino, Salvatore

    2011-01-01

    Some of the ATP-binding cassette (ABC) transporters like P-glycoprotein (P-gp; ABCB1, MDR1), BCRP (ABCG2) and MRPs (ABCCs) that are present at the blood-brain barrier (BBB) influence the brain pharmacokinetics (PK) of their substrates by restricting their uptake or enhancing their clearance from the brain into the blood, which has consequences for their CNS pharmacodynamics (PD). Opioid drugs have been invaluable tools for understanding the PK-PD relationships of these ABC-transporters. The effects of morphine, methadone and loperamide on the CNS are modulated by P-gp. This review examines the ways in which other opioid drugs and some of their active metabolites interact with ABC transporters and suggests new mechanisms that may be involved in the variability of the response of the CNS to these drugs like carrier-mediated system belonging to the solute carrier (SLC) superfamily. Exposure to opioids may also alter the expression of ABC transporters. P-gp can be overproduced during morphine treatment, suggesting that the drug has a direct or, more likely, an indirect action. Variations in cerebral neurotransmitters during exposure to opioids and the release of cytokines during pain could be new endogenous stimuli affecting transporter synthesis. This review concludes with an analysis of the pharmacotherapeutic and clinical impacts of the interactions between ABC transporters and opioids.

  20. Oxidative Stress in HIV Infection and Alcohol Use: Role of Redox Signals in Modulation of Lipid Rafts and ATP-Binding Cassette Transporters.

    PubMed

    Thangavel, Samikkannu; Mulet, Carmen T; Atluri, Venkata S R; Agudelo, Marisela; Rosenberg, Rhonda; Devieux, Jessy G; Nair, Madhavan P N

    2018-02-01

    Human immunodeficiency virus (HIV) infection induces oxidative stress and alcohol use accelerates disease progression, subsequently causing immune dysfunction. However, HIV and alcohol impact on lipid rafts-mediated immune dysfunction remains unknown. In this study, we investigate the modulation by which oxidative stress induces reactive oxygen species (ROS) affecting redox expression, lipid rafts caveiloin-1, ATP-binding cassette (ABC) transporters, and transcriptional sterol regulatory element-binding protein (SREBP) gene and protein modification and how these mechanisms are associated with arachidonic acid (AA) metabolites in HIV positive alcohol users, and how they escalate immune dysfunction. In both alcohol using HIV-positive human subjects and in vitro studies of alcohol with HIV-1 gp120 protein in peripheral blood mononuclear cells, increased ROS production significantly affected redox expression in glutathione synthetase (GSS), super oxide dismutase (SOD), and glutathione peroxidase (GPx), and subsequently impacted lipid rafts Cav-1, ABC transporters ABCA1, ABCG1, ABCB1, and ABCG4, and SREBP transcription. The increased level of rate-limiting enzyme 3-hydroxy-3-methylglutaryl HMG-CoA reductase (HMGCR), subsequently, inhibited 7-dehydrocholesterol reductase (DHCR-7). Moreover, the expression of cyclooxygenase-2 (COX-2) and lipoxygenase-5 (5-LOX) mRNA and protein modification tentatively increased the levels of prostaglandin E2 synthases (PGE 2 ) in plasma when compared with either HIV or alcohol alone. This article suggests for the first time that the redox inhibition affects lipid rafts, ABC-transporter, and SREBP transcription and modulates AA metabolites, serving as an important intermediate signaling network during immune cell dysfunction in HIV-positive alcohol users. These findings indicate that HIV infection induces oxidative stress and redox inhibition, affecting lipid rafts and ABC transports, subsequently upregulating AA metabolites and leading to

  1. Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development

    PubMed Central

    Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia ur; Liu, Liping; Qin, Yuan

    2017-01-01

    Pineapple (Ananas comosus L.) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs, 20 ABCBs, 16 ABCCs, 2 ABCDs, one ABCEs, 5 ABCFs, 42 ABCGs and 9 ABCIs). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4, AcABCC7, AcABCC9, AcABCG26, AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production. PMID:29312399

  2. Genome-Wide Identification and Expression Profiling of ATP-Binding Cassette (ABC) Transporter Gene Family in Pineapple (Ananas comosus (L.) Merr.) Reveal the Role of AcABCG38 in Pollen Development.

    PubMed

    Chen, Piaojuan; Li, Yi; Zhao, Lihua; Hou, Zhimin; Yan, Maokai; Hu, Bingyan; Liu, Yanhui; Azam, Syed Muhammad; Zhang, Ziyan; Rahman, Zia Ur; Liu, Liping; Qin, Yuan

    2017-01-01

    Pineapple ( Ananas comosus L .) cultivation commonly relies on asexual reproduction which is easily impeded by many factors in agriculture production. Sexual reproduction might be a novel approach to improve the pineapple planting. However, genes controlling pineapple sexual reproduction are still remain elusive. In different organisms a conserved superfamily proteins known as ATP binding cassette (ABC) participate in various biological processes. Whereas, till today the ABC gene family has not been identified in pineapple. Here 100 ABC genes were identified in the pineapple genome and grouped into eight subfamilies (5 ABCAs , 20 ABCB s, 16 ABCCs , 2 ABCDs , one ABCEs , 5 ABCFs , 42 ABCGs and 9 ABCIs ). Gene expression profiling revealed the dynamic expression pattern of ABC gene family in various tissues and different developmental stages. AcABCA5, AcABCB6, AcABCC4 , AcABCC7 , AcABCC9 , AcABCG26 , AcABCG38 and AcABCG42 exhibited preferential expression in ovule and stamen. Over-expression of AcABCG38 in the Arabidopsis double mutant abcg1-2abcg16-2 partially restored its pollen abortion defects, indicating that AcABCG38 plays important roles in pollen development. Our study on ABC gene family in pineapple provides useful information for developing sexual pineapple plantation which could be utilized to improve pineapple agricultural production.

  3. ABC transporter content diversity in Streptococcus pneumoniae impacts competence regulation and bacteriocin production.

    PubMed

    Wang, Charles Y; Patel, Nisha; Wholey, Wei-Yun; Dawid, Suzanne

    2018-06-19

    The opportunistic pathogen Streptococcus pneumoniae (pneumococcus) uses natural genetic competence to increase its adaptability through horizontal gene transfer. One method of acquiring DNA is through predation of neighboring strains with antimicrobial peptides called "bacteriocins." Competence and production of the major family of pneumococcal bacteriocins, pneumocins, are regulated by the quorum-sensing systems com and blp , respectively. In the classical paradigm, the ABC transporters ComAB and BlpAB each secretes its own system's signaling pheromone and in the case of BlpAB also secretes the pneumocins. While ComAB is found in all pneumococci, only 25% of strains encode an intact version of BlpAB [BlpAB(+)] while the rest do not [BlpAB(-)]. Contrary to the classical paradigm, it was previously shown that BlpAB(-) strains can activate blp through ComAB-mediated secretion of the blp pheromone during brief periods of competence. To better understand the full extent of com - blp crosstalk, we examined the contribution of each transporter to competence development and pneumocin secretion. We found that BlpAB(+) strains have a greater capacity for competence activation through BlpAB-mediated secretion of the com pheromone. Similarly, we show that ComAB and BlpAB are promiscuous and both can secrete pneumocins. Consequently, differences in pneumocin secretion between BlpAB(+) and BlpAB(-) strains derive from the regulation and kinetics of transporter expression rather than substrate specificity. We speculate that BlpAB(-) strains (opportunists) use pneumocins mainly in a narrowly tailored role for DNA acquisition and defense during competence while BlpAB(+) strains (aggressors) expand their use for the general inhibition of rival strains. Copyright © 2018 the Author(s). Published by PNAS.

  4. Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli

    PubMed Central

    Ruiz, Natividad; Gronenberg, Luisa S.; Kahne, Daniel; Silhavy, Thomas J.

    2008-01-01

    The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM. PMID:18375759

  5. Identification of two inner-membrane proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia coli.

    PubMed

    Ruiz, Natividad; Gronenberg, Luisa S; Kahne, Daniel; Silhavy, Thomas J

    2008-04-08

    The outer membrane (OM) of most Gram-negative bacteria contains lipopolysaccharide (LPS) in the outer leaflet. LPS, or endotoxin, is a molecule of important biological activities. In the host, LPS elicits a potent immune response, while in the bacterium, it plays a crucial role by establishing a barrier to limit entry of hydrophobic molecules. Before LPS is assembled at the OM, it must be synthesized at the inner membrane (IM) and transported across the aqueous periplasmic compartment. Much is known about the biosynthesis of LPS but, until recently, little was known about its transport and assembly. We applied a reductionist bioinformatic approach that takes advantage of the small size of the proteome of the Gram-negative endosymbiont Blochmannia floridanus to search for novel factors involved in OM biogenesis. This led to the discovery of two essential Escherichia coli IM proteins of unknown function, YjgP and YjgQ, which are required for the transport of LPS to the cell surface. We propose that these two proteins, which we have renamed LptF and LptG, respectively, are the missing transmembrane components of the ABC transporter that, together with LptB, functions to extract LPS from the IM en route to the OM.

  6. Characterization of human ATP-binding cassette protein subfamily D reconstituted into proteoliposomes.

    PubMed

    Okamoto, Takumi; Kawaguchi, Kosuke; Watanabe, Shiro; Agustina, Rina; Ikejima, Toshiki; Ikeda, Keisuke; Nakano, Minoru; Morita, Masashi; Imanaka, Tsuneo

    2018-02-19

    In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1‒3 are located on peroxisomal membrane and play an important role in the transportation of various fatty acid-CoA derivatives, including very long chain fatty acid-CoA, into peroxisomes. ABCD4 is located on lysosomal membrane and is suggested to be involved in the transport of vitamin B 12 from lysosomes to the cytosol. However, the precise transport mechanism by which these ABC transporters facilitate the import or export of substrate has yet to be well elucidated. In this study, the overexpression of human ABCD1‒4 in the methylotrophic yeast Pichia pastoris and a purification procedure were developed. The detergent-solubilized proteins were reconstituted into liposomes. ABCD1‒4 displayed stable ATPase activity, which was inhibited by AlF 3 . Furthermore, ABCD1‒4 were found to possess an equal levels of acyl-CoA thioesterase activity. Proteoliposomes is expected to be an aid in the further biochemical characterization of ABCD transporters. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. In vitro methanol production from methyl coenzyme M using the Methanosarcina barkeri MtaABC protein complex.

    PubMed

    Dong, Ming; Gonzalez, Tara D; Klems, Meghan M; Steinberg, Lisa M; Chen, Wilfred; Papoutsakis, Eleftherios T; Bahnson, Brian J

    2017-09-01

    Methanol:coenzyme M methyltransferase is an enzyme complex composed of three subunits, MtaA, MtaB, and MtaC, found in methanogenic archaea and is needed for their growth on methanol ultimately producing methane. MtaABC catalyzes the energetically favorable methyl transfer from methanol to coenzyme M to form methyl coenzyme M. Here we demonstrate that this important reaction for possible production of methanol from the anaerobic oxidation of methane can be reversed in vitro. To this effect, we have expressed and purified the Methanosarcina barkeri MtaABC enzyme, and developed an in vitro functional assay that demonstrates MtaABC can catalyze the energetically unfavorable (ΔG° = 27 kJ/mol) reverse reaction starting from methyl coenzyme M and generating methanol as a product. Demonstration of an in vitro ability of MtaABC to produce methanol may ultimately enable the anaerobic oxidation of methane to produce methanol and from methanol alternative fuel or fuel-precursor molecules. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1243-1249, 2017. © 2017 American Institute of Chemical Engineers.

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

    NASA Astrophysics Data System (ADS)

    Liao, Jielou

    2004-03-01

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

  9. Inhibition of Human Drug Transporter Activities by the Pyrethroid Pesticides Allethrin and Tetramethrin

    PubMed Central

    Chedik, Lisa; Bruyere, Arnaud; Le Vee, Marc; Stieger, Bruno; Denizot, Claire; Parmentier, Yannick; Potin, Sophie; Fardel, Olivier

    2017-01-01

    Pyrethroids are widely-used chemical insecticides, to which humans are commonly exposed, and known to alter functional expression of drug metabolizing enzymes. Limited data have additionally suggested that drug transporters, that constitute key-actors of the drug detoxification system, may also be targeted by pyrethroids. The present study was therefore designed to analyze the potential regulatory effects of these pesticides towards activities of main ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, using transporter-overexpressing cells. The pyrethroids allethrin and tetramethrin were found to inhibit various ABC and SLC drug transporters, including multidrug resistance-associated protein (MRP) 2, breast cancer resistance protein (BCRP), organic anion transporter polypeptide (OATP) 1B1, organic anion transporter (OAT) 3, multidrug and toxin extrusion transporter (MATE) 1, organic cation transporter (OCT) 1 and OCT2, with IC50 values however ranging from 2.6 μM (OCT1 inhibition by allethrin) to 77.6 μM (OAT3 inhibition by tetramethrin) and thus much higher than pyrethroid concentrations (in the nM range) reached in environmentally pyrethroid-exposed humans. By contrast, allethrin and tetramethrin cis-stimulated OATP2B1 activity and failed to alter activities of OATP1B3, OAT1 and MATE2-K, whereas P-glycoprotein activity was additionally moderately inhibited. Twelve other pyrethoids used at 100 μM did not block activities of the various investigated transporters, or only moderately inhibited some of them (inhibition by less than 50%). In silico analysis of structure-activity relationships next revealed that molecular parameters, including molecular weight and lipophilicity, are associated with transporter inhibition by allethrin/tetramethrin and successfully predicted transporter inhibition by the pyrethroids imiprothrin and prallethrin. Taken together, these data fully demonstrated that two pyrethoids, i.e., allethrin and tetramethrin, can

  10. In Vitro Analysis of Metabolite Transport Proteins.

    PubMed

    Roell, Marc-Sven; Kuhnert, Franziska; Zamani-Nour, Shirin; Weber, Andreas P M

    2017-01-01

    The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges.Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.

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

    PubMed Central

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

    2014-01-01

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

  12. ATP-Binding Cassette Efflux Transporters in Human Placenta

    PubMed Central

    Ni, Zhanglin; Mao, Qingcheng

    2010-01-01

    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

  13. The ABC and AUSSAT.

    ERIC Educational Resources Information Center

    McGarritty, Ian

    1985-01-01

    Discusses the Australian Broadcasting Corporation's (ABC) utilization of the AUSSAT telecommunications satellite to extend its television and radio transmission range to reach remote Australian audiences; the satellite's program gathering and interchange capabilities; and ABC's generation of other benefits to offset cost of satellite services.…

  14. Transport of Proteins through Nanopores

    NASA Astrophysics Data System (ADS)

    Luan, Binquan

    In biological cells, a malfunctioned protein (such as misfolded or damaged) is degraded by a protease in which an unfoldase actively drags the protein into a nanopore-like structure and then a peptidase cuts the linearized protein into small fragments (i.e. a recycling process). Mimicking this biological process, many experimental studies have focused on the transport of proteins through a biological protein pore or a synthetic solid-state nanopore. Potentially, the nanopore-based sensors can provide a platform for interrogating proteins that might be disease-related or be targeted by a new drug molecule. The single-profile of a protein chain inside an extremely small nanopore might even permit the sequencing of the protein. Here, through all-atom molecular dynamics simulations, I will show various types of protein transport through a nanopore and reveal the nanoscale mechanics/energetics that plays an important role governing the protein transport.

  15. ABC Transporter Genes and Risk of Type 2 Diabetes

    PubMed Central

    Schou, Jesper; Tybjærg-Hansen, Anne; Møller, Holger J.; Nordestgaard, Børge G.; Frikke-Schmidt, Ruth

    2012-01-01

    OBJECTIVE Alterations of pancreatic β-cell cholesterol content may contribute to β-cell dysfunction. Two important determinants of intracellular cholesterol content are the ATP-binding cassette (ABC) transporters A1 (ABCA1) and -G1 (ABCG1). Whether genetic variation in ABCA1 and ABCG1 predicts risk of type 2 diabetes in the general population is unknown. RESEARCH DESIGN AND METHODS We tested whether genetic variation in the promoter and coding regions of ABCA1 and ABCG1 predicted risk of type 2 diabetes in the general population. Twenty-seven variants, identified by previous resequencing of both genes, were genotyped in the Copenhagen City Heart Study (CCHS) (n = 10,185). Two loss-of-function mutations (ABCA1 N1800H and ABCG1 g.-376C>T) (n = 322) and a common variant (ABCG1 g.-530A>G) were further genotyped in the Copenhagen General Population Study (CGPS) (n = 30,415). RESULTS Only one of the variants examined, ABCG1 g.-530A>G, predicted a decreased risk of type 2 diabetes in the CCHS (P for trend = 0.05). Furthermore, when validated in the CGPS or in the CCHS and CGPS combined (n = 40,600), neither the two loss-of-function mutations (ABCA1 N1800H, ABCG1 g.-376C>T) nor ABCG1 g.-530A>G were associated with type 2 diabetes (P values >0.57 and >0.30, respectively). CONCLUSIONS Genetic variations in ABCA1 and ABCG1 were not associated with increased risk of type 2 diabetes in the general population. These data were obtained in general population samples harboring the largest number of heterozygotes for loss-of-function mutations in ABCA1 and ABCG1. PMID:23139370

  16. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    NASA Astrophysics Data System (ADS)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  17. Differential Protein Expression in Streptococcus uberis under Planktonic and Biofilm Growth Conditions ▿ †

    PubMed Central

    Crowley, R. C.; Leigh, J. A.; Ward, P. N.; Lappin-Scott, H. M.; Bowler, L. D.

    2011-01-01

    The bovine pathogen Streptococcus uberis was assessed for biofilm growth. The transition from planktonic to biofilm growth in strain 0140J correlated with an upregulation of several gene products that have been shown to be important for pathogenesis, including a glutamine ABC transporter (SUB1152) and a lactoferrin binding protein (gene lbp; protein SUB0145). PMID:21075893

  18. Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

    PubMed

    Sletvold, H; Johnsen, P J; Hamre, I; Simonsen, G S; Sundsfjord, A; Nielsen, K M

    2008-07-01

    Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

  19. Expression, purification and characterization of GAPDH-ChSase ABC I from Proteus vulgaris in Escherichia coli.

    PubMed

    Li, Ye; Chen, Zhenya; Zhou, Zhao; Yuan, Qipeng

    2016-12-01

    Chondroitinases (ChSases) are a family of polysaccharide lyases that can depolymerize high molecular weight chondroitin sulfate (CS) and dermatan sulfate (DS). In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is stably expressed in different cells like normal cells and cancer cells and the expression is relatively insensitive to experimental conditions, was expressed as a fusion protein with ChSase ABC I. Results showed that the expression level and enzyme activity of GAPDH-ChSase ABC I were about 2.2 and 3.0 times higher than those of ChSase ABC I. By optimization of fermentation conditions, higher productivity of ChSase ABC I was achieved as 880 ± 61 IU/g wet cell weight compared with the reported ones. The optimal temperature and pH of GAPDH-ChSase ABC I were 40 °C and 7.5, respectively. GAPDH-ChSase ABC I had a kcat/Km of 131 ± 4.1 L/μmol s and the catalytic efficiency was decreased as compared to ChSase ABC I. The relative activity of GAPDH-ChSase ABC I remained 89% after being incubated at 30 °C for 180 min and the thermostability of ChSase ABC I was enhanced by GAPDH when it was incubated at 30, 35, 40 and 45 °C. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Role of the Fur Regulon in Iron Transport in Bacillus subtilis

    PubMed Central

    Ollinger, Juliane; Song, Kyung-Bok; Antelmann, Haike; Hecker, Michael; Helmann, John D.

    2006-01-01

    The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding ∼40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT. PMID:16672620

  1. Role of the Fur regulon in iron transport in Bacillus subtilis.

    PubMed

    Ollinger, Juliane; Song, Kyung-Bok; Antelmann, Haike; Hecker, Michael; Helmann, John D

    2006-05-01

    The Bacillus subtilis ferric uptake regulator (Fur) protein mediates the iron-dependent repression of at least 20 operons encoding approximately 40 genes. We investigated the physiological roles of Fur-regulated genes by the construction of null mutations in 14 transcription units known or predicted to function in siderophore biosynthesis or iron uptake. We demonstrate that ywbLMN, encoding an elemental iron uptake system orthologous to the copper oxidase-dependent Fe(III) uptake system of Saccharomyces cerevisiae, is essential for growth in low iron minimal medium lacking citric acid. 2,3-Dihydroxybenzoyl-glycine (Itoic acid), the siderophore precursor produced by laboratory strains of B. subtilis, is of secondary importance. In the presence of citrate, the YfmCDEF ABC transporter is required for optimal growth. B. subtilis is unable to grow in minimal medium containing the iron chelator EDDHA unless the ability to synthesize the intact bacillibactin siderophore is restored (by the introduction of a functional sfp gene) or exogenous siderophores are provided. Utilization of the catecholate siderophores bacillibactin and enterobactin requires the FeuABC importer and the YusV ATPase. Utilization of hydroxamate siderophores requires the FhuBGC ABC transporter together with the FhuD (ferrichrome) or YxeB (ferrioxamine) substrate-binding proteins. Growth with schizokinen or arthrobactin is at least partially dependent on the YfhA YfiYZ importer and the YusV ATPase. We have also investigated the effects of a fur mutation on the proteome and documented the derepression of 11 Fur-regulated proteins, including a newly identified thioredoxin reductase homolog, YcgT.

  2. High-throughput screening identifies Ceefourin 1 and Ceefourin 2 as highly selective inhibitors of multidrug resistance protein 4 (MRP4).

    PubMed

    Cheung, Leanna; Flemming, Claudia L; Watt, Fujiko; Masada, Nanako; Yu, Denise M T; Huynh, Tony; Conseil, Gwenaëlle; Tivnan, Amanda; Polinsky, Alexander; Gudkov, Andrei V; Munoz, Marcia A; Vishvanath, Anasuya; Cooper, Dermot M F; Henderson, Michelle J; Cole, Susan P C; Fletcher, Jamie I; Haber, Michelle; Norris, Murray D

    2014-09-01

    Multidrug resistance protein 4 (MRP4/ABCC4), a member of the ATP-binding cassette (ABC) transporter superfamily, is an organic anion transporter capable of effluxing a wide range of physiologically important signalling molecules and drugs. MRP4 has been proposed to contribute to numerous functions in both health and disease; however, in most cases these links remain to be unequivocally established. A major limitation to understanding the physiological and pharmacological roles of MRP4 has been the absence of specific small molecule inhibitors, with the majority of established inhibitors also targeting other ABC transporter family members, or inhibiting the production, function or degradation of important MRP4 substrates. We therefore set out to identify more selective and well tolerated inhibitors of MRP4 that might be used to study the many proposed functions of this transporter. Using high-throughput screening, we identified two chemically distinct small molecules, Ceefourin 1 and Ceefourin 2, that inhibit transport of a broad range of MRP4 substrates, yet are highly selective for MRP4 over other ABC transporters, including P-glycoprotein (P-gp), ABCG2 (Breast Cancer Resistance Protein; BCRP) and MRP1 (multidrug resistance protein 1; ABCC1). Both compounds are more potent MRP4 inhibitors in cellular assays than the most widely used inhibitor, MK-571, requiring lower concentrations to effect a comparable level of inhibition. Furthermore, Ceefourin 1 and Ceefourin 2 have low cellular toxicity, and high microsomal and acid stability. These newly identified inhibitors should be of great value for efforts to better understand the biological roles of MRP4, and may represent classes of compounds with therapeutic application. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Regulation of Human γδ T Cells by BTN3A1 Protein Stability and ATP-Binding Cassette Transporters

    PubMed Central

    Rhodes, David A.; Chen, Hung-Chang; Williamson, James C.; Hill, Alfred; Yuan, Jack; Smith, Sam; Rhodes, Harriet; Trowsdale, John; Lehner, Paul J.; Herrmann, Thomas; Eberl, Matthias

    2018-01-01

    Activation of human Vγ9/Vδ2 T cells by “phosphoantigens” (pAg), the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach these problems, we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 T cell receptor (TCR) transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules, such as periplakin or RhoB, did not account for the functional variation between HeLa-L and HeLa-M cells. Instead, the data implicate a checkpoint controlling BTN3A1 stability and protein trafficking, acting at an early time point in its maturation. In addition, plasma membrane profiling was used to identify proteins upregulated in HMB-PP-treated HeLa-M. ABCG2, a member of the ATP-binding cassette (ABC) transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated with

  4. Peroxisomal ATP-binding cassette transporters form mainly tetramers

    PubMed Central

    Geillon, Flore; Gondcaille, Catherine; Raas, Quentin; Dias, Alexandre M. M.; Pecqueur, Delphine; Truntzer, Caroline; Lucchi, Géraldine; Ducoroy, Patrick; Falson, Pierre; Savary, Stéphane; Trompier, Doriane

    2017-01-01

    ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane. PMID:28258215

  5. Determination of proteins induced in response to jasmonic acid and salicylic acid in resistant and susceptible cultivars of tomato.

    PubMed

    Afroz, Amber; Khan, Muhammad Rashid; Komatsu, Setsuko

    2010-07-01

    Jasmonic acid (JA) and salicylic acid (SA) are signaling molecules that play key roles in the regulation of metabolic processes, reproduction, and defense against pathogens. The proteomics approach was used to identify proteins that are induced by JA and SA in the tomato cultivars Roma and Pant Bahr, which are susceptible and resistant to bacterial wilt, respectively. Threonine deaminase and leucine amino peptidase were upregulated, and ribulose-1,5-bisphosphate carboxylase/oxygenase small chain was downregulated by time-course application of JA. Translationally controlled tumor protein was upregulated by time-course application of SA. Protein disulfide isomerase was upregulated by application of either JA or SA. Proteins related to defense, energy, and protein destination/storage are suspected to be responsible for the susceptibility or resistance of the cultivars. Furthermore, in Roma, iron ABC transporter was upregulated by JA and down-regulated by SA. Iron ABC transporter plays a part in the signal transduction of both JA and SA in cultivars of tomato that are resistant to bacterial wilt.

  6. Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of Holliday junction resolution

    PubMed Central

    van Gool, Alain J.; Hajibagheri, Nasser M.A.; Stasiak, Andrzej; West, Stephen C.

    1999-01-01

    Genetic recombination can lead to the formation of intermediates in which DNA molecules are linked by Holliday junctions. Movement of a junction along DNA, by a process known as branch migration, leads to heteroduplex formation, whereas resolution of a junction completes the recombination process. Holliday junctions can be resolved in either of two ways, yielding products in which there has, or has not, been an exchange of flanking markers. The ratio of these products is thought to be determined by the frequency with which the two isomeric forms (conformers) of the Holliday junction are cleaved. Recent studies with enzymes that process Holliday junctions in Escherichia coli, the RuvABC proteins, however, indicate that protein binding causes the junction to adopt an open square-planar configuration. Within such a structure, DNA isomerization can have little role in determining the orientation of resolution. To determine the role that junction-specific protein assembly has in determining resolution bias, a defined in vitro system was developed in which we were able to direct the assembly of the RuvABC resolvasome. We found that the bias toward resolution in one orientation or the other was determined simply by the way in which the Ruv proteins were positioned on the junction. Additionally, we provide evidence that supports current models on RuvABC action in which Holliday junction resolution occurs as the resolvasome promotes branch migration. PMID:10421637

  7. ABCB1 and ABCC1-like transporters in immune system cells from sea urchins Echinometra lucunter and Echinus esculentus and oysters Crassostrea gasar and Crassostrea gigas.

    PubMed

    Marques-Santos, Luis Fernando; Hégaret, Hélène; Lima-Santos, Leonardo; Queiroga, Fernando Ramos; da Silva, Patricia Mirella

    2017-11-01

    ABC transporters activity and expression have been associated with the multixenobiotic resistance phenotype (MXR). The activity of these proteins leads to a reduction in the intracellular concentration of several xenobiotics, thus reducing their toxicity. However, little attention has been given to the expression of ABC transporters in marine invertebrates and few studies have investigated their role in immune system cells of sea urchins and shellfish bivalves. The aim of the present study was to investigate the activity of the ABC transporters ABCB1 and ABCC1 in immune system cells of sea urchins (coelomocytes) and oysters (hemocytes) from different climatic regions (Brazil and France). Sea urchins and oysters were collected at Paraíba coast; Brazil (Echinometra lucunter and Crassostrea gasar) and Rade of Brest; France (Echinus esculentus and Crassostrea gigas). Coelomocytes and hemocytes were stained with the ABC transporter substrate calcein-AM and dye accumulation analyzed under flow cytometry. Reversin 205 (ABCB1 transporter blocker) and MK571 (ABCC1 transporter blocker) were used as pharmacological tools to investigate ABC transporter activity. A different pattern of calcein accumulation was observed in coelomocytes: phagocytes > colorless spherulocytes > vibrate cells > red spherulocytes. The treatment with MK571 increased calcein fluorescence levels in coelomocytes from both species. However, reversin 205 treatment was not able to increase calcein fluorescence in E. esculentus coelomocytes. These data suggest that ABCC1-like transporter activity is present in both sea urchin species, but ABCB1-like transporter activity might only be present in E. lucunter coelomocytes. The activity of ABCC1-like transporter was observed in all cell types from both bivalve species. However, reversin 205 only increased calcein accumulation in hyalinocytes of the oyster C. gasar, suggesting the absence of ABCB1-like transporter activity in all other cell types

  8. Celecoxib sensitizes imatinib-resistant K562 cells to imatinib by inhibiting MRP1-5, ABCA2 and ABCG2 transporters via Wnt and Ras signaling pathways.

    PubMed

    Dharmapuri, Gangappa; Doneti, Ravinder; Philip, Gundala Harold; Kalle, Arunasree M

    2015-07-01

    Imatinib mesylate, a tyrosine kinase inhibitor, is very effective in the treatment of chronic myeloid leukemia (CML). However, development of resistance to imatinib therapy is also a very common mechanism observed with long-term administration of the drug. Our previous studies have highlighted the role of cyclooxygenase-2 (COX-2) in regulating the expression of multidrug resistant protein-1 (MDR1), P-gp, in imatinib-resistant K562 cells (IR-K562) via PGE2-cAMP-PKC-NF-κB pathway and inhibition of COX-2 by celecoxib, a COX-2 specific inhibitor, inhibits this pathway and reverses the drug resistance. Studies have identified that not only MDR1 but other ATP-binding cassette transport proteins (ABC transporters) are involved in the development of imatinib resistance. Here, we tried to study the role of COX-2 in the regulation of other ABC transporters such as MRP1, MRP2, MRP3, ABCA2 and ABCG2 that have been already implicated in imatinib resistance development. The results of the study clearly indicated that overexpression of COX-2 lead to upregulation of MRP family proteins in IR-K562 cells and celecoxib down-regulated the ABC transporters through Wnt and MEK signaling pathways. The study signifies that celecoxib in combination with the imatinib can be a good alternate treatment strategy for the reversal of imatinib resistance. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough.

    PubMed

    Ramos, Ana Raquel; Grein, Fabian; Oliveira, Gonçalo P; Venceslau, Sofia S; Keller, Kimberly L; Wall, Judy D; Pereira, Inês A C

    2015-07-01

    Flavin-based electron bifurcation (FBEB) is an important mechanism for the energy metabolism of anaerobes. A new family of NADH dehydrogenases, the flavin oxidoreductase (FlxABCD, previously called FloxABCD), was proposed to perform FBEB in sulphate-reducing organisms coupled with heterodisulfide reductase (HdrABC). We found that the hdrABC-flxABCD gene cluster is widespread among anaerobic bacteria, pointing to a general and important role in their bioenergetics. In this work, we studied FlxABCD of Desulfovibrio vulgaris Hildenborough. The hdr-flx genes are part of the same transcriptional unit and are increased in transcription during growth in ethanol-sulfate, and to a less extent during pyruvate fermentation. Two mutant strains were generated: one where expression of the hdr-flx genes was interrupted and another lacking the flxA gene. Both strains were unable to grow with ethanol-sulfate, whereas growth was restored in a flxA-complemented strain. The mutant strains also produced very reduced amounts of ethanol compared with the wild type during pyruvate fermentation. Our results show that in D. vulgaris, the FlxABCD-HdrABC proteins are essential for NADH oxidation during growth on ethanol, probably involving a FBEB mechanism that leads to reduction of ferredoxin and the small protein DsrC, while in fermentation they operate in reverse, reducing NAD(+) for ethanol production. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  10. THE ROLE OF MULTIDRUG RESISTANCE ASSOCIATED PROTEIN (MRP) IN THE BLOOD-BRAIN BARRIER AND OPIOID ANALGESIA

    PubMed Central

    Su, Wendy; Pasternak, Gavril W.

    2013-01-01

    The blood brain barrier protects the brain from circulating compounds and drugs. The ATP-binding cassette (ABC) transporter P-glycoprotein (Pgp) is involved with the barrier, both preventing the influx of agent from the blood into the brain and facilitating the efflux of compounds from the brain into the blood, raising the possibility of a similar role for other transporters. Multidrug resistance associated protein (MRP), a 190 kDa protein similar to Pgp is also ABC transport that has been implicated in the blood brain barrier. The current study explores its role in opioid action. Immunohistochemically, it is localized in the choroid plexus in ratsand can be selectively downregulated by antisense treatment at both the level of mRNA, as shown by RT-PCR, and protein, as demonstrated immunohistochemically. Behaviorally, downregulation of MRP significantly enhances the analgesic potency of systemic morphine in MRP knockout mice and in antisense-treated rats by lowering the blood brain barrier. Following intracerebroventricular administration, a number of compounds, including some opioids, are rapidly secreted from the brain into the blood where they contribute to the overall analgesic effects by activating peripheral systems. MRP plays a role in this efflux. Downregulating MRP expression leads to a corresponding decrease in the transport and a diminished analgesic response from opioids administered intracerebroventricularly. Thus, the transporter protein MRP plays a role in maintaining the blood-brain barrier and modulates the activity of opioids. PMID:23508590

  11. ABC's of Construction. Final Report.

    ERIC Educational Resources Information Center

    Greater Baton Rouge Chamber of Commerce, LA.

    The ABC's of Construction project was a demonstration project designed to integrate basic skills training with an industry-developed vocational-craft training program. The program was located at the central training facility of the Pelican Chapter of Associated Builders and Contractors (ABC), an organization made up of nearly 300 member companies…

  12. Statistical Hypothesis Testing in Intraspecific Phylogeography: NCPA versus ABC

    PubMed Central

    Templeton, Alan R.

    2009-01-01

    Nested clade phylogeographic analysis (NCPA) and approximate Bayesian computation (ABC) have been used to test phylogeographic hypotheses. Multilocus NCPA tests null hypotheses, whereas ABC discriminates among a finite set of alternatives. The interpretive criteria of NCPA are explicit and allow complex models to be built from simple components. The interpretive criteria of ABC are ad hoc and require the specification of a complete phylogeographic model. The conclusions from ABC are often influenced by implicit assumptions arising from the many parameters needed to specify a complex model. These complex models confound many assumptions so that biological interpretations are difficult. Sampling error is accounted for in NCPA, but ABC ignores important sources of sampling error that creates pseudo-statistical power. NCPA generates the full sampling distribution of its statistics, but ABC only yields local probabilities, which in turn make it impossible to distinguish between a good fitting model, a non-informative model, and an over-determined model. Both NCPA and ABC use approximations, but convergences of the approximations used in NCPA are well defined whereas those in ABC are not. NCPA can analyze a large number of locations, but ABC cannot. Finally, the dimensionality of tested hypothesis is known in NCPA, but not for ABC. As a consequence, the “probabilities” generated by ABC are not true probabilities and are statistically non-interpretable. Accordingly, ABC should not be used for hypothesis testing, but simulation approaches are valuable when used in conjunction with NCPA or other methods that do not rely on highly parameterized models. PMID:19192182

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

    PubMed Central

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

    2015-01-01

    A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53–36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins.—Gulati, S., Balderes, D., Kim, C., Guo, Z. A., Wilcox, L., Area-Gomez, E., Snider, J., Wolinski, H., Stagljar, I., Granato, J. T., Ruggles, K. V., DeGiorgis, J. A., Kohlwein, S. D., Schon, E. A., Sturley, S. L. ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification. PMID:26220175

  14. Do You Know Your ABC?

    ERIC Educational Resources Information Center

    Neale, Claire

    2013-01-01

    Within primary schools, the core subjects of literacy and numeracy are highly regarded, and rightly so, as children need to learn to read, write and be numerically literate. This means that all children learn their ABCs at an early age, But, what about the "other ABC"--"Airway, Breathing and Circulation?" Accidents and medical…

  15. Retinal-specific ATP-binding cassette transporter (ABCR/ABCA4) is expressed at the choroid plexus in rat brain.

    PubMed

    Bhongsatiern, Jiraganya; Ohtsuki, Sumio; Tachikawa, Masanori; Hori, Satoko; Terasaki, Tetsuya

    2005-03-01

    ATP-binding cassette (ABC) transporter A4 is a member of the ABC transporter subfamily A which has been reported to be exclusively expressed in the retina. In contrast, a previous report has suggested a possible relationship between ABCA4 and CNS function. The purpose of the present study was to investigate the localization of ABCA4 mRNA and protein in rat brain. In situ hybridization analysis revealed that ABCA4 mRNA was localized in the lateral ventricles. RT-PCR analysis detected ABCA4 mRNA in isolated rat choroid plexus and conditionally immortalized rat choroid plexus epithelial cells (TR-CSFB). Furthermore, ABCA4 protein was also detected in the isolated rat choroid plexus at about 250 kDa by western blot analysis, and its apparent molecular size was reduced by N-glycosidase F treatment. These results suggest that glycosylated ABCA4 protein is expressed in rat choroid plexus epithelial cells. ABCA4 may play a role in the function of the blood-cerebrospinal fluid barrier and affect CSF conditions.

  16. Architectures of Lipid Transport Systems for the Bacterial Outer Membrane.

    PubMed

    Ekiert, Damian C; Bhabha, Gira; Isom, Georgia L; Greenan, Garrett; Ovchinnikov, Sergey; Henderson, Ian R; Cox, Jeffery S; Vale, Ronald D

    2017-04-06

    How phospholipids are trafficked between the bacterial inner and outer membranes through the hydrophilic space of the periplasm is not known. We report that members of the mammalian cell entry (MCE) protein family form hexameric assemblies with a central channel capable of mediating lipid transport. The E. coli MCE protein, MlaD, forms a ring associated with an ABC transporter complex in the inner membrane. A soluble lipid-binding protein, MlaC, ferries lipids between MlaD and an outer membrane protein complex. In contrast, EM structures of two other E. coli MCE proteins show that YebT forms an elongated tube consisting of seven stacked MCE rings, and PqiB adopts a syringe-like architecture. Both YebT and PqiB create channels of sufficient length to span the periplasmic space. This work reveals diverse architectures of highly conserved protein-based channels implicated in the transport of lipids between the membranes of bacteria and some eukaryotic organelles. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Glaucarubinone sensitizes KB cells to paclitaxel by inhibiting ABC transporters via ROS-dependent and p53-mediated activation of apoptotic signaling pathways

    PubMed Central

    Karthikeyan, Subburayan; Hoti, Sugeerappa Laxmanappa; Nazeer, Yasin; Hegde, Harsha Vasudev

    2016-01-01

    Multidrug resistance (MDR) is considered to be the major contributor to failure of chemotherapy in oral squamous cell carcinoma (SCC). This study was aimed to explore the effects and mechanisms of glaucarubinone (GLU), one of the major quassinoids from Simarouba glauca DC, in potentiating cytotoxicity of paclitaxel (PTX), an anticancer drug in KB cells. Our data showed that the administration of GLU pre-treatment significantly enhanced PTX anti-proliferative effect in ABCB1 over-expressing KB cells. The Rh 123 drug efflux studies revealed that there was a significant transport function inhibition by GLU-PTX treatment. Interestingly, it was also found that this enhanced anticancer efficacy of GLU was associated with PTX-induced cell arrest in the G2/M phase of cell cycle. Further, the combined treatment of GLU-PTX had significant decrease in the expression levels of P-gp, MRPs, and BCRP in resistant KB cells at both mRNA and protein levels. Furthermore, the combination treatments showed significant reactive oxygen species (ROS) production, chromatin condensation and reduced mitochondrial membrane potential in resistant KB cells. The results from DNA fragmentation analysis also demonstrated the GLU induced apoptosis in KB cells and its synergy with PTX. Importantly, GLU and/or PTX triggered apoptosis through the activation of pro-apoptotic proteins such as p53, Bax, and caspase-9. Our findings demonstrated for the first time that GLU causes cell death in human oral cancer cells via the ROS-dependent suppression of MDR transporters and p53-mediated activation of the intrinsic mitochondrial pathway of apoptosis. Additionally, the present study also focussed on investigation of the protective effect of GLU and combination drugs in human normal blood lymphocytes. Normal blood lymphocytes assay indicated that GLU is able to induce selective toxicity in cancer cells and in silico molecular docking studies support the choice of GLU as ABC inhibitor to enhance PTX efficacy

  18. An ATP-Binding Cassette Transporter and Two rRNA Methyltransferases Are Involved in Resistance to Avilamycin in the Producer Organism Streptomyces viridochromogenes Tü57

    PubMed Central

    Weitnauer, Gabriele; Gaisser, Sibylle; Trefzer, Axel; Stockert, Sigrid; Westrich, Lucy; Quiros, Luis M.; Mendez, Carmen; Salas, Jose A.; Bechthold, Andreas

    2001-01-01

    Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 μg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 μg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA. PMID:11181344

  19. An ATP-binding cassette transporter and two rRNA methyltransferases are involved in resistance to avilamycin in the producer organism Streptomyces viridochromogenes Tü57.

    PubMed

    Weitnauer, G; Gaisser, S; Trefzer, A; Stockert, S; Westrich, L; Quiros, L M; Mendez, C; Salas, J A; Bechthold, A

    2001-03-01

    Three different resistance factors from the avilamycin biosynthetic gene cluster of Streptomyces viridochromogenes Tü57, which confer avilamycin resistance when expressed in Streptomyces lividans TK66, were isolated. Analysis of the deduced amino acid sequences showed that AviABC1 is similar to a large family of ATP-binding transporter proteins and that AviABC2 resembles hydrophobic transmembrane proteins known to act jointly with the ATP-binding proteins. The deduced amino acid sequence of aviRb showed similarity to those of other rRNA methyltransferases, and AviRa did not resemble any protein in the databases. Independent expression in S. lividans TK66 of aviABC1 plus aviABC2, aviRa, or aviRb conferred different levels of resistance to avilamycin: 5, 10, or 250 microg/ml, respectively. When either aviRa plus aviRb or aviRa plus aviRb plus aviABC1 plus aviABC2 was coexpressed in S. lividans TK66, avilamycin resistance levels reached more than 250 microg/ml. Avilamycin A inhibited poly(U)-directed polyphenylalanine synthesis in an in vitro system using ribosomes of S. lividans TK66(pUWL201) (GWO), S. lividans TK66(pUWL201-Ra) (GWRa), or S. lividans TK66(pUWL201-Rb) (GWRb), whereas ribosomes of S. lividans TK66 containing pUWL201-Ra+Rb (GWRaRb) were highly resistant. aviRa and aviRb were expressed in Escherichia coli, and both enzymes were purified as fusion proteins to near homogeneity. Both enzymes showed rRNA methyltransferase activity using a mixture of 16S and 23S rRNAs from E. coli as the substrate. Coincubation experiments revealed that the enzymes methylate different positions of rRNA.

  20. Neuroprotective effect of chondroitinase ABC on primary and secondary brain injury after stroke in hypertensive rats.

    PubMed

    Chen, Xin-ran; Liao, Song-jie; Ye, Lan-xiang; Gong, Qiong; Ding, Qiao; Zeng, Jin-sheng; Yu, Jian

    2014-01-16

    Focal cerebral infarction causes secondary damage in the ipsilateral ventroposterior thalamic nucleus (VPN). Chondroitin sulfate proteoglycans (CSPGs) are a family of putative inhibitory components, and its degradation by chondroitinase ABC (ChABC) promotes post-injury neurogenesis. This study investigated the role of ChABC in the primary and secondary injury post stroke in hypertension. Renovascular hypertensive Sprague-Dawley rats underwent middle cerebral artery occlusion (MCAO), and were subjected to continuous intra-infarct infusion of ChABC (0.12 U/d for 7 days) 24 h later. Neurological function was evaluated by a modified neurologic severity score. Neurons were counted in the peri-infarct region and the ipsilateral VPN 8 and 14 days after MCAO by Nissl staining and NeuN labeling. The expressions of CSPGs, growth-associated protein-43 (GAP-43) and synaptophysin (SYN) were detected with immunofluorescence or Western blotting. The intra-infarct infusion of ChABC, by degrading accumulated CSPGs, rescued neuronal loss and increased the levels of GAP-43 and SYN in both the ipsilateral cortex and VPN, indicating enhancd neuron survival as well as augmented axonal growth and synaptic plasticity, eventually improving overall neurological function. The study demonstrated that intra-infarct ChABC infusion could salvage the brain from both primary and secondary injury by the intervention on the neuroinhibitory environment post focal cerebral infarction. © 2013 Published by Elsevier B.V.

  1. The trehalose-specific transporter LpqY-SugABC is required for antimicrobial and anti-biofilm activity of trehalose analogues in Mycobacterium smegmatis.

    PubMed

    Wolber, Jeffrey M; Urbanek, Bailey L; Meints, Lisa M; Piligian, Brent F; Lopez-Casillas, Irene C; Zochowski, Kailey M; Woodruff, Peter J; Swarts, Benjamin M

    2017-10-10

    Mycobacteria, including the bacterial pathogen that causes human tuberculosis, possess distinctive pathways for synthesizing and utilizing the non-mammalian disaccharide trehalose. Trehalose metabolism is essential for mycobacterial viability and has been linked to in vitro biofilm formation, which may bear relevance to in vivo drug tolerance. Previous research has shown that some trehalose analogues bearing modifications at the 6-position inhibit growth of various mycobacterial species. In this work, 2-, 5-, and 6-position-modified trehalose analogues were synthesized using our previously reported one-step chemoenzymatic method and shown to inhibit growth and biofilm formation in the two-to three-digit micromolar range in Mycobacterium smegmatis. The trehalose-specific ABC transporter LpqY-SugABC was essential for antimicrobial and anti-biofilm activity, suggesting that inhibition by monosubstituted trehalose analogues requires cellular uptake and does not proceed via direct action on extracellular targets such as antigen 85 acyltransferases or trehalose dimycolate hydrolase. Although the potency of the described compounds in in vitro growth and biofilm assays is moderate, this study reports the first trehalose-based mycobacterial biofilm inhibitors and reinforces the concept of exploiting unique sugar uptake pathways to deliver inhibitors and other chemical cargo to mycobacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

    PubMed

    Pelkonen, Laura; Sato, Kazuki; Reinisalo, Mika; Kidron, Heidi; Tachikawa, Masanori; Watanabe, Michitoshi; Uchida, Yasuo; Urtti, Arto; Terasaki, Tetsuya

    2017-03-06

    The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na + /K + ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.

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

    PubMed

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

    2014-05-23

    Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed Central

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

    2015-01-01

    Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool. PMID:26472925

  5. Overcoming the heterologous bias: An in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Puri, Nidhi; Manoharlal, Raman; Sharma, Monika

    2011-01-07

    Research highlights: {yields} First report to demonstrate an in vivo expression system of an ABC multidrug transporter CgCdr1p of C. glabrata. {yields} First report on the structure and functional characterization of CgCdr1p. {yields} Functional conservation of divergent but typical residues of CgCdr1p. {yields} CgCdr1p elicits promiscuity towards substrates and has a large drug binding pocket with overlapping specificities. -- Abstract: We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplishedmore » by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the arte

  6. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle.

    PubMed

    Reidy, P T; Walker, D K; Dickinson, J M; Gundermann, D M; Drummond, M J; Timmerman, K L; Cope, M B; Mukherjea, R; Jennings, K; Volpi, E; Rasmussen, B B

    2014-06-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. Copyright © 2014 the American Physiological Society.

  7. Soy-dairy protein blend and whey protein ingestion after resistance exercise increases amino acid transport and transporter expression in human skeletal muscle

    PubMed Central

    Reidy, P. T.; Walker, D. K.; Dickinson, J. M.; Gundermann, D. M.; Drummond, M. J.; Timmerman, K. L.; Cope, M. B.; Mukherjea, R.; Jennings, K.; Volpi, E.

    2014-01-01

    Increasing amino acid availability (via infusion or ingestion) at rest or postexercise enhances amino acid transport into human skeletal muscle. It is unknown whether alterations in amino acid availability, from ingesting different dietary proteins, can enhance amino acid transport rates and amino acid transporter (AAT) mRNA expression. We hypothesized that the prolonged hyperaminoacidemia from ingesting a blend of proteins with different digestion rates postexercise would enhance amino acid transport into muscle and AAT expression compared with the ingestion of a rapidly digested protein. In a double-blind, randomized clinical trial, we studied 16 young adults at rest and after acute resistance exercise coupled with postexercise (1 h) ingestion of either a (soy-dairy) protein blend or whey protein. Phenylalanine net balance and transport rate into skeletal muscle were measured using stable isotopic methods in combination with femoral arteriovenous blood sampling and muscle biopsies obtained at rest and 3 and 5 h postexercise. Phenylalanine transport into muscle and mRNA expression of select AATs [system L amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, system A amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, cationic amino acid transporter 1/SLC7A1] increased to a similar extent in both groups (P < 0.05). However, the ingestion of the protein blend resulted in a prolonged and positive net phenylalanine balance during postexercise recovery compared with whey protein (P < 0.05). Postexercise myofibrillar protein synthesis increased similarly between groups. We conclude that, while both protein sources enhanced postexercise AAT expression, transport into muscle, and myofibrillar protein synthesis, postexercise ingestion of a protein blend results in a slightly prolonged net amino acid balance across the leg compared with whey protein. PMID:24699854

  8. Tunneling explains efficient electron transport via protein junctions.

    PubMed

    Fereiro, Jerry A; Yu, Xi; Pecht, Israel; Sheves, Mordechai; Cuevas, Juan Carlos; Cahen, David

    2018-05-15

    Metalloproteins, proteins containing a transition metal ion cofactor, are electron transfer agents that perform key functions in cells. Inspired by this fact, electron transport across these proteins has been widely studied in solid-state settings, triggering the interest in examining potential use of proteins as building blocks in bioelectronic devices. Here, we report results of low-temperature (10 K) electron transport measurements via monolayer junctions based on the blue copper protein azurin (Az), which strongly suggest quantum tunneling of electrons as the dominant charge transport mechanism. Specifically, we show that, weakening the protein-electrode coupling by introducing a spacer, one can switch the electron transport from off-resonant to resonant tunneling. This is a consequence of reducing the electrode's perturbation of the Cu(II)-localized electronic state, a pattern that has not been observed before in protein-based junctions. Moreover, we identify vibronic features of the Cu(II) coordination sphere in transport characteristics that show directly the active role of the metal ion in resonance tunneling. Our results illustrate how quantum mechanical effects may dominate electron transport via protein-based junctions.

  9. Direct observation of electrogenic NH4(+) transport in ammonium transport (Amt) proteins.

    PubMed

    Wacker, Tobias; Garcia-Celma, Juan J; Lewe, Philipp; Andrade, Susana L A

    2014-07-08

    Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4(+) scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4(+)/NH3 transport is used instead in acid-base and pH homeostasis in kidney or NH4(+)/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters.

  10. Direct observation of electrogenic NH4+ transport in ammonium transport (Amt) proteins

    PubMed Central

    Wacker, Tobias; Garcia-Celma, Juan J.; Lewe, Philipp; Andrade, Susana L. A.

    2014-01-01

    Ammonium transport (Amt) proteins form a ubiquitous family of integral membrane proteins that specifically shuttle ammonium across membranes. In prokaryotes, archaea, and plants, Amts are used as environmental NH4+ scavengers for uptake and assimilation of nitrogen. In the eukaryotic homologs, the Rhesus proteins, NH4+/NH3 transport is used instead in acid–base and pH homeostasis in kidney or NH4+/NH3 (and eventually CO2) detoxification in erythrocytes. Crystal structures and variant proteins are available, but the inherent challenges associated with the unambiguous identification of substrate and monitoring of transport events severely inhibit further progress in the field. Here we report a reliable in vitro assay that allows us to quantify the electrogenic capacity of Amt proteins. Using solid-supported membrane (SSM)-based electrophysiology, we have investigated the three Amt orthologs from the euryarchaeon Archaeoglobus fulgidus. Af-Amt1 and Af-Amt3 are electrogenic and transport the ammonium and methylammonium cation with high specificity. Transport is pH-dependent, with a steep decline at pH values of ∼5.0. Despite significant sequence homologies, functional differences between the three proteins became apparent. SSM electrophysiology provides a long-sought-after functional assay for the ubiquitous ammonium transporters. PMID:24958855

  11. 4th International Consensus Conference on Advanced Breast Cancer (ABC4), Lisbon, November 4, 2017 : ABC4 Consensus: Assessment by a Panel of German Experts.

    PubMed

    Untch, Michael; Würstlein, Rachel; Marschner, Norbert; Lüftner, Diana; Augustin, Doris; Briest, Susanne; Ettl, Johannes; Haidinger, Renate; Müller, Lothar; Müller, Volkmar; Ruckhäberle, Eugen; Harbeck, Nadia; Thomssen, Christoph

    2018-05-01

    The fourth international advanced breast cancer consensus conference (ABC4) on the diagnosis and treatment of advanced breast cancer (ABC) headed by Professor Fatima Cardoso was once again held in Lisbon on November 2 - 4, 2017. To simplify matters, the abbreviation ABC will be used hereinafter in the text. In clinical practice, the abbreviation corresponds to metastatic breast cancer or locally far-advanced disease. This year the focus was on new developments in the treatment of ABC. Topics discussed included the importance of CDK4/6 inhibition in hormone receptor (HR)-positive ABC, the use of dual antibody blockade to treat HER2-positive ABC, PARP inhibition in triple-negative ABC and the potential therapeutic outcomes. Another major area discussed at the conference was BRCA-associated breast cancer, the treatment of cerebral metastasis, and individualized treatment decisions based on molecular testing (so-called precision medicine). As in previous years, close cooperation with representatives from patient organizations from around the world is an important aspect of the ABC conference. This cooperation was reinforced and expanded at the ABC4 conference. A global alliance was founded at the conclusion of the consensus conference, which aims to promote and coordinate the measures considered necessary by patient advocates worldwide. Because the panel of experts was composed of specialists from all over the world, it was inevitable that the ABC consensus also reflected country-specific features. As in previous years, a team of German breast cancer specialists who closely followed the consensus voting of the ABC panelists in Lisbon and intensively discussed the votes has therefore commented on the consensus in the context of the current German guidelines on the diagnosis and treatment of breast cancer 1 ,  2 used in clinical practice in Germany. The ABC consensus is based on the votes of the ABC panelists in Lisbon.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    2010-07-27

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

  13. The minimal-ABC trees with B1-branches.

    PubMed

    Dimitrov, Darko; Du, Zhibin; Fonseca, Carlos M da

    2018-01-01

    The atom-bond connectivity index (or, for short, ABC index) is a molecular structure descriptor bridging chemistry to graph theory. It is probably the most studied topological index among all numerical parameters of a graph that characterize its topology. For a given graph G = (V, E), the ABC index of G is defined as [Formula: see text], where di denotes the degree of the vertex i, and ij is the edge incident to the vertices i and j. A combination of physicochemical and the ABC index properties are commonly used to foresee the bioactivity of different chemical composites. Additionally, the applicability of the ABC index in chemical thermodynamics and other areas of chemistry, such as in dendrimer nanostars, benzenoid systems, fluoranthene congeners, and phenylenes is well studied in the literature. While finding of the graphs with the greatest ABC-value is a straightforward assignment, the characterization of the tree(s) with minimal ABC index is a problem largely open and has recently given rise to numerous studies and conjectures. A B1-branch of a graph is a pendent path of order 2. In this paper, we provide an important step forward to the full characterization of these minimal trees. Namely, we show that a minimal-ABC tree contains neither 4 nor 3 B1-branches. The case when the number of B1-branches is 2 is also considered.

  14. Functional Tat transport of unstructured, small, hydrophilic proteins.

    PubMed

    Richter, Silke; Lindenstrauss, Ute; Lücke, Christian; Bayliss, Richard; Brüser, Thomas

    2007-11-16

    The twin-arginine translocation (Tat) system is a protein translocation system that is adapted to the translocation of folded proteins across biological membranes. An understanding of the folding requirements for Tat substrates is of fundamental importance for the elucidation of the transport mechanism. We now demonstrate for the first time Tat transport for fully unstructured proteins, using signal sequence fusions to naturally unfolded FG repeats from the yeast Nsp1p nuclear pore protein. The transport of unfolded proteins becomes less efficient with increasing size, consistent with only a single interaction between the system and the substrate. Strikingly, the introduction of six residues from the hydrophobic core of a globular protein completely blocked translocation. Physiological data suggest that hydrophobic surface patches abort transport at a late stage, most likely by membrane interactions during transport. This study thus explains the observed restriction of the Tat system to folded globular proteins on a molecular level.

  15. Yang-Mills theory and the ABC conjecture

    NASA Astrophysics Data System (ADS)

    He, Yang-Hui; Hu, Zhi; Probst, Malte; Read, James

    2018-05-01

    We establish a precise correspondence between the ABC Conjecture and 𝒩 = 4 super-Yang-Mills theory. This is achieved by combining three ingredients: (i) Elkies’ method of mapping ABC-triples to elliptic curves in his demonstration that ABC implies Mordell/Faltings; (ii) an explicit pair of elliptic curve and associated Belyi map given by Khadjavi-Scharaschkin; and (iii) the fact that the bipartite brane-tiling/dimer model for a gauge theory with toric moduli space is a particular dessin d’enfant in the sense of Grothendieck. We explore this correspondence for the highest quality ABC-triples as well as large samples of random triples. The conjecture itself is mapped to a statement about the fundamental domain of the toroidal compactification of the string realization of 𝒩 = 4 SYM.

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

    PubMed

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

    2016-01-01

    Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (<0.5 μg/mL) and GO (<20 μg/mL) did not decrease cell viability, generate reactive oxygen species, or disrupt mitochondrial function. However, graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 μg/mL graphene and 0.05 μg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

  17. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  18. Caenorhabditis elegans ABCRNAi Transporters Interact Genetically With rde-2 and mut-7

    PubMed Central

    Sundaram, Prema; Han, Wang; Cohen, Nancy; Echalier, Benjamin; Albin, John; Timmons, Lisa

    2008-01-01

    RNA interference (RNAi) mechanisms are conserved and consist of an interrelated network of activities that not only respond to exogenous dsRNA, but also perform endogenous functions required in the fine tuning of gene expression and in maintaining genome integrity. Not surprisingly, RNAi functions have widespread influences on cellular function and organismal development. Previously, we observed a reduced capacity to mount an RNAi response in nine Caenorhabditis elegans mutants that are defective in ABC transporter genes (ABCRNAi mutants). Here, we report an exhaustive study of mutants, collectively defective in 49 different ABC transporter genes, that allowed for the categorization of one additional transporter into the ABCRNAi gene class. Genetic complementation tests reveal functions for ABCRNAi transporters in the mut-7/rde-2 branch of the RNAi pathway. These second-site noncomplementation interactions suggest that ABCRNAi proteins and MUT-7/RDE-2 function together in parallel pathways and/or as multiprotein complexes. Like mut-7 and rde-2, some ABCRNAi mutants display transposon silencing defects. Finally, our analyses reveal a genetic interaction network of ABCRNAi gene function with respect to this part of the RNAi pathway. From our results, we speculate that the coordinated activities of ABCRNAi transporters, through their effects on endogenous RNAi-related mechanisms, ultimately affect chromosome function and integrity. PMID:18245353

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Hijacking membrane transporters for arsenic phytoextraction

    PubMed Central

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

    2012-01-01

    Arsenic is a toxic metalloid and recognized carcinogen. Arsenate and arsenite are the most common arsenic species available for uptake by plants. As an inorganic phosphate (Pi) analog, arsenate is acquired by plant roots through endogenous Pi transport systems. Inside the cell, arsenate is reduced to the thiol-reactive form arsenite. Glutathione (GSH)-conjugates of arsenite may be extruded from the cell or sequestered in vacuoles by members of the ATP-binding cassette (ABC) family of transporters. In the present study we sought to enhance both plant arsenic uptake through Pi transporter overexpression, and plant arsenic tolerance through ABC transporter overexpression. We demonstrate that Arabidopsis thaliana plants overexpressing the high-affinity Pi transporter family members, AtPht1;1 or AtPht1;7, are hypersensitive to arsenate due to increased arsenate uptake. These plants do not exhibit increased sensitivity to arsenite. Co-overexpression of the yeast ABC transporter YCF1 in combination with AtPht1;1 or AtPht1;7 suppresses the arsenate-sensitive phenotype while further enhancing arsenic uptake. Taken together, our results support an arsenic transport mechanism in which arsenate uptake is increased through Pi transporter overexpression, and arsenic tolerance is enhanced through YCF1-mediated vacuolar sequestration. This work substantiates the viability of coupling enhanced uptake and vacuolar sequestration as a means for developing a prototypical engineered arsenic hyperaccumulator. PMID:23108027

  2. Transcription of putative tonoplast transporters in response to glyphosate and paraquat stress in Conyza bonariensis and Conyza canadensis and selection of reference genes for qRT-PCR.

    PubMed

    Moretti, Marcelo L; Alárcon-Reverte, Rocio; Pearce, Stephen; Morran, Sarah; Hanson, Bradley D

    2017-01-01

    Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines

  3. Transcription of putative tonoplast transporters in response to glyphosate and paraquat stress in Conyza bonariensis and Conyza canadensis and selection of reference genes for qRT-PCR

    PubMed Central

    Alárcon-Reverte, Rocio; Pearce, Stephen; Morran, Sarah; Hanson, Bradley D.

    2017-01-01

    Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines

  4. High-Affinity Vanadate Transport System in the Cyanobacterium Anabaena variabilis ATCC 29413

    PubMed Central

    Pratte, Brenda S.; Thiel, Teresa

    2006-01-01

    High-affinity vanadate transport systems have not heretofore been identified in any organism. Anabaena variabilis, which can fix nitrogen by using an alternative V-dependent nitrogenase, transported vanadate well. The concentration of vanadate giving half-maximum V-nitrogenase activity when added to V-starved cells was about 3 × 10−9 M. The genes for an ABC-type vanadate transport system, vupABC, were found in A. variabilis about 5 kb from the major cluster of genes encoding the V-nitrogenase, and like those genes, the vupABC genes were repressed by molybdate; however, unlike the V-nitrogenase genes the vanadate transport genes were expressed in vegetative cells. A vupB mutant failed to grow by using V-nitrogenase unless high levels of vanadate were provided, suggesting that there was also a low-affinity vanadate transport system that functioned in the vupB mutant. The vupABC genes belong to a family of putative metal transport genes that include only one other characterized transport system, the tungstate transport genes of Eubacterium acidaminophilum. Similar genes are not present in the complete genomes of other bacterial strains that have a V-nitrogenase, including Azotobacter vinelandii, Rhodopseudomonas palustris, and Methanosarcina barkeri. PMID:16385036

  5. Effect of levofloxacin, pazufloxacin, enrofloxacin, and meloxicam on the immunolocalization of ABCG-2 transporter protein in rabbit retina.

    PubMed

    Khan, Adil Mehraj; Rampal, Satyavan; Sood, Naresh Kumar

    2018-03-01

    Adenosine triphosphate-binding cassette (ABC) sub-family G member-2 (ABCG-2) is a transporter protein, implicated for multi-drug efflux from tissues. This study evaluated the effect of fluoroquinolones; levofloxacin, pazufloxacin and enrofloxacin, and non-steroidal anti-inflammatory drug, meloxicam; on the immunolocalization of ABCG-2 transporter protein of rabbit retinas. Thirty-two male rabbits were randomly divided in to eight groups. Control group was gavaged, 2% benzyl alcohol in 5% dextrose since these chemicals are excipients of the drug preparations used in the treatment groups of this study. Four groups were exclusively gavaged, levofloxacin hemihydrate (10 mg/kg body weight b.i.d 12 h), pazufloxacin mesylate (10 mg/kg body weight b.i.d 12 h), enrofloxacin (20 mg/kg body weight o.d.), and meloxicam (0.2 mg/kg body weight o.d.), respectively. Three other groups were co-gavaged meloxicam with above fluoroquinolones, respectively. These drugs were administered for 21 days. ABCG-2 immunolocalization was mild in the retinas of control and levofloxacin-alone-treated groups. The immunolocalization intensity was significantly higher in meloxicam-alone-treated group when compared to control and levofloxacin-alone-treated groups. Immunolocalization of this transporter increased in the levofloxacin-meloxicam co-treated group when compared to the levofloxacin-alone-treated group. Highest immunolocalization was observed in the enrofloxacin-meloxicam co-treated group although the immunolocalization of all treatment groups, except the levofloxacin-alone-treated group, was significantly higher than the control and levofloxacin-alone-treated groups.

  6. The xenoestrogens, bisphenol A and para-nonylphenol, decrease the expression of the ABCG2 transporter protein in human term placental explant cultures.

    PubMed

    Sieppi, E; Vähäkangas, K; Rautio, A; Ietta, F; Paulesu, L; Myllynen, P

    2016-07-05

    Many endogenous and xenobiotic compounds are substrates and regulators of human placental ABC transporters. ABCG2 is protecting fetus against foreign chemicals. Environmental xenoestrogens, like bisphenol A (BPA) and p-nonylphenol (p-NP), mimic natural estrogens and can affect hormonal systems. Effects of BPA, p-NP, DES (diethylstilbestrol) and estradiol (E2), on ABCG2 expression were studied using human first trimester and term placental explants. Role of estrogen receptors (ER) in the effects of chemicals was studied by ER antagonist. Term placenta expressed less ABCG2 protein. In term placentas BPA (p < 0.05), p-NP (p < 0.01) and E2 (p < 0.05) decreased the ABCG2 protein expression after 48 h exposure while after 24 h exposure, only E2 decreased the expression (p < 0.05). The chemicals did not affect ABCG2 in first trimester placentas. The ER antagonist affected differently the responses of chemicals. In conclusion, environmental xenoestrogens downregulate placental ABCG2 protein expression depending on gestational age. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. ABC1 Consensus Conference - a German Perspective: First International Consensus Conference on Advanced Breast Cancer (ABC1), Lisbon, November 5, 2011.

    PubMed

    Thomssen, Christoph; Marschner, Norbert; Untch, Michael; Decker, Thomas; Hegewisch-Becker, Susanna; Jackisch, Christian; Janni, Wolfgang; Hans-Joachim, Lück; von Minckwitz, Gunter; Scharl, Anton; Schneeweiss, Andreas; Tesch, Hans; Welt, Anja; Harbeck, Nadia

    2012-02-01

    A group of German breast cancer experts (medical oncologists and gynaecologists) reviewed and commented on the results of the first international 'Advanced Breast Cancer First Consensus Conference' (ABC1) for the diagnosis and treatment of advanced breast cancer. The ABC1 Conference is an initiative of the European School of Oncology (ESO) Metastatic Breast Cancer Task Force in cooperation with the EBCC (European Breast Cancer Conference), ESMO (European Society of Medical Oncology) and the American JNCI (Journal of the National Cancer Institute). The main focus of the ABC1 Conference was metastatic breast cancer (stage IV). The ABC1 consensus is based on the vote of 33 breast cancer experts from different countries and has been specified as a guideline for therapeutic practice by the German expert group. It is the objective of the ABC1 consensus as well as of the German comments to provide an internationally standardized and evidence-based foundation for qualified decision-making in the treatment of metastatic breast cancer.

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

    PubMed

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

    2014-10-01

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

  9. Multidrug Resistance Proteins (MRPs/ABCCs) in Cancer Chemotherapy and Genetic Diseases

    PubMed Central

    Chen, Zhe-Sheng; Tiwari, Amit K.

    2011-01-01

    The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins that are best known for their ability to transport a wide variety of exogenous and endogenous substances across membranes against a concentration gradient via ATP hydrolysis. There are seven subfamilies of human ABC transporters, one of the largest being the ‘C’ subfamily (gene symbol ABCC). Nine ABCC subfamily members, the so-called Multidrug Resistance Proteins (MRPs) 1-9, have been implicated in mediating multidrug resistance in tumor cells to varying degrees as the efflux extrude chemotherapeutic compounds (or their metabolites) from malignant cells. Some of the MRPs are also known to either influence drug disposition in normal tissues or modulate the elimination of drugs (or their metabolites) via hepatobiliary or renal excretory pathways. In addition, the cellular efflux of physiologically important organic anions such as leukotriene C4 and cAMP is mediated by one or more of the MRPs. Finally, mutations in several MRPs are associated with human genetic disorders. In this review article, the current biochemical and physiological knowledge of MRP1-MRP9 in cancer chemotherapy and human genetic disease is summarized. The mutations in MRP2/ABCC2 leading to conjugated hyperbilirubinemia (Dubin-Johnson syndrome) and in MRP6/ABCC6 leading to the connective tissue disorder Pseudoxanthoma elasticum are also discussed. PMID:21740521

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

    PubMed

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

    2017-08-01

    Drug resistance is a major problem in anticancer therapy. ATP-binding cassette (ABC) transporters have a role in the multidrug resistance. A new regimen of chemotherapy has been proposed, called "metronomic chemotherapy". Metronomic chemotherapy is the frequent, regular administration of drug doses designed to maintain low, but active, concentrations of chemotherapeutic drugs over prolonged periods of time, without causing serious toxicities. Metronomic chemotherapy regimens were developed to optimize the antitumor efficacy of agents that target the tumor vasculature instead of tumor cells, and to reduce toxicity of antineoplastic drugs [1]. Nevertheless, recent studies revealed that ABC transporters are expressed at a higher level in the endothelium in the tumor. To avoid resistance to metronomic anti-angiogenic chemotherapy, ABC transporter inhibition of tumor endothelial cells may be a promising strategy. In this mini-review, we discuss the possible mechanism of resistance to metronomic chemotherapy from the viewpoint of tumor endothelial cell biology, focusing on ABC transporters. Copyright © 2017. Published by Elsevier B.V.

  11. Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.).

    PubMed

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-04-01

    Biopesticides or transgenic crops based on Cry toxins from the soil bacterium Bacillus thuringiensis (Bt) effectively control agricultural insect pests. The sustainable use of Bt biopesticides and Bt crops is threatened, however, by the development of Cry resistance in the target pests. The diamondback moth, Plutella xylostella (L.), is the first pest that developed resistance to a Bt biopesticide in the field, and a recent study has shown that the resistance of P. xylostella to Cry1Ac is caused by a mutation in an ATP-binding cassette (ABC) transporter gene (ABCC2). In this study, we report that down-regulation of a novel ABC transporter gene from ABCG subfamily (Pxwhite) is associated with Cry1Ac resistance in P. xylostella. The full-length cDNA sequence of Pxwhite was cloned and analyzed. Spatial-temporal expression detection revealed that Pxwhite was expressed in all tissues and developmental stages, and highest expressed in Malpighian tubule tissue and in egg stage. Sequence variation analysis of Pxwhite indicated the absence of constant non-synonymous mutations between susceptible and resistant strains, whereas midgut transcript analysis showed that Pxwhite was remarkably reduced in all resistant strains and further reduced when larvae of the moderately resistant SZ-R strain were subjected to selection with Cry1Ac toxin. Furthermore, RNA interference (RNAi)-mediated suppression of Pxwhite gene expression significantly reduced larval susceptibility to Cry1Ac toxin, and genetic linkage analysis confirmed that down-regulation of Pxwhite gene is tightly linked to Cry1Ac resistance in P. xylostella. To our knowledge, this is the first report indicating that Pxwhite gene is involved in Cry1Ac resistance in P. xylostella. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Structure of a pantothenate transporter and implications for ECF module sharing and energy coupling of group II ECF transporters

    PubMed Central

    Zhang, Minhua; Bao, Zhihao; Zhao, Qin; Guo, Hui; Xu, Ke; Wang, Chengcheng

    2014-01-01

    Energy-coupling factor (ECF) transporters are a unique group of ATP-binding cassette (ABC) transporters responsible for micronutrient uptake from the environment. Each ECF transporter is composed of an S component (or EcfS protein) and T/A/A′ components (or EcfT/A/A′ proteins; ECF module). Among the group II ECF transporters, several EcfS proteins share one ECF module; however, the underlying mechanism remains unknown. Here we report the structure of a group II ECF transporter–pantothenate transporter from Lactobacillus brevis (LbECF-PanT), which shares the ECF module with the folate and hydroxymethylpyrimidine transporters (LbECF-FolT and LbECF-HmpT). Structural and mutational analyses revealed the residues constituting the pantothenate-binding pocket. We found that although the three EcfS proteins PanT, FolT, and HmpT are dissimilar in sequence, they share a common surface area composed of the transmembrane helices 1/2/6 (SM1/2/6) to interact with the coupling helices 2/3 (CH2/3) of the same EcfT. CH2 interacts mainly with SM1 via hydrophobic interactions, which may modulate the sliding movement of EcfS. CH3 binds to a hydrophobic surface groove formed by SM1, SM2, and SM6, which may transmit the conformational changes from EcfA/A′ to EcfS. We also found that the residues at the intermolecular surfaces in LbECF-PanT are essential for transporter activity, and that these residues may mediate intermolecular conformational transmission and/or affect transporter complex stability. In addition, we found that the structure of EcfT is conformationally dynamic, which supports its function as a scaffold to mediate the interaction of the ECF module with various EcfS proteins to form different transporter complexes. PMID:25512487

  13. Reexamining the role of choline transporter-like (Ctlp) proteins in choline transport.

    PubMed

    Zufferey, Rachel; Santiago, Teresa C; Brachet, Valerie; Ben Mamoun, Choukri

    2004-02-01

    In Saccharomyces cerevisiae, choline enters the cell via a single high-affinity transporter, Hnmlp. hnm1delta cells lacking HNM1 gene are viable. However, they are unable to transport choline suggesting that no additional active choline transporters are present in this organism. A complementation study of a choline auxotrophic mutant, ctrl-ise (hnm1-ise), using a cDNA library from Torpedo marmorata electric lobe identified a membrane protein named Torpedo marmorata choline transporter-like, tCtl1p. tCtllp was proposed to mediate a high-affinity choline transport (O'Regan et al., 1999, Proc. Natl. Acad. Sci.). Homologs of tCtl1p have been identified in other organisms, including yeast (Pns1p, YOR161c) and are postulated to function as choline transporters. Here we provide several lines of evidence indicating that Ctlp proteins are not involved in choline transport. Loss of PNS1 has no effect on choline transport and overexpression of either PNS1 or tCTL1 does not restore choline uptake activity of choline transport-defective mutants. The data presented here call into question the role of proteins of the CTL family in choline transport and suggest that the mechanism by which tCTL1 complements hnm1-ise mutant is independent of its ability to transport choline.

  14. Searching for the fastest dynamo: laminar ABC flows.

    PubMed

    Alexakis, Alexandros

    2011-08-01

    The growth rate of the dynamo instability as a function of the magnetic Reynolds number R(M) is investigated by means of numerical simulations for the family of the Arnold-Beltrami-Childress (ABC) flows and for two different forcing scales. For the ABC flows that are driven at the largest available length scale, it is found that, as the magnetic Reynolds number is increased: (a) The flow that results first in a dynamo is the 2 1/2-dimensional flow for which A=B and C=0 (and all permutations). (b) The second type of flow that results in a dynamo is the one for which A=B≃2C/5 (and permutations). (c) The most symmetric flow, A=B=C, is the third type of flow that results in a dynamo. (d) As R(M) is increased, the A=B=C flow stops being a dynamo and transitions from a local maximum to a third-order saddle point. (e) At larger R(M), the A=B=C flow reestablishes itself as a dynamo but remains a saddle point. (f) At the largest examined R(M), the growth rate of the 2 1/2-dimensional flows starts to decay, the A=B=C flow comes close to a local maximum again, and the flow A=B≃2C/5 (and permutations) results in the fastest dynamo with growth rate γ≃0.12 at the largest examined R(M). For the ABC flows that are driven at the second largest available length scale, it is found that (a) the 2 1/2-dimensional flows A=B,C=0 (and permutations) are again the first flows that result in a dynamo with a decreased onset. (b) The most symmetric flow, A=B=C, is the second type of flow that results in a dynamo. It is, and it remains, a local maximum. (c) At larger R(M), the flow A=B≃2C/5 (and permutations) appears as the third type of flow that results in a dynamo. As R(M) is increased, it becomes the flow with the largest growth rate. The growth rates appear to have some correlation with the Lyapunov exponents, but constructive refolding of the field lines appears equally important in determining the fastest dynamo flow.

  15. Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death*

    PubMed Central

    Chowdhury, Sayan; Mukhopadhyay, Rupkatha; Saha, Sourav; Mishra, Amartya; Sengupta, Souvik; Roy, Syamal; Majumder, Hemanta K.

    2014-01-01

    In parasites, ATP-binding cassette (ABC) transporters represent an important family of proteins related to drug resistance and other biological activities. Resistance of leishmanial parasites to therapeutic drugs continues to escalate in developing countries, and in many instances, it is due to overexpressed ABC efflux pumps. Progressively adapted baicalein (BLN)-resistant parasites (pB25R) show overexpression of a novel ABC transporter, which was classified as ABCC2 or Leishmania donovani multidrug resistance protein 2 (LdMRP2). The protein is primarily localized in the flagellar pocket region and in internal vesicles. Overexpressed LdABCC2 confers substantial BLN resistance to the parasites by rapid drug efflux. The BLN-resistant promastigotes when transformed into amastigotes in macrophage cells cannot be cured by treatment of macrophages with BLN. Amastigote resistance is concomitant with the overexpression of macrophage MRP2 transporter. Reporter analysis and site-directed mutagenesis assays demonstrated that antioxidant response element 1 is activated upon infection. The expression of this phase II detoxifying gene is regulated by NFE2-related factor 2 (Nrf2)-mediated antioxidant response element activation. In view of the fact that the signaling pathway of phosphoinositol 3-kinase controls microfilament rearrangement and translocation of actin-associated proteins, the current study correlates with the intricate pathway of phosphoinositol 3-kinase-mediated nuclear translocation of Nrf2, which activates MRP2 expression in macrophages upon infection by the parasites. In contrast, phalloidin, an agent that prevents depolymerization of actin filaments, inhibits Nrf2 translocation and Mrp2 gene activation by pB25R infection. Taken together, these results provide insight into the mechanisms by which resistant clinical isolates of L. donovani induce intracellular events relevant to drug resistance. PMID:24706751

  16. Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides.

    PubMed

    Narita, Shin-ichiro; Tokuda, Hajime

    2009-07-07

    Seven Lpt proteins (A through G) are thought to be involved in lipopolysaccharide transport from the inner to outer membrane of Escherichia coli. LptB belongs to the ATP-binding cassette transporter superfamily. Although the lptB gene lacks neighboring genes encoding membrane subunits, bioinformatic analyses recently indicated that two distantly located consecutive genes, lptF and lptG, could encode membrane subunits. To examine this possibility, LptB was expressed with LptF and LptG. We report here that both LptF and LptG formed a complex with LptB. Furthermore, an inner membrane protein, LptC, which had been implicated in lipopolysaccharide transport, was also included in this complex.

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

    PubMed

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

    2017-07-01

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

  18. Attenuated mutant strain of Salmonella Typhimurium lacking the ZnuABC transporter contrasts tumor growth promoting anti-cancer immune response.

    PubMed

    Chirullo, Barbara; Ammendola, Serena; Leonardi, Leonardo; Falcini, Roberto; Petrucci, Paola; Pistoia, Claudia; Vendetti, Silvia; Battistoni, Andrea; Pasquali, Paolo

    2015-07-10

    Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens.We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors.

  19. Transcriptional regulation, metal binding properties and structure of Pden1597, an unusual zinc transport protein from Paracoccus denitrificans

    DOE PAGES

    Handali, Melody; Neupane, Durga P.; Roychowdhury, Hridindu; ...

    2015-03-18

    Here, ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn 2+ and Mn 2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn 2+ or Mn 2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties,more » and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn 2+ over Mn 2+ specificity, the Zn 2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn 2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn 2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn 2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn 2+ specificity.« less

  20. Transcriptional Regulation, Metal Binding Properties and Structure of Pden1597, an Unusual Zinc Transport Protein from Paracoccus denitrificans*

    PubMed Central

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

    2015-01-01

    ATP-binding cassette (ABC) transporters of the cluster 9 family are ubiquitous among bacteria and essential for acquiring Zn2+ and Mn2+ from the environment or, in the case of pathogens, from the host. These rely on a substrate-binding protein (SBP) to coordinate the relevant metal with high affinity and specificity and subsequently release it to a membrane permease for translocation into the cytoplasm. Although a number of cluster 9 SBP structures have been determined, the structural attributes conferring Zn2+ or Mn2+ specificity remain ambiguous. Here we describe the gene expression profile, in vitro metal binding properties, and crystal structure of a new cluster 9 SBP from Paracoccus denitrificans we have called AztC. Although all of our results strongly indicate Zn2+ over Mn2+ specificity, the Zn2+ ion is coordinated by a conserved Asp residue only observed to date as a metal ligand in Mn2+-specific SBPs. The unusual sequence properties of this protein are shared among close homologues, including members from the human pathogens Klebsiella pneumonia and Enterobacter aerogenes, and would seem to suggest a subclass of Zn2+-specific transporters among the cluster 9 family. In any case, the unusual coordination environment of AztC expands the already considerable range of those available to Zn2+-specific SBPs and highlights the presence of a His-rich loop as the most reliable indicator of Zn2+ specificity. PMID:25787075

  1. The Origin and Early Evolution of Membrane Proteins

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Schweighofter, Karl; Wilson, Michael A.

    2006-01-01

    The origin and early evolution of membrane proteins, and in particular ion channels, are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Prokarya, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

  2. Jump into a New Fold—A Homology Based Model for the ABCG2/BCRP Multidrug Transporter

    PubMed Central

    László, Laura; Sarkadi, Balázs

    2016-01-01

    ABCG2/BCRP is a membrane protein, involved in xenobiotic and endobiotic transport in key pharmacological barriers and drug metabolizing organs, in the protection of stem cells, and in multidrug resistance of cancer. Pharmacogenetic studies implicated the role of ABCG2 in response to widely used medicines and anticancer agents, as well as in gout. Its Q141K variant exhibits decreased functional expression thus increased drug accumulation and decreased urate secretion. Still, there has been no reliable molecular model available for this protein, as the published structures of other ABC transporters could not be properly fitted to the ABCG2 topology and experimental data. The recently published high resolution structure of a close homologue, the ABCG5-ABCG8 heterodimer, revealed a new ABC transporter fold, unique for ABCG proteins. Here we present a structural model of the ABCG2 homodimer based on this fold and detail the experimental results supporting this model. In order to describe the effect of mutations on structure and dynamics, and characterize substrate recognition and cholesterol regulation we performed molecular dynamics simulations using full length ABCG2 protein embedded in a membrane bilayer and in silico docking simulations. Our results show that in the Q141K variant the introduced positive charge diminishes the interaction between the nucleotide binding and transmembrane domains and the R482G variation alters the orientation of transmembrane helices. Moreover, the R482 position, which plays a role the substrate specificity of the transporter, is located in one of the substrate binding pockets identified by the in silico docking calculations. In summary, the ABCG2 model and in silico simulations presented here may have significant impact on understanding drug distribution and toxicity, as well as drug development against cancer chemotherapy resistance or gout. PMID:27741279

  3. Multixenobiotic resistance in Mytilus edulis: Molecular and functional characterization of an ABCG2- type transporter in hemocytes and gills.

    PubMed

    Ben Cheikh, Yosra; Xuereb, Benoit; Boulangé-Lecomte, Céline; Le Foll, Frank

    2018-02-01

    Among the cellular protection arsenal, ABC transporters play an important role in xenobiotic efflux in marine organisms. Two pumps belonging to B and C subfamily has been identified in Mytilus edulis. In this study, we investigated the presence of the third major subtype ABCG2/BCRP protein in mussel tissues. Transcript was expressed in hemocytes and with higher level in gills. Molecular characterization revealed that mussel ABCG2 transporter shares the sequence and organizational structure with mammalian and molluscan orthologs. Overall identity of the predicted amino acid sequence with corresponding homologs from other organisms was between 49% and 98%. Moreover, protein efflux activity was demonstrated using a combination of fluorescent allocrites and specific inhibitors. The accumulation of bodipy prazosin and pheophorbide A was heterogeneous in gills and hemocytes. Most of the used blockers enhanced probe accumulation at different levels, most significantly for bodipy prazosin. Moreover, Mrp classical blocker MK571 showed a polyspecificity. In conclusion, our data demonstrate that several ABC transporters contribute to MXR phenotype in the blue mussel including ABCG2 that forms an active pump in hemocytes and gills. Efforts are needed to distinguish between the different members and to explore their single function and specificity towards allocrites and chemosensitizers. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. LhnR and upstream operon LhnABC in Agrobacterium vitis regulate the induction of tobacco hypersensitive responses, grape necrosis and swarming motility.

    PubMed

    Zheng, Desen; Hao, Guixia; Cursino, Luciana; Zhang, Hongsheng; Burr, Thomas J

    2012-09-01

    The characterization of Tn5 transposon insertional mutants of Agrobacterium vitis strain F2/5 revealed a gene encoding a predicted LysR-type transcriptional regulator, lhnR (for 'LysR-type regulator associated with HR and necrosis'), and an immediate upstream operon consisting of three open reading frames (lhnABC) required for swarming motility, surfactant production and the induction of a hypersensitive response (HR) on tobacco and necrosis on grape. The operon lhnABC is unique to A. vitis among the sequenced members in Rhizobiaceae. Mutagenesis of lhnR and lhnABC by gene disruption and complementation of ΔlhnR and ΔlhnABC confirmed their roles in the expression of these phenotypes. Mutation of lhnR resulted in complete loss of HR, swarming motility, surfactant production and reduced necrosis, whereas mutation of lhnABC resulted in loss of swarming motility, delayed and reduced HR development and reduced surfactant production and necrosis. The data from promoter-green fluorescent protein (gfp) fusions showed that lhnR suppresses the expression of lhnABC and negatively autoregulates its own expression. It was also shown that lhnABC negatively affects its own expression and positively affects the transcription of lhnR. lhnR and lhnABC constitute a regulatory circuit that coordinates the transcription level of lhnR, resulting in the expression of swarming, surfactant, HR and necrosis phenotypes. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

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

    PubMed Central

    2012-01-01

    Background The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic

  6. Whole genome re-sequencing identifies a mutation in an ABC transporter (mdr2) in a Plasmodium chabaudi clone with altered susceptibility to antifolate drugs☆

    PubMed Central

    Martinelli, Axel; Henriques, Gisela; Cravo, Pedro; Hunt, Paul

    2011-01-01

    In malaria parasites, mutations in two genes of folate biosynthesis encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) modify responses to antifolate therapies which target these enzymes. However, the involvement of other genes which modify the availability of exogenous folate, for example, has been proposed. Here, we used short-read whole-genome re-sequencing to determine the mutations in a clone of the rodent malaria parasite, Plasmodium chabaudi, which has altered susceptibility to both sulphadoxine and pyrimethamine. This clone bears a previously identified S106N mutation in dhfr and no mutation in dhps. Instead, three additional point mutations in genes on chromosomes 2, 13 and 14 were identified. The mutated gene on chromosome 13 (mdr2 K392Q) encodes an ABC transporter. Because Quantitative Trait Locus analysis previously indicated an association of genetic markers on chromosome 13 with responses to individual and combined antifolates, MDR2 is proposed to modulate antifolate responses, possibly mediated by the transport of folate intermediates. PMID:20858498

  7. A Perspective on Efflux Transport Proteins in the Liver

    PubMed Central

    Kock, K; Brouwer, K.L.R

    2013-01-01

    Detailed knowledge regarding the influence of hepatic transport proteins on drug disposition has advanced at a rapid pace over the past decade. Efflux transport proteins located in the basolateral and apical (canalicular) membranes of hepatocytes play an important role in the hepatic elimination of many endogenous and exogenous compounds, including drugs and metabolites. This review focuses on the role of these efflux transporters in hepatic drug excretion. The impact of these proteins as underlying factors for disease is highlighted, and the importance of hepatic efflux proteins in the efficacy and toxicity of drugs is discussed. In addition, a brief overview of methodology to evaluate the function of hepatic efflux transport proteins is provided. Current challenges in predicting the impact of altered efflux protein function on systemic, intestinal and hepatocyte exposure to drugs and metabolites are highlighted. PMID:22948894

  8. Regulation of the ATPase activity of ABCE1 from Pyrococcus abyssi by Fe-S cluster status and Mg²⁺: implication for ribosomal function.

    PubMed

    Sims, Lynn M; Igarashi, Robert Y

    2012-08-15

    Ribosomal function is dependent on multiple proteins. The ABCE1 ATPase, a unique ABC superfamily member that bears two Fe₄S₄ clusters, is crucial for ribosomal biogenesis and recycling. Here, the ATPase activity of the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg²⁺. Typically ATPases require Mg²⁺, as is true for PabABCE1, but Mg²⁺ also acts as a negative allosteric effector that modulates ATP affinity of PabABCE1. Physiological [Mg²⁺] inhibits the PabABCE1 ATPase (K(i) of ∼1 μM) for both apo- and holo-PabABCE1. Comparative kinetic analysis of Mg²⁺ inhibition shows differences in degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent ATP K(m) of apo-PabABCE1 increases >30-fold from ∼30 μM to over 1 mM with M²⁺. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge (φ) to being dependent on φ with cellular [Mg²⁺]. These findings uncover intricate overlapping effects by both [Mg²⁺] and the status of Fe-S clusters that regulate ABCE1's ATPase activity with implications to ribosomal function. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Production and characterization of single-chain antibody (scFv) against 3ABC non-structural protein in Escherichia coli for sero-diagnosis of Foot and Mouth Disease virus.

    PubMed

    Sharma, Gaurav K; Mahajan, Sonalika; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati K; Pattnaik, Bramhadev

    2014-11-01

    Differentiation of Foot-and-Mouth Disease infected from vaccinated animals is essential for effective implementation of vaccination based control programme. Detection of antibodies against 3ABC non-structural protein of FMD virus by immunodiagnostic assays provides reliable indication of FMD infection. Sero-monitoring of FMD in the large country like India is a big task where thousands of serum samples are annually screened. Currently, monoclonal or polyclonal antibodies are widely used in these immunodiagnostic assays. Considering the large population of livestock in the country, an economical and replenishable alternative of these antibodies was required. In this study, specific short chain variable fragment (scFv) antibody against 3B region of 3ABC poly-protein was developed. High level of scFv expression in Escherichia coli system was obtained by careful optimization in four different strains. Two formats of enzyme immunoassays (sandwich and competitive ELISAs) were optimized using scFv with objective to differentiate FMD infected among the vaccinated population. The assays were statistically validated by testing 2150 serum samples. Diagnostic sensitivity/specificity of sandwich and competitive ELISAs were determined by ROC method as 92.2%/95.5% and 89.5%/93.5%, respectively. This study demonstrated that scFv is a suitable alternate for immunodiagnosis of FMD on large scale. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  10. EHD proteins: Key conductors of endocytic transport

    PubMed Central

    Naslavsky, Naava; Caplan, Steve

    2010-01-01

    Regulation of endocytic transport is controlled by an elaborate network of proteins. Rab GTP-binding proteins and their effectors have well-defined roles in mediating specific endocytic transport steps, but until recently, less was known about the four mammalian dynamin-like C-terminal Eps15 Homology Domain (EHD) proteins that also regulate endocytic events. In recent years, however, great strides have been made in understanding the structure and function of these unique proteins. Indeed, a growing body of literature addresses EHD protein structure, interactions with binding partners, functions in mammalian cells, and the generation of various new model systems. Accordingly, this is now an opportune time to pause and review the function and mechanisms of action of EHD proteins, and to highlight some of the challenges and future directions for the field. PMID:21067929

  11. Membrane transporters studied by EPR spectroscopy: structure determination and elucidation of functional dynamics.

    PubMed

    Mullen, Anna; Hall, Jenny; Diegel, Janika; Hassan, Isa; Fey, Adam; MacMillan, Fraser

    2016-06-15

    During their mechanistic cycles membrane transporters often undergo extensive conformational changes, sampling a range of orientations, in order to complete their function. Such membrane transporters present somewhat of a challenge to conventional structural studies; indeed, crystallization of membrane-associated proteins sometimes require conditions that vary vastly from their native environments. Moreover, this technique currently only allows for visualization of single selected conformations during any one experiment. EPR spectroscopy is a magnetic resonance technique that offers a unique opportunity to study structural, environmental and dynamic properties of such proteins in their native membrane environments, as well as readily sampling their substrate-binding-induced dynamic conformational changes especially through complementary computational analyses. Here we present a review of recent studies that utilize a variety of EPR techniques in order to investigate both the structure and dynamics of a range of membrane transporters and associated proteins, focusing on both primary (ABC-type transporters) and secondary active transporters which were key interest areas of the late Professor Stephen Baldwin to whom this review is dedicated. © 2016 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  12. CREATING AN IPHONE APPLICATION FOR COLLECTING CONTINUOUS ABC DATA

    PubMed Central

    Whiting, Seth W; Dixon, Mark R

    2012-01-01

    This paper provides an overview and task analysis for creating a continuous ABC data-collection application using Xcode on a Mac computer. Behavior analysts can program an ABC data collection system, complete with a customized list of target clients, antecedents, behaviors, and consequences to be recorded, and have the data automatically sent to an e-mail account after observations have concluded. Further suggestions are provided to customize the ABC data- collection system for individual preferences and clinical needs. PMID:23060682

  13. ABCs of Being Smart: S Is for Supporting

    ERIC Educational Resources Information Center

    Foster, Joanne

    2014-01-01

    Joanne Foster's article "R We There Yet?" was first published in "Parenting for High Potential" ("PHP") in 2006, which became the springboard for the "ABCs of Being Smart" series of columns. At that time, Foster invited "PHP" readers to think about their own versions of the "ABCs of Being…

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

    PubMed

    Döring, Barbara; Petzinger, Ernst

    2014-08-01

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

  15. Regulation of ATP-binding Cassette Transporters and Cholesterol Efflux by Glucose in Primary Human Monocytes and Murine Bone Marrow-derived Macrophages

    PubMed Central

    Spartano, N. L.; Lamon-Fava, S.; Matthan, N. R.; Ronxhi, J.; Greenberg, A. S.; Obin, M. S.; Lichtenstein, A. H.

    2014-01-01

    Purpose Individuals with type 2 diabetes mellitus are at increased risk of developing atherosclerosis. This may be partially attributable to suppression of macrophage ATP-binding cassette (ABC) transporter mediated cholesterol efflux by sustained elevated blood glucose concentrations. 2 models were used to assess this potential relationship: human monocytes/leukocytes and murine bone marrow-derived macrophages (BMDM). Methods 10 subjects (4 F/6 M, 50–85 years, BMI 25–35 kg/m2) underwent an oral glucose challenge. Baseline and 1- and 2-h post-challenge ABC-transporter mRNA expression was determined in monocytes, leukocytes and peripheral blood mononuclear cells (PBMC). In a separate study, murine-BMDM were exposed to 5 mmol/L D-glucose (control) or additional 20 mmol/L D-or L-glucose and 25 ug/mL oxidized low density lipoprotein (oxLDL). High density lipoprotein (HDL)-mediated cholesterol efflux and ABC-transporter (ABCA1 and ABCG1) expression were determined. Results Baseline ABCA1and ABCG1 expression was lower (> 50 %) in human monocytes and PBMC than leukocytes (p < 0.05). 1 h post-challenge leukocyte ABCA1 and ABCG1 expression increased by 37 % and 30 %, respectively (p < 0.05), and began to return to baseline thereafter. There was no significant change in monocyte ABC-transporter expression. In murine BMDM, higher glucose concentrations suppressed HDL-mediated cholesterol efflux (10 %; p < 0.01) without significantly affecting ABCA1 and ABCG1 expression. Data demonstrate that leukocytes are not a reliable indicator of monocyte ABC-transporter expression. Conclusions Human monocyte ABC-transporter gene expression was unresponsive to a glucose challenge. Correspondingly, in BMDM, hyperglycemia attenuated macrophage cholesterol efflux in the absence of altered ABC-transporter expression, suggesting that hyperglycemia, per se, suppresses cholesterol transporter activity. This glucose-related impairment in cholesterol efflux may potentially contribute to

  16. Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos.

    PubMed

    Bošnjak, Ivana; Pleić, Ivana Lepen; Borra, Marco; Mladineo, Ivona

    2013-12-01

    A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub-lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2) and trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters-P-glycoprotein (ABCB1/P-gp) and sulfonylurea-receptor-like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the two-cell, as well as an overall decrease during the blastulae stage. Calcein-AM efflux activity assay indicated the activation of multidrug resistance-associated protein/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the two-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI-inducible ABC membrane transporters, coexpressed in the zygote, two-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

  17. Characterization of mammalian glucose transport proteins using photoaffinity labeling techniques

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wadzinski, B.E.

    1989-01-01

    A carrier-free radioiodinated phenylazide derivative of forskolin, 3-iodo-4-azidophenethylamido-7-O-succinyl-deacetyl-forskolin (({sup 125}I)IAPS-forskolin), has been shown to be a highly selective photoaffinity probe for the human erythrocyte glucose transported and the glucose transport proteins found in several mammalian tissues and cultured cells where the glucose transport protein is present at a low concentration. The photoincorporation of ({sup 125}I)IAPS-forskolin into these glucose transporters was blocked by D- (but not L-) glucose, cytochalasin B, and forskolin. In addition to labeling the mammalian glucose transport proteins, ({sup 125}I)IAPS-forskolin also labeled the L-arabinose transporter from E. coli. In muscle and adipose tissues, glucose transport is markedly increasedmore » in response to insulin. ({sup 125}I)IAPS-forskolin was shown to selectivity tag the glucose transporter in membranes derived from these cells. In addition, the covalent derivatization of the transport protein in subcellular fractions of the adipocyte has provided a means to study the hormonal regulation of glucose transport. ({sup 125}I)IAPS-forskolin has also been used to label the purified human erythrocyte glucose transporter. The site of insertion has therefore been localized by analysis of the radiolabeled peptides which were produced following chemical and proteolytic digestion of the labeled transport protein.« less

  18. The Tp0684 (MglB-2) Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology.

    PubMed

    Brautigam, Chad A; Deka, Ranjit K; Liu, Wei Z; Norgard, Michael V

    2016-01-01

    Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein's topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum's physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.

  19. Mechanistic logic underlying the axonal transport of cytosolic proteins

    PubMed Central

    Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

    2011-01-01

    Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

  20. Two endoplasmic reticulum (ER) membrane proteins that facilitate ER-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins.

    PubMed

    Barz, W P; Walter, P

    1999-04-01

    Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for "delayed GPI-anchored protein transport"), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Delta dgt1Delta cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non-GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Delta dgt1Delta cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Delta dgt1Delta cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins.

  1. Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

    PubMed

    Puri, Nidhi; Manoharlal, Raman; Sharma, Monika; Sanglard, Dominique; Prasad, Rajendra

    2011-01-07

    We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded. Copyright © 2010 Elsevier Inc. All rights reserved.

  2. Optimization of Straight Cylindrical Turning Using Artificial Bee Colony (ABC) Algorithm

    NASA Astrophysics Data System (ADS)

    Prasanth, Rajanampalli Seshasai Srinivasa; Hans Raj, Kandikonda

    2017-04-01

    Artificial bee colony (ABC) algorithm, that mimics the intelligent foraging behavior of honey bees, is increasingly gaining acceptance in the field of process optimization, as it is capable of handling nonlinearity, complexity and uncertainty. Straight cylindrical turning is a complex and nonlinear machining process which involves the selection of appropriate cutting parameters that affect the quality of the workpiece. This paper presents the estimation of optimal cutting parameters of the straight cylindrical turning process using the ABC algorithm. The ABC algorithm is first tested on four benchmark problems of numerical optimization and its performance is compared with genetic algorithm (GA) and ant colony optimization (ACO) algorithm. Results indicate that, the rate of convergence of ABC algorithm is better than GA and ACO. Then, the ABC algorithm is used to predict optimal cutting parameters such as cutting speed, feed rate, depth of cut and tool nose radius to achieve good surface finish. Results indicate that, the ABC algorithm estimated a comparable surface finish when compared with real coded genetic algorithm and differential evolution algorithm.

  3. An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease.

    PubMed Central

    Urban, M; Bhargava, T; Hamer, J E

    1999-01-01

    Cells tolerate exposure to cytotoxic compounds through the action of ATP-driven efflux pumps belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. Phytopathogenic fungi encounter toxic environments during plant invasion as a result of the plant defense response. Here we demonstrate the requirement for an ABC transporter during host infection by the fungal plant pathogen Magnaporthe grisea. The ABC1 gene was identified in an insertional mutagenesis screen for pathogenicity mutants. The ABC1 insertional mutant and a gene-replacement mutant arrest growth and die shortly after penetrating either rice or barley epidermal cells. The ABC1-encoded protein is similar to yeast ABC transporters implicated in multidrug resistance, and ABC1 gene transcripts are inducible by toxic drugs and a rice phytoalexin. However, abc1 mutants are not hypersensitive to antifungal compounds. The non-pathogenic, insertional mutation in ABC1 occurs in the promoter region and dramatically reduces transcript induction by metabolic poisons. These data strongly suggest that M.grisea requires the up-regulation of specific ABC transporters for pathogenesis; most likely to protect itself against plant defense mechanisms. PMID:9927411

  4. A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

    PubMed Central

    2012-01-01

    Background Copper is an essential catalytic co-factor for metabolically important cellular enzymes, such as cytochrome-c oxidase. Eukaryotic cells acquire copper through a copper transport protein and distribute intracellular copper using molecular chaperones. The copper chelator, neocuproine, inhibits Plasmodium falciparum ring-to-trophozoite transition in vitro, indicating a copper requirement for malaria parasite development. How the malaria parasite acquires or secretes copper still remains to be fully elucidated. Methods PlasmoDB was searched for sequences corresponding to candidate P. falciparum copper-requiring proteins. The amino terminal domain of a putative P. falciparum copper transport protein was cloned and expressed as a maltose binding fusion protein. The copper binding ability of this protein was examined. Copper transport protein-specific anti-peptide antibodies were generated in chickens and used to establish native protein localization in P. falciparum parasites by immunofluorescence microscopy. Results Six P. falciparum copper-requiring protein orthologs and a candidate P. falciparum copper transport protein (PF14_0369), containing characteristic copper transport protein features, were identified in PlasmoDB. The recombinant amino terminal domain of the transport protein bound reduced copper in vitro and within Escherichia coli cells during recombinant expression. Immunolocalization studies tracked the copper binding protein translocating from the erythrocyte plasma membrane in early ring stage to a parasite membrane as the parasites developed to schizonts. The protein appears to be a PEXEL-negative membrane protein. Conclusion Plasmodium falciparum parasites express a native protein with copper transporter characteristics that binds copper in vitro. Localization of the protein to the erythrocyte and parasite plasma membranes could provide a mechanism for the delivery of novel anti-malarial compounds. PMID:23190769

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

    PubMed

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

    2017-03-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Herrou, Julien; Willett, Jonathan W.; Czyż, Daniel M.

    ABSTRACT Brucella abortusσ E1is an EcfG family sigma factor that regulates the transcription of dozens of genes in response to diverse stress conditions and is required for maintenance of chronic infection in a mouse model. A putative ATP-binding cassette transporter operon,bab1_0223-bab1_0226, is among the most highly activated gene sets in the σ E1regulon. The proteins encoded by the operon resemble quaternary ammonium-compatible solute importers but are most similar in sequence to the broadly conserved YehZYXW system, which remains largely uncharacterized. Transcription ofyehZYXWis activated by the general stress sigma factor σ SinEnterobacteriaceae, which suggests a functional role for this transport systemmore » in bacterial stress response across the classesAlphaproteobacteriaandGammaproteobacteria. We present evidence thatB. abortusYehZYXW does not function as an importer of known compatible solutes under physiological conditions and does not contribute to the virulence defect of a σ E1-null strain. The solein vitrophenotype associated with genetic disruption of this putative transport system is reduced growth in the presence of high Li +ion concentrations. A crystal structure ofB. abortusYehZ revealed a class II periplasmic binding protein fold with significant structural homology toArchaeoglobus fulgidusProX, which binds glycine betaine. However, the structure of the YehZ ligand-binding pocket is incompatible with high-affinity binding to glycine betaine. This is consistent with weak measured binding of YehZ to glycine betaine and related compatible solutes. We conclude that YehZYXW is a conserved, stress-regulated transport system that is phylogenetically and functionally distinct from quaternary ammonium-compatible solute importers. IMPORTANCEBrucella abortusσ E1regulates transcription in response to stressors encountered in its mammalian host and is necessary for maintenance of chronic infection in a mouse model. The functions of the majority of

  7. Integrated regulation of motor-driven organelle transport by scaffolding proteins.

    PubMed

    Fu, Meng-meng; Holzbaur, Erika L F

    2014-10-01

    Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Application of activity-based costing (ABC) for a Peruvian NGO healthcare provider.

    PubMed

    Waters, H; Abdallah, H; Santillán, D

    2001-01-01

    This article describes the application of activity-based costing (ABC) to calculate the unit costs of the services for a health care provider in Peru. While traditional costing allocates overhead and indirect costs in proportion to production volume or to direct costs, ABC assigns costs through activities within an organization. ABC uses personnel interviews to determine principal activities and the distribution of individual's time among these activities. Indirect costs are linked to services through time allocation and other tracing methods, and the result is a more accurate estimate of unit costs. The study concludes that applying ABC in a developing country setting is feasible, yielding results that are directly applicable to pricing and management. ABC determines costs for individual clinics, departments and services according to the activities that originate these costs, showing where an organization spends its money. With this information, it is possible to identify services that are generating extra revenue and those operating at a loss, and to calculate cross subsidies across services. ABC also highlights areas in the health care process where efficiency improvements are possible. Conclusions about the ultimate impact of the methodology are not drawn here, since the study was not repeated and changes in utilization patterns and the addition of new clinics affected applicability of the results. A potential constraint to implementing ABC is the availability and organization of cost information. Applying ABC efficiently requires information to be readily available, by cost category and department, since the greatest benefits of ABC come from frequent, systematic application of the methodology in order to monitor efficiency and provide feedback for management. The article concludes with a discussion of the potential applications of ABC in the health sector in developing countries.

  9. Creating an iPhone application for collecting continuous ABC data.

    PubMed

    Whiting, Seth W; Dixon, Mark R

    2012-01-01

    This paper provides an overview and task analysis for creating a continuous ABC data-collection application using Xcode on a Mac computer. Behavior analysts can program an ABC data collection system, complete with a customized list of target clients, antecedents, behaviors, and consequences to be recorded, and have the data automatically sent to an e-mail account after observations have concluded. Further suggestions are provided to customize the ABC data- collection system for individual preferences and clinical needs.

  10. An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins

    PubMed Central

    Lange, Heike; Lisowsky, Thomas; Gerber, Jana; Mühlenhoff, Ulrich; Kispal, Gyula; Lill, Roland

    2001-01-01

    Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process. PMID:11493598

  11. An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins.

    PubMed

    Lange, H; Lisowsky, T; Gerber, J; Mühlenhoff, U; Kispal, G; Lill, R

    2001-08-01

    Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR ('augmenter of liver regeneration'), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.

  12. ABC versus CAB for cardiopulmonary resuscitation: a prospective, randomized simulator-based trial.

    PubMed

    Marsch, Stephan; Tschan, Franziska; Semmer, Norbert K; Zobrist, Roger; Hunziker, Patrick R; Hunziker, Sabina

    2013-09-06

    After years of advocating ABC (Airway-Breathing-Circulation), current guidelines of cardiopulmonary resuscitation (CPR) recommend CAB (Circulation-Airway-Breathing). This trial compared ABC with CAB as initial approach to CPR from the arrival of rescuers until the completion of the first resuscitation cycle. 108 teams, consisting of two physicians each, were randomized to receive a graphical display of either the ABC algorithm or the CAB algorithm. Subsequently teams had to treat a simulated cardiac arrest. Data analysis was performed using video recordings obtained during simulations. The primary endpoint was the time to completion of the first resuscitation cycle of 30 compressions and two ventilations. The time to execution of the first resuscitation measure was 32 ± 12 seconds in ABC teams and 25 ± 10 seconds in CAB teams (P = 0.002). 18/53 ABC teams (34%) and none of the 55 CAB teams (P = 0.006) applied more than the recommended two initial rescue breaths which caused a longer duration of the first cycle of 30 compressions and two ventilations in ABC teams (31 ± 13 vs.23 ± 6 sec; P = 0.001). Overall, the time to completion of the first resuscitation cycle was longer in ABC teams (63 ± 17 vs. 48 ± 10 sec; P <0.0001). This randomized controlled trial found CAB superior to ABC with an earlier start of CPR and a shorter time to completion of the first 30:2 resuscitation cycle. These findings endorse the change from ABC to CAB in international resuscitation guidelines.

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

    PubMed Central

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

    2014-01-01

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

  14. Proteomics-based identification of midgut proteins correlated with Cry1Ac resistance in Plutella xylostella (L.).

    PubMed

    Xia, Jixing; Guo, Zhaojiang; Yang, Zezhong; Zhu, Xun; Kang, Shi; Yang, Xin; Yang, Fengshan; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Xu, Weijun; Zhang, Youjun

    2016-09-01

    The diamondback moth, Plutella xylostella (L.), is a worldwide pest of cruciferous crops and can rapidly develop resistance to many chemical insecticides. Although insecticidal crystal proteins (i.e., Cry and Cyt toxins) derived from Bacillus thuringiensis (Bt) have been useful alternatives to chemical insecticides for the control of P. xylostella, resistance to Bt in field populations of P. xylostella has already been reported. A better understanding of the resistance mechanisms to Bt should be valuable in delaying resistance development. In this study, the mechanisms underlying P. xylostella resistance to Bt Cry1Ac toxin were investigated using two-dimensional differential in-gel electrophoresis (2D-DIGE) and ligand blotting for the first time. Comparative analyses of the constitutive expression of midgut proteins in Cry1Ac-susceptible and -resistant P. xylostella larvae revealed 31 differentially expressed proteins, 21 of which were identified by mass spectrometry. Of these identified proteins, the following fell into diverse eukaryotic orthologous group (KOG) subcategories may be involved in Cry1Ac resistance in P. xylostella: ATP-binding cassette (ABC) transporter subfamily G member 4 (ABCG4), trypsin, heat shock protein 70 (HSP70), vacuolar H(+)-ATPase, actin, glycosylphosphatidylinositol anchor attachment 1 protein (GAA1) and solute carrier family 30 member 1 (SLC30A1). Additionally, ligand blotting identified the following midgut proteins as Cry1Ac-binding proteins in Cry1Ac-susceptible P. xylostella larvae: ABC transporter subfamily C member 1 (ABCC1), solute carrier family 36 member 1 (SLC36A1), NADH dehydrogenase iron-sulfur protein 3 (NDUFS3), prohibitin and Rap1 GTPase-activating protein 1. Collectively, these proteomic results increase our understanding of the molecular resistance mechanisms to Bt Cry1Ac toxin in P. xylostella and also demonstrate that resistance to Bt Cry1Ac toxin is complex and multifaceted. Copyright © 2016 Elsevier B.V. All

  15. Two Endoplasmic Reticulum (ER) Membrane Proteins That Facilitate ER-to-Golgi Transport of Glycosylphosphatidylinositol-anchored Proteins

    PubMed Central

    Barz, Wolfgang P.; Walter, Peter

    1999-01-01

    Many eukaryotic cell surface proteins are anchored in the lipid bilayer through glycosylphosphatidylinositol (GPI). GPI anchors are covalently attached in the endoplasmic reticulum (ER). The modified proteins are then transported through the secretory pathway to the cell surface. We have identified two genes in Saccharomyces cerevisiae, LAG1 and a novel gene termed DGT1 (for “delayed GPI-anchored protein transport”), encoding structurally related proteins with multiple membrane-spanning domains. Both proteins are localized to the ER, as demonstrated by immunofluorescence microscopy. Deletion of either gene caused no detectable phenotype, whereas lag1Δ dgt1Δ cells displayed growth defects and a significant delay in ER-to-Golgi transport of GPI-anchored proteins, suggesting that LAG1 and DGT1 encode functionally redundant or overlapping proteins. The rate of GPI anchor attachment was not affected, nor was the transport rate of several non–GPI-anchored proteins. Consistent with a role of Lag1p and Dgt1p in GPI-anchored protein transport, lag1Δ dgt1Δ cells deposit abnormal, multilayered cell walls. Both proteins have significant sequence similarity to TRAM, a mammalian membrane protein thought to be involved in protein translocation across the ER membrane. In vivo translocation studies, however, did not detect any defects in protein translocation in lag1Δ dgt1Δ cells, suggesting that neither yeast gene plays a role in this process. Instead, we propose that Lag1p and Dgt1p facilitate efficient ER-to-Golgi transport of GPI-anchored proteins. PMID:10198056

  16. Interaction of Extracellular Domain 2 of the Human Retina-specific ATP-binding Cassette Transporter (ABCA4) with All-trans-retinal*

    PubMed Central

    Biswas-Fiss, Esther E.; Kurpad, Deepa S.; Joshi, Kinjalben; Biswas, Subhasis B.

    2010-01-01

    The retina-specific ATP-binding cassette (ABC) transporter, ABCA4, is essential for transport of all-trans-retinal from the rod outer segment discs in the retina and is associated with a broad range of inherited retinal diseases, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. A unique feature of the ABCA subfamily of ABC transporters is the presence of highly conserved, long extracellular loops or domains (ECDs) with unknown function. The high degree of sequence conservation and mapped disease-associated mutations in these domains suggests an important physiological significance. Conformational analysis using CD spectroscopy of purified, recombinant ECD2 protein demonstrated that it has an ordered and stable structure composed of 27 ± 3% α-helix, 20 ± 3% β-pleated sheet, and 53 ± 3% coil. Significant conformational changes were observed in disease-associated mutant proteins. Using intrinsic tryptophan fluorescence emission spectrum of ECD2 polypeptide and fluorescence anisotropy, we have demonstrated that this domain specifically interacts with all-trans-retinal. Furthermore, the retinal interaction appeared preferential for the all-trans-isomer and was directly measurable through fluorescence anisotropy analysis. Our results demonstrate that the three macular degeneration-associated mutations lead to significant changes in the secondary structure of the ECD2 domain of ABCA4, as well as in its interaction with all-trans-retinal. PMID:20404325

  17. Anticipated Benefits of Care (ABC): psychometrics and predictive value in psychiatric disorders.

    PubMed

    Warden, D; Trivedi, M H; Carmody, T J; Gollan, J K; Kashner, T M; Lind, L; Crismon, M L; Rush, A J

    2010-06-01

    Attitudes and expectations about treatment have been associated with symptomatic outcomes, adherence and utilization in patients with psychiatric disorders. No measure of patients' anticipated benefits of treatment on domains of everyday functioning has previously been available. The Anticipated Benefits of Care (ABC) is a new, 10-item questionnaire used to measure patient expectations about the impact of treatment on domains of everyday functioning. The ABC was collected at baseline in adult out-patients with major depressive disorder (MDD) (n=528), bipolar disorder (n=395) and schizophrenia (n=447) in the Texas Medication Algorithm Project (TMAP). Psychometric properties of the ABC were assessed, and the association of ABC scores with treatment response at 3 months was evaluated. Evaluation of the ABC's internal consistency yielded Cronbach's alpha of 0.90-0.92 for patients across disorders. Factor analysis showed that the ABC was unidimensional for all patients and for patients with each disorder. For patients with MDD, lower anticipated benefits of treatment was associated with less symptom improvement and lower odds of treatment response [odds ratio (OR) 0.72, 95% confidence interval (CI) 0.57-0.87, p=0.0011]. There was no association between ABC and symptom improvement or treatment response for patients with bipolar disorder or schizophrenia, possibly because these patients had modest benefits with treatment. The ABC is the first self-report that measures patient expectations about the benefits of treatment on everyday functioning, filling an important gap in available assessments of attitudes and expectations about treatment. The ABC is simple, easy to use, and has acceptable psychometric properties for use in research or clinical settings.

  18. Object Detection Based on Template Matching through Use of Best-So-Far ABC

    PubMed Central

    2014-01-01

    Best-so-far ABC is a modified version of the artificial bee colony (ABC) algorithm used for optimization tasks. This algorithm is one of the swarm intelligence (SI) algorithms proposed in recent literature, in which the results demonstrated that the best-so-far ABC can produce higher quality solutions with faster convergence than either the ordinary ABC or the current state-of-the-art ABC-based algorithm. In this work, we aim to apply the best-so-far ABC-based approach for object detection based on template matching by using the difference between the RGB level histograms corresponding to the target object and the template object as the objective function. Results confirm that the proposed method was successful in both detecting objects and optimizing the time used to reach the solution. PMID:24812556

  19. Expression and activity of multidrug resistance protein 1 in a murine thymoma cell line

    PubMed Central

    Echevarria-Lima, Juliana; Kyle-Cezar, Fernanda; Leite, Daniela F P; Capella, Luiz; Capella, Márcia A M; Rumjanek, Vivian M

    2005-01-01

    Multidrug resistance proteins [MRPs and P-glycoprotein (Pgp)] are members of the family of ATP-binding cassette (ABC) transport proteins, originally described as being involved in the resistance against anti-cancer agents in tumour cells. These proteins act as ATP-dependent efflux pumps and have now been described in normal cells where they exert physiological roles. The aim of this work was to investigate the expression and activity of MRP and Pgp in the thymoma cell line, EL4. It was observed that EL4 cells expressed mRNA for MRP1, but not for MRP2, MRP3 or Pgp. The activity of ABC transport proteins was evaluated by using the efflux of the fluorescent probes carboxy-2′-7′-dichlorofluorescein diacetate (CFDA) and rhodamine 123 (Rho 123). EL4 cells did not retain CFDA intracellularly, and MRP inhibitors (probenecid, indomethacin and MK 571) decreased MRP1 activity in a concentration-dependent manner. As expected, EL4 cells accumulated Rho 123, and the presence of cyclosporin A and verapamil did not modify this accumulation. Most importantly, when EL4 cells were incubated in the presence of the MRP1 inhibitors indomethacin and MK 571 for 6 days, they started to express CD4 and CD8 molecules on their surface, producing double-positive cells and CD8 single-positive cells. Our results suggest that MRP activity is important for the maintenance of the undifferentiated state in this cell type. This finding might have implications in the physiological process of normal thymocyte maturation. PMID:15804283

  20. Synthetic single domain antibodies for the conformational trapping of membrane proteins

    PubMed Central

    Arnold, Fabian M; Stohler, Peter; Bocquet, Nicolas; Hug, Melanie N; Huber, Sylwia; Siegrist, Martin; Hetemann, Lisa; Gera, Jennifer; Gmür, Samira; Spies, Peter; Gygax, Daniel

    2018-01-01

    Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins. PMID:29792401

  1. Structural and functional insights into the lipopolysaccharide ABC transporter LptB2FG.

    PubMed

    Dong, Haohao; Zhang, Zhengyu; Tang, Xiaodi; Paterson, Neil G; Dong, Changjiang

    2017-08-09

    The cell surface of most Gram-negative bacteria contains lipopolysaccharide that is essential for their viability and drug resistance. A 134-kDa protein complex LptB 2 FG is unique among ATP-binding cassette transporters because it extracts lipopolysaccharide from the external leaflet of the inner membrane and propels it along a filament that extends across the periplasm to directly deliver lipopolysaccharide into the external leaflet of the outer membrane. Here we report the crystal structure of the lipopolysaccharide transporter LptB 2 FG from Klebsiella pneumoniae, in which both LptF and LptG are composed of a β-jellyroll-like periplasmic domain and six α-helical segments in the transmembrane domain. LptF and LptG form a central cavity containing highly conserved hydrophobic residues. Structural and functional studies suggest that LptB 2 FG uses an alternating lateral access mechanism to extract lipopolysaccharide and traffic it along the hydrophobic cavity toward the transporter's periplasmic domains.Lipopolysaccharides (LPS) are synthesized at the periplasmic side of the inner membrane of Gram-negative bacteria and are then extracted by the LptB 2 FG complex during the first step of LPS transport to the outer membrane. Here the authors present the LptB 2 FG structure, which supports an alternating lateral access mechanism for LPS extraction.

  2. IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes

    DOE PAGES

    Hadjithomas, Michalis; Chen, I-Min A.; Chu, Ken; ...

    2016-11-29

    Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic genemore » clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery.« less

  3. IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hadjithomas, Michalis; Chen, I-Min A.; Chu, Ken

    Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic genemore » clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery.« less

  4. The ABCs of Sex Ed.

    ERIC Educational Resources Information Center

    Sroka, Stephen R.

    2002-01-01

    Cites statistics on extent of sexually transmitted diseases and pregnancies among adolescents; describes ideological dispute over how to teach sex education; advocates teaching the ABCs of sex education: Abstinence, Be Monogamous, and Condoms. (PKP)

  5. Abrasive wear behavior of heat-treated ABC-silicon carbide

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Xiao Feng; Lee, Gun Y.; Chen, Da

    2002-06-17

    Hot-pressed silicon carbide, containing aluminum, boron, and carbon additives (ABC-SiC), was subjected to three-body and two-body wear testing using diamond abrasives over a range of sizes. In general, the wear resistance of ABC-SiC, with suitable heat treatment, was superior to that of commercial SiC.

  6. The naphthoquinones, vitamin K3 and its structural analog plumbagin, are substrates of the multidrug resistance-linked ABC drug transporter ABCG2

    PubMed Central

    Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V.

    2008-01-01

    Vitamin K3 (Menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2 which are essential for blood clotting. The naturally occurring structural analog of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We, here, report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette (ABC) drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone, but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). They inhibited the binding of [125I]-Iodoarylazidoprazosin (IAAP), a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC50 values of 7.3 and 22.6 μM, respectively, but had no effect on the binding of this photoaffinity analog to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of this transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared to the control cells, suggesting that they are substrates of this transporter. Collectively, these data demonstrate for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function. PMID:18065489

  7. Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gao, Jinlan; Li, Xiaolu; Tsinghua-Peking Joint Center for Life Sciences, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, People's Republic of China

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer We truncated the signal peptide of OppA{sub TTE0054} to make it express in Escherichia coli as a soluble protein. Black-Right-Pointing-Pointer Crystals of OppA{sub TTE0054} were grown by sitting-drop vapor diffusion method. Black-Right-Pointing-Pointer The crystal of OppA{sub TTE0054} diffracted to 2.25 A. -- Abstract: Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS-PAGEmore » after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 A. The crystal belonged to space group C222{sub 1}, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 A, and {alpha} = {beta} = {gamma} = 90 Degree-Sign .« less

  8. A Manual for Implementation of ABC Video Duplication Projects.

    ERIC Educational Resources Information Center

    Hill, Joseph, Ed.

    The ABC (Appalachian BOCES Consortium) consists of 10 BOCES (Boards of Cooperative Educational Services) which serve the 14 southern counties of New York State designated as Appalachia. Each year since 1974, the ABC has participated in regional video duplication projects, which have yielded a total of nearly 4,000 video titles. The complexity of…

  9. Downregulation of miR-29a/b/c in placenta accreta inhibits apoptosis of implantation site intermediate trophoblast cells by targeting MCL1.

    PubMed

    Gu, Yongzhong; Bian, Yuehong; Xu, Xiaofei; Wang, Xietong; Zuo, Changting; Meng, Jinlai; Li, Hongyan; Zhao, Shigang; Ning, Yunnan; Cao, Yongzhi; Huang, Tao; Yan, Junhao; Chen, Zi-Jiang

    2016-12-01

    Placenta accreta is defined as abnormal adhesion of placental villi to the uterine myometrium. Although this condition has become more common as a result of the increasing rate of cesarean sections, the underlying causative mechanism(s) remain elusive. Because microRNA-29a/b/c (miR-29a/b/c) have been shown to play important roles in placental development, this study evaluated the roles of these microRNAs in placenta accreta. Expression of miR-29a/b/c and myeloid cell leukemia-1 (MCL1) were quantified in patient tissues and HTR8/SVneo trophoblast cells using the real-time quantitative polymerase chain reaction. Western blotting was used to analyze expression of the MCL1 protein in HTR8/SVneo trophoblast cells with altered expression of miR-29a/b/c. To determine their role in apoptosis, miR-29a/b/c were overexpressed in HTR-8/SVneo cells, and levels of apoptosis were analyzed by flow cytometry. Luciferase activity assays were used to determine whether MCL1 is a target gene of miR-29a/b/c. Expression of miR-29a/b/c was significantly lower in creta sites compared to noncreta sites (p = 0.018, 0.041, and 0.022, respectively), but expression of MCL1 was upregulated in creta sites (p = 0.039). MCL1 expression was significantly downregulated in HTR-8/SVneo cells overexpressing miR-29a/b/c (p = 0.002, 0.008, and 0.013, respectively). Luciferase activity assays revealed that miR-29a/b/c directly target the 3' untranslated region of MCL1 in 293T cells. Over-expression of miR-29a/b/c induced apoptosis in the HTR-8/SVneo trophoblast cell line. Moreover, histopathological evaluation revealed that the number of implantation site intermediate trophoblast (ISIT) cells was increased in creta sites and that these cells were positive for MCL1. Our results demonstrate that in placenta accreta, miR-29a/b/c inhibits apoptosis of ISIT cells by targeting MCL1. These findings provide new insights into the pathogenesis of placenta accreta. Copyright © 2016 Elsevier Ltd. All rights

  10. TryTransDB: A web-based resource for transport proteins in Trypanosomatidae.

    PubMed

    Sonar, Krushna; Kabra, Ritika; Singh, Shailza

    2018-03-12

    TryTransDB is a web-based resource that stores transport protein data which can be retrieved using a standalone BLAST tool. We have attempted to create an integrated database that can be a one-stop shop for the researchers working with transport proteins of Trypanosomatidae family. TryTransDB (Trypanosomatidae Transport Protein Database) is a web based comprehensive resource that can fire a BLAST search against most of the transport protein sequences (protein and nucleotide) from Trypanosomatidae family organisms. This web resource further allows to compute a phylogenetic tree by performing multiple sequence alignment (MSA) using CLUSTALW suite embedded in it. Also, cross-linking to other databases helps in gathering more information for a certain transport protein in a single website.

  11. Deciphering the Routes of invasion of Drosophila suzukii by Means of ABC Random Forest.

    PubMed

    Fraimout, Antoine; Debat, Vincent; Fellous, Simon; Hufbauer, Ruth A; Foucaud, Julien; Pudlo, Pierre; Marin, Jean-Michel; Price, Donald K; Cattel, Julien; Chen, Xiao; Deprá, Marindia; François Duyck, Pierre; Guedot, Christelle; Kenis, Marc; Kimura, Masahito T; Loeb, Gregory; Loiseau, Anne; Martinez-Sañudo, Isabel; Pascual, Marta; Polihronakis Richmond, Maxi; Shearer, Peter; Singh, Nadia; Tamura, Koichiro; Xuéreb, Anne; Zhang, Jinping; Estoup, Arnaud

    2017-04-01

    Deciphering invasion routes from molecular data is crucial to understanding biological invasions, including identifying bottlenecks in population size and admixture among distinct populations. Here, we unravel the invasion routes of the invasive pest Drosophila suzukii using a multi-locus microsatellite dataset (25 loci on 23 worldwide sampling locations). To do this, we use approximate Bayesian computation (ABC), which has improved the reconstruction of invasion routes, but can be computationally expensive. We use our study to illustrate the use of a new, more efficient, ABC method, ABC random forest (ABC-RF) and compare it to a standard ABC method (ABC-LDA). We find that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well. All introduced populations passed through bottlenecks, and analyses reveal five distinct admixture events. These findings can inform hypotheses concerning how this species evolved between different and independent source and invasive populations. Methodological comparisons indicate that ABC-RF and ABC-LDA show concordant results if ABC-LDA is based on a large number of simulated datasets but that ABC-RF out-performs ABC-LDA when using a comparable and more manageable number of simulated datasets, especially when analyzing complex introduction scenarios. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. MetaABC--an integrated metagenomics platform for data adjustment, binning and clustering.

    PubMed

    Su, Chien-Hao; Hsu, Ming-Tsung; Wang, Tse-Yi; Chiang, Sufeng; Cheng, Jen-Hao; Weng, Francis C; Kao, Cheng-Yan; Wang, Daryi; Tsai, Huai-Kuang

    2011-08-15

    MetaABC is a metagenomic platform that integrates several binning tools coupled with methods for removing artifacts, analyzing unassigned reads and controlling sampling biases. It allows users to arrive at a better interpretation via series of distinct combinations of analysis tools. After execution, MetaABC provides outputs in various visual formats such as tables, pie and bar charts as well as clustering result diagrams. MetaABC source code and documentation are available at http://bits2.iis.sinica.edu.tw/MetaABC/ CONTACT: dywang@gate.sinica.edu.tw; hktsai@iis.sinica.edu.tw Supplementary data are available at Bioinformatics online.

  13. A two-dimensional electrophoretic profile of the proteins secreted by Herbaspirillum seropedicae strain Z78.

    PubMed

    Chaves, Daniela Fojo Seixas; de Souza, Emanuel Maltempi; Monteiro, Rose Adele; de Oliveira Pedrosa, Fábio

    2009-11-02

    Herbaspirillum seropedicae is an endophytic bacterium that associates with rice, sugarcane and other economically important crops. Secreted proteins play a key role in the plant-bacterial interaction. Using 2D electrophoresis and peptide mass fingerprint mass spectrometry, 63 protein spots representing 41 different secreted proteins were identified during growth of H. seropedicae under nitrogen-sufficient conditions. In silico analysis showed that 25.4% of the proteins had signal peptides and 15.9% were predicted to be non-classically secreted. Among the most abundant were flagellar components and ABC-type transport system proteins. Nine secreted proteins had also been identified in the cellular proteome, suggesting that they also play a role in the extracellular environment. No type III secreted proteins were detected by comparison of the wild type strain with an hrcN mutant strain.

  14. Phase behavior of model ABC triblock copolymers

    NASA Astrophysics Data System (ADS)

    Chatterjee, Joon

    The phase behavior of poly(isoprene-b-styrene- b-ethylene oxide) (ISO), a model ABC triblock copolymer has been studied. This class of materials exhibit self-assembly, forming a large array of ordered morphologies at length scales of 5-100 nm. The formation of stable three-dimensionally continuous network morphologies is of special interest in this study. Since these nanostructures considerably impact the material properties, fundamental knowledge for designing ABC systems have high technological importance for realizing applications in the areas of nanofabrication, nanoporous media, separation membranes, drug delivery and high surface area catalysts. A comprehensive framework was developed to describe the phase behavior of the ISO triblock copolymers at weak to intermediate segregation strengths spanning a wide range of composition. Phases were characterized through a combination of characterization techniques, including small angle x-ray scattering, dynamic mechanical spectroscopy, transmission electron microscopy, and birefringence measurements. Combined with previous investigations on ISO, six different stable ordered state symmetries have been identified: lamellae (LAM), Fddd orthorhombic network (O70), double gyroid (Q230), alternating gyroid (Q214), hexagonal (HEX), and body-centered cubic (BCC). The phase map was found to be somewhat asymmetric around the fI = fO isopleth. This work provides a guide for theoretical studies and gives insight into the intricate effects of various parameters on the self-assembly of ABC triblock copolymers. Experimental SAXS data evaluated with a simple scattering intensity model show that local mixing varies continuously across the phase map between states of two- and three-domain segregation. Strategies of blending homopolymers with ISO triblock copolymer were employed for studying the swelling properties of a lamellar state. Results demonstrate that lamellar domains swell or shrink depending upon the type of homopolymer that

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

    USDA-ARS?s Scientific Manuscript database

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

  16. Structures of FolT in substrate-bound and substrate-released conformations reveal a gating mechanism for ECF transporters

    NASA Astrophysics Data System (ADS)

    Zhao, Qin; Wang, Chengcheng; Wang, Chengyuan; Guo, Hui; Bao, Zhihao; Zhang, Minhua; Zhang, Peng

    2015-07-01

    Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.

  17. Involvement of LeMRP, an ATP-binding cassette transporter, in shikonin transport and biosynthesis in Lithospermum erythrorhizon.

    PubMed

    Zhu, Y; Chu, S-J; Luo, Y-L; Fu, J-Y; Tang, C-Y; Lu, G-H; Pang, Y-J; Wang, X-M; Yang, R-W; Qi, J-L; Yang, Y-H

    2018-03-01

    Shikonin and its derivatives are important medicinal secondary metabolites accumulating in roots of Lithospermum erythrorhizon. Although some membrane proteins have been identified as transporters of secondary metabolites, the mechanisms underlying shikonin transport and accumulation in L. erythrorhizon cells still remain largely unknown. In this study, we isolated a cDNA encoding LeMRP, an ATP-binding cassette transporter from L. erythrorhizon, and further investigated its functions in the transport and biosynthesis of shikonin using the yeast transformation and transgenic hairy root methods, respectively. Real-time PCR was applied for expression analyses of LeMRP and shikonin biosynthetic enzyme genes. Functional analysis of LeMRP using the heterologous yeast cell expression system showed that LeMRP could be involved in shikonin transport. Transgenic hairy roots of L. erythrorhizon demonstrated that LeMRP overexpressing hairy roots produced more shikonin than the empty vector (EV) control. Real-time PCR results revealed that the enhanced shikonin biosynthesis in the overexpression lines was mainly caused by highly up-regulated expression of genes coding key enzymes (LePAL, HMGR, Le4CL and LePGT) involved in shikonin biosynthesis. Conversely, LeMRP RNAi decreased the accumulation of shikonin and effectively down-regulated expression level of the above genes. Typical inhibitors of ABC proteins, such as azide and buthionine sulphoximine, dramatically inhibited accumulation of shikonin in hairy roots. Our findings provide evidence for the important direct or indirect role of LeMRP in transmembrane transport and biosynthesis of shikonin. © 2017 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.

  18. Purification and characterization of chondroitinase ABC from Acinetobacter sp. C26.

    PubMed

    Zhu, Changliang; Zhang, Jingliang; Zhang, Jing; Jiang, Yanhui; Shen, Zhaopeng; Guan, Huashi; Jiang, Xiaolu

    2017-02-01

    An extracellular chondroitinase ABC (ChSase ABC, EC 4.2.2.4) produced by cultivating Acinetobacter sp. C26, was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q-Sepharose Fast Flow and Sephadex G-100 chromatography. The 76kDa enzyme was purified 48.09-fold to homogeneity with specific activity of 348.64U/mg, Using the chondroitin sulfate A (CS-A) as substrate, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) of ChSase ABC were found to be 10.471μmol/min/ml and 0.105mg/ml, respectively. The enzyme showed the highest activity at the optimal conditions of pH 6.0 and 42 ∘C, respectively. This enzyme was stable at pH 5-10, 5-9 and 5-7 at 4°C, 37°C and 42°C, respectively. Investigation about thermal stability of ChSase ABC displayed that it was stable at 37°C. ChSase ABC activity was increased in presence of Na + , K + , Mn 2+ , 1,10-phenanthrolin and strongly inhibited by Cu 2+ , Hg 2+ , Al 3+ and SDS. These properties suggested that ChSase ABC from Acinetobacter sp. C26 bring promising prospects in medical and industry applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Comparative proteome analysis of Actinoplanes sp. SE50/110 grown with maltose or glucose shows minor differences for acarbose biosynthesis proteins but major differences for saccharide transporters.

    PubMed

    Wendler, Sergej; Otto, Andreas; Ortseifen, Vera; Bonn, Florian; Neshat, Armin; Schneiker-Bekel, Susanne; Wolf, Timo; Zemke, Till; Wehmeier, Udo F; Hecker, Michael; Kalinowski, Jörn; Becher, Dörte; Pühler, Alfred

    2016-01-10

    Actinoplanes sp. SE50/110 is known for the production of the α-glucosidase inhibitor and anti-diabetic drug acarbose. Acarbose (acarviosyl-maltose) is produced as the major product when the bacterium is grown in medium with maltose, while acarviosyl-glucose is the major product when glucose is the sole carbon source in the medium. In this study, a state-of-the-art proteomics approach was applied combining subcellular fractionation, in vivo metabolic labeling and shotgun mass spectrometry to analyze differences in the proteome of Actinoplanes sp. SE50/110 cultures grown in minimal medium containing either maltose or glucose as the sole carbon source. To study proteins in distinct subcellular locations, a cytosolic, an enriched membrane, a membrane shaving and an extracellular fraction were included in the analysis. Altogether, quantitative proteome data was obtained for 2497 proteins representing about 30% of the ca. 8270 predicted proteins of Actinoplanes sp. SE50/110. When comparing protein quantities of maltose- to glucose-grown cultures, differences were observed for saccharide transport and metabolism proteins, whereas differences for acarbose biosynthesis gene cluster proteins were almost absent. The maltose-inducible α-glucosidase/maltase MalL as well as the ABC-type saccharide transporters AglEFG, MalEFG and MstEAF had significantly higher quantities in the maltose growth condition. The only highly abundant saccharide transporter in the glucose condition was the monosaccharide transporter MstEAF, which may indicate that MstEAF is the major glucose importer. Taken all findings together, the previously observed formation of acarviosyl-maltose and acarviosyl-glucose is more closely connected to the transport of saccharides than to a differential expression of the acarbose gene cluster. Diabetes is a global pandemic accounting for about 11% of the worldwide healthcare expenditures (>600 billion US dollars) and is projected to affect 592 million people by 2035

  20. Mdr65 decreases toxicity of multiple insecticides in Drosophila melanogaster.

    PubMed

    Sun, Haina; Buchon, Nicolas; Scott, Jeffrey G

    2017-10-01

    ABC transporters are ubiquitous membrane-bound proteins, present in both prokaryotes and eukaryotes. The major function of eukaryotic ABC transporters is to mediate the efflux of a variety of substrates (including xenobiotics) out of cells. ABC transporters have been widely investigated in humans, particularly for their involvement in multidrug resistance (MDR). Considerably less is known about their roles in transport and/or excretion in insects. ABC transporters are only known to function as exporters in insects. Drosophila melanogaster has 56 ABC transporter genes, including eight which are phylogenetically most similar to the human Mdr genes (ABCB1 clade). We investigated the role of ABC transporters in the ABCB1 clade in modulating the susceptibility to insecticides. We took advantage of the GAL4/UAS system in D. melanogaster to knockdown the expression levels of Mdr65, Mdr50, Mdr49 and ABCB6 using transgenic UAS-RNAi lines and conditional driver lines. The most notable effects were increased sensitivities to nine different insecticides by silencing of Mdr65. Furthermore, a null mutation of Mdr65 decreased the malathion, malaoxon and fipronil LC 50 values by a factor of 1.9, 2.1 and 3.9, respectively. Altogether, this data demonstrates the critical role of ABC transporters, particularly Mdr65, in altering the toxicity of specific, structurally diverse, insecticides in D. melanogaster. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Characterisation of single domain ATP-binding cassette protien homologues of Theileria parva.

    PubMed

    Kibe, M K; Macklin, M; Gobright, E; Bishop, R; Urakawa, T; ole-MoiYoi, O K

    2001-09-01

    Two distinct genes encoding single domain, ATP-binding cassette transport protein homologues of Theileria parva were cloned and sequenced. Neither of the genes is tandemly duplicated. One gene, TpABC1, encodes a predicted protein of 593 amino acids with an N-terminal hydrophobic domain containing six potential membrane-spanning segments. A single discontinuous ATP-binding element was located in the C-terminal region of TpABC1. The second gene, TpABC2, also contains a single C-terminal ATP-binding motif. Copies of TpABC2 were present at four loci in the T. parva genome on three different chromosomes. TpABC1 exhibited allelic polymorphism between stocks of the parasite. Comparison of cDNA and genomic sequences revealed that TpABC1 contained seven short introns, between 29 and 84 bp in length. The full-length TpABC1 protein was expressed in insect cells using the baculovirus system. Application of antibodies raised against the recombinant antigen to western blots of T. parva piroplasm lysates detected an 85 kDa protein in this life-cycle stage.

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

    PubMed Central

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

    2015-01-01

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

  3. Overexpression of Both ERG11 and ABC2 Genes Might Be Responsible for Itraconazole Resistance in Clinical Isolates of Candida krusei

    PubMed Central

    He, Xiaoyuan; Zhao, Mingfeng; Chen, Jinyan; Wu, Rimao; Zhang, Jianlei; Cui, Rui; Jiang, Yanyu; Chen, Jie; Cao, Xiaoli; Xing, Yi; Zhang, Yuchen; Meng, Juanxia; Deng, Qi; Sui, Tao

    2015-01-01

    Objective To study the main molecular mechanisms responsible for itraconazole resistance in clinical isolates of Candida krusei. Methods The 14α-demethylases encoded by ERG11 gene in the 16 C.krusei clinical isolates were amplified by polymerase chain reaction (PCR), and their nucleotide sequences were determined to detect point mutations. Meanwhile, ERG11 and efflux transporters (ABC1 and ABC2) genes were determined by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for their expression in itraconazole-resistant (R), itraconazole-susceptible dose dependent (SDD) and itraconazole-susceptible (S) C.krusei at the mRNA level. Results We found 7-point mutations in ERG11 gene of all the C.krusei clinical isolates, including 6 synonymous mutations and 1 missense mutation (C44T). However, the missense mutation was found in the three groups. The mRNA levels of ERG11 gene in itraconazole-resistant isolates showed higher expression compared with itraconazole-susceptible dose dependent and itraconazole-susceptible ones (P = 0.015 and P = 0.002 respectively). ABC2 gene mRNA levels in itraconazole-resistant group was significantly higher than the other two groups, and the levels of their expression in the isolates appeared to increase with the decrease of susceptibility to itraconazole (P = 0.007 in SDD compared with S, P = 0.016 in SDD with R, and P<0.001 in S with R respectively). While ABC1 gene presented lower expression in itraconazole resistant strains. However, the mRNA levels of ERG11, ABC1 and ABC2 in a C.krusei (CK10) resistant to both itraconazole and voriconazole were expressed highest in all the itraconazole-resistant isolates. Conclusions There are ERG11 gene polymorphisms in clinical isolates of C.krusei. ERG11 gene mutations may not be involved in the development of itraconazole resistance in C.krusei. ERG11 and ABC2 overexpression might be responsible for the acquired itraconazole resistance of these clinical isolates. PMID

  4. IMG-ABC: new features for bacterial secondary metabolism analysis and targeted biosynthetic gene cluster discovery in thousands of microbial genomes.

    PubMed

    Hadjithomas, Michalis; Chen, I-Min A; Chu, Ken; Huang, Jinghua; Ratner, Anna; Palaniappan, Krishna; Andersen, Evan; Markowitz, Victor; Kyrpides, Nikos C; Ivanova, Natalia N

    2017-01-04

    Secondary metabolites produced by microbes have diverse biological functions, which makes them a great potential source of biotechnologically relevant compounds with antimicrobial, anti-cancer and other activities. The proteins needed to synthesize these natural products are often encoded by clusters of co-located genes called biosynthetic gene clusters (BCs). In order to advance the exploration of microbial secondary metabolism, we developed the largest publically available database of experimentally verified and predicted BCs, the Integrated Microbial Genomes Atlas of Biosynthetic gene Clusters (IMG-ABC) (https://img.jgi.doe.gov/abc/). Here, we describe an update of IMG-ABC, which includes ClusterScout, a tool for targeted identification of custom biosynthetic gene clusters across 40 000 isolate microbial genomes, and a new search capability to query more than 700 000 BCs from isolate genomes for clusters with similar Pfam composition. Additional features enable fast exploration and analysis of BCs through two new interactive visualization features, a BC function heatmap and a BC similarity network graph. These new tools and features add to the value of IMG-ABC's vast body of BC data, facilitating their in-depth analysis and accelerating secondary metabolite discovery. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Computational models for predicting interactions with membrane transporters.

    PubMed

    Xu, Y; Shen, Q; Liu, X; Lu, J; Li, S; Luo, C; Gong, L; Luo, X; Zheng, M; Jiang, H

    2013-01-01

    Membrane transporters, including two members: ATP-binding cassette (ABC) transporters and solute carrier (SLC) transporters are proteins that play important roles to facilitate molecules into and out of cells. Consequently, these transporters can be major determinants of the therapeutic efficacy, toxicity and pharmacokinetics of a variety of drugs. Considering the time and expense of bio-experiments taking, research should be driven by evaluation of efficacy and safety. Computational methods arise to be a complementary choice. In this article, we provide an overview of the contribution that computational methods made in transporters field in the past decades. At the beginning, we present a brief introduction about the structure and function of major members of two families in transporters. In the second part, we focus on widely used computational methods in different aspects of transporters research. In the absence of a high-resolution structure of most of transporters, homology modeling is a useful tool to interpret experimental data and potentially guide experimental studies. We summarize reported homology modeling in this review. Researches in computational methods cover major members of transporters and a variety of topics including the classification of substrates and/or inhibitors, prediction of protein-ligand interactions, constitution of binding pocket, phenotype of non-synonymous single-nucleotide polymorphisms, and the conformation analysis that try to explain the mechanism of action. As an example, one of the most important transporters P-gp is elaborated to explain the differences and advantages of various computational models. In the third part, the challenges of developing computational methods to get reliable prediction, as well as the potential future directions in transporter related modeling are discussed.

  6. Monitoring the function of membrane transport proteins in detergent-solubilized form

    PubMed Central

    Quick, Matthias; Javitch, Jonathan A.

    2007-01-01

    Transport proteins constitute ≈10% of most proteomes and play vital roles in the translocation of solutes across membranes of all organisms. Their (dys)function is implicated in many disorders, making them frequent targets for pharmacotherapy. The identification of substrates for members of this large protein family, still replete with many orphans of unknown function, has proven difficult, in part because high-throughput screening is greatly complicated by endogenous transporters present in many expression systems. In addition, direct structural studies require that transporters be extracted from the membrane with detergent, thereby precluding transport measurements because of the lack of a vectorial environment and necessitating reconstitution into proteoliposomes for activity measurements. Here, we describe a direct scintillation proximity-based radioligand-binding assay for determining transport protein function in crude cell extracts and in purified form. This rapid and universally applicable assay with advantages over cell-based platforms will greatly facilitate the identification of substrates for many orphan transporters and allows monitoring the function of transport proteins in a nonmembranous environment. PMID:17360689

  7. A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance

    PubMed Central

    Walter, Stephanie; Kahla, Amal; Arunachalam, Chanemoughasoundharam; Perochon, Alexandre; Khan, Mojibur R.; Scofield, Steven R.; Doohan, Fiona M.

    2015-01-01

    The mycotoxin deoxynivalenol (DON) acts as a disease virulence factor for Fusarium fungi, and tolerance of DON enhances wheat resistance to Fusarium head blight (FHB) disease. Two variants of an ATP-binding cassette (ABC) family C transporter gene were cloned from DON-treated wheat mRNA, namely TaABCC3.1 and TaABCC3.2. These represent two of three putative genes identified on chromosomes 3A, 3B, and 3D of the wheat genome sequence. Variant TaABCC3.1 represents the DON-responsive transcript previously associated with DON resistance in wheat. PCR-based mapping and in silico sequence analyses located TaABCC3.1 to the short arm of wheat chromosome 3B (not within the FHB resistance quantitative trait locus Fhb1). In silico analyses of microarray data indicated that TaABCC3 genes are expressed in reproductive tissue and roots, and in response to the DON producer Fusarium graminearum. Gene expression studies showed that TaABCC3.1 is activated as part of the early host response to DON and in response to the FHB defence hormone jasmonic acid. Virus-induced gene silencing (VIGS) confirmed that TaABCC3 genes contributed to DON tolerance. VIGS was performed using two independent viral construct applications: one specifically targeted TaABCC3.1 for silencing, while the other targeted this gene and the chromosome 3A homeologue. In both instances, VIGS resulted in more toxin-induced discoloration of spikelets, compared with the DON effects in non-silenced spikelets at 14 d after toxin treatment (≥2.2-fold increase, P<0.05). Silencing by both VIGS constructs enhanced head ripening, and especially so in DON-treated heads. VIGS of TaABCC3 genes also reduced the grain number by more than 28% (P<0.05), both with and without DON treatment, and the effects were greater for the construct that targeted the two homeologues. Hence, DON-responsive TaABCC3 genes warrant further study to determine their potential as disease resistance breeding targets and their function in grain formation

  8. Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury.

    PubMed

    Lee, Hyunjung; McKeon, Robert J; Bellamkonda, Ravi V

    2010-02-23

    Chondroitin sulfate proteoglycans (CSPGs) are a major class of axon growth inhibitors that are up-regulated after spinal cord injury (SCI) and contribute to regenerative failure. Chondroitinase ABC (chABC) digests glycosaminoglycan chains on CSPGs and can thereby overcome CSPG-mediated inhibition. But chABC loses its enzymatic activity rapidly at 37 degrees C, necessitating the use of repeated injections or local infusions for a period of days to weeks. These infusion systems are invasive, infection-prone, and clinically problematic. To overcome this limitation, we have thermostabilized chABC and developed a system for its sustained local delivery in vivo, obviating the need for chronically implanted catheters and pumps. Thermostabilized chABC remained active at 37 degrees C in vitro for up to 4 weeks. CSPG levels remained low in vivo up to 6 weeks post-SCI when thermostabilized chABC was delivered by a hydrogel-microtube scaffold system. Axonal growth and functional recovery following the sustained local release of thermostabilized chABC versus a single treatment of unstabilized chABC demonstrated significant differences in CSPG digestion. Animals treated with thermostabilized chABC in combination with sustained neurotrophin-3 delivery showed significant improvement in locomotor function and enhanced growth of cholera toxin B subunit-positive sensory axons and sprouting of serotonergic fibers. Therefore, improving chABC thermostability facilitates minimally invasive, sustained, local delivery of chABC that is potentially effective in overcoming CSPG-mediated regenerative failure. Combination therapy with thermostabilized chABC with neurotrophic factors enhances axonal regrowth, sprouting, and functional recovery after SCI.

  9. SU-E-T-401: Feasibility Study of Using ABC to Gate Lung SBRT Treatment

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cao, D; Xie, X; Shepard, D

    2014-06-01

    Purpose: The current SBRT treatment techniques include free breathing (FB) SBRT and gated FB SBRT. Gated FB SBRT has smaller target and less lung toxicity with longer treatment time. The recent development of direct connectivity between the ABC and linac allowing for automated beam gating. In this study, we have examined the feasibility of using ABC system to gate the lung SBRT treatment. Methods: A CIRS lung phantom with a 3cm sphere-insert and a moving chest plate was used in this study. Sinusoidal motion was used for the FB pattern. An ABC signal was imported to simulate breath holds. 4D-CTmore » was taken in FB mode and average-intensity-projection (AIP) was used to create FB and 50% gated FB SBRT planning CT. A manually gated 3D CT scan was acquired for ABC gated SBRT planning.An SBRT plan was created for each treatment option. A surface-mapping system was used for 50% gating and ABC system was used for ABC gating. A manually gated CBCT scan was also performed to verify setup. Results: Among three options, the ABC gated plan has the smallest PTV of 35.94cc, which is 35% smaller comparing to that of the FB plan. Consequently, the V20 of the left lung reduced by 15% and 23% comparing to the 50% gated FB and FB plans, respectively. The FB plan took 4.7 minutes to deliver, while the 50% gated FB plan took 18.5 minutes. The ABC gated plan delivery took only 10.6 minutes. A stationary target with 3cm diameter was also obtained from the manually gated CBCT scan. Conclusion: A strategy for ABC gated lung SBRT was developed. ABC gating can significantly reduce the lung toxicity while maintaining the target coverage. Comparing to the 50% gated FB SBRT, ABC gated treatment can also provide less lung toxicity as well as improved delivery efficiency. This research is funded by Elekta.« less

  10. Creating an iPhone Application for Collecting Continuous ABC Data

    ERIC Educational Resources Information Center

    Whiting, Seth W.; Dixon, Mark R.

    2012-01-01

    This paper provides an overview and task analysis for creating a continuous ABC data- collection application using Xcode on a Mac computer. Behavior analysts can program an ABC data collection system, complete with a customized list of target clients, antecedents, behaviors, and consequences to be recorded, and have the data automatically sent to…

  11. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    PubMed

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  12. Laboratory investigation of grouted coupler connection details for ABC bridge projects.

    DOT National Transportation Integrated Search

    2015-08-01

    With an ever increasing desire to utilize accelerated bridge construction (ABC) techniques, it is becoming critical that bridge : designers and contractors have confidence in typical details. The Keg Creek Bridge on US 6 in Iowa was a recent ABC exam...

  13. Automated brainstem co-registration (ABC) for MRI.

    PubMed

    Napadow, Vitaly; Dhond, Rupali; Kennedy, David; Hui, Kathleen K S; Makris, Nikos

    2006-09-01

    Group data analysis in brainstem neuroimaging is predicated on accurate co-registration of anatomy. As the brainstem is comprised of many functionally heterogeneous nuclei densely situated adjacent to one another, relatively small errors in co-registration can manifest in increased variance or decreased sensitivity (or significance) in detecting activations. We have devised a 2-stage automated, reference mask guided registration technique (Automated Brainstem Co-registration, or ABC) for improved brainstem co-registration. Our approach utilized a brainstem mask dataset to weight an automated co-registration cost function. Our method was validated through measurement of RMS error at 12 manually defined landmarks. These landmarks were also used as guides for a secondary manual co-registration option, intended for outlier individuals that may not adequately co-register with our automated method. Our methodology was tested on 10 healthy human subjects and compared to traditional co-registration techniques (Talairach transform and automated affine transform to the MNI-152 template). We found that ABC had a significantly lower mean RMS error (1.22 +/- 0.39 mm) than Talairach transform (2.88 +/- 1.22 mm, mu +/- sigma) and the global affine (3.26 +/- 0.81 mm) method. Improved accuracy was also found for our manual-landmark-guided option (1.51 +/- 0.43 mm). Visualizing individual brainstem borders demonstrated more consistent and uniform overlap for ABC compared to traditional global co-registration techniques. Improved robustness (lower susceptibility to outliers) was demonstrated with ABC through lower inter-subject RMS error variance compared with traditional co-registration methods. The use of easily available and validated tools (AFNI and FSL) for this method should ease adoption by other investigators interested in brainstem data group analysis.

  14. Role of non-receptor protein kinases in spermatid transport during spermatogenesis*

    PubMed Central

    Wan, H. T.; Mruk, Dolores D.; Tang, Elizabeth I.; Xiao, Xiang; Cheng, Yan-ho; Wong, Elissa W.P.; Wong, Chris K. C.; Cheng, C. Yan

    2014-01-01

    Non-receptor protein tyrosine kinases are cytoplasmic kinases that activate proteins by phosphorylating target protein tyrosine residues, in turn affecting multiple functions in eukaryotic cells. Herein, we focus on the role of non-receptor protein tyrosine kinases, most notably, FAK, c-Yes and c-Src, in the transport of spermatids across the seminiferous epithelium during spermatogenesis. Since spermatids, which are formed from spermatocytes via meiosis, are immotile haploid cells, they must be transported by Sertoli cells across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Without the timely transport of spermatids across the epithelium, the release of sperms at spermiation fails to occur, leading to infertility. Thus, the molecular event pertinent to spermatid transport is crucial to spermatogenesis. Herein, we provide a critical discussion based on recent findings in the field. We also provide a hypothetical model on spermatid transport, and the role of non-receptor protein tyrosine kinases in this event. We also highlight areas of research that deserve attention by investigators in the field. PMID:24727349

  15. Dietary protein affects urea transport across rat urothelia.

    PubMed

    Spector, David A; Deng, Jie; Stewart, Kerry J

    2012-10-01

    Recent evidence suggests that regulated solute transport occurs across mammalian lower urinary tract epithelia (urothelia). To study the effects of dietary protein on net urothelial transport of urea, creatinine, and water, we used an in vivo rat bladder model designed to mimic physiological conditions. We placed groups of rats on 3-wk diets differing only by protein content (40, 18, 6, and 2%) and instilled 0.3 ml of collected urine in the isolated bladder of anesthetized rats. After 1 h dwell, retrieved urine volumes were unchanged, but mean urea nitrogen (UN) and creatinine concentrations fell 17 and 4%, respectively, indicating transurothelial urea and creatinine reabsorption. The fall in UN (but not creatinine) concentration was greatest in high protein (40%) rats, 584 mg/dl, and progressively less in rats receiving lower protein content: 18% diet, 224 mg/dl; 6% diet, 135 mg/dl; and 2% diet, 87 mg/dl. The quantity of urea reabsorbed was directly related to a urine factor, likely the concentration of urea in the instilled urine. In contrast, the percentage of instilled urea reabsorbed was greater in the two dietary groups receiving the lowest protein (26 and 23%) than in those receiving higher protein (11 and 9%), suggesting the possibility that a bladder/urothelial factor, also affected by dietary protein, may have altered bladder permeability. These findings demonstrate significant regulated urea transport across the urothelium, resulting in alteration of urine excreted by the kidneys, and add to the growing evidence that the lower urinary tract may play an unappreciated role in mammalian solute homeostasis.

  16. Anger and the ABC model underlying Rational-Emotive Behavior Therapy.

    PubMed

    Ziegler, Daniel J; Smith, Phillip N

    2004-06-01

    The ABC model underlying Ellis's Rational-Emotive Behavior Therapy predicts that people who think more irrationally should display greater trait anger than do people who think less irrationally. This study tested this prediction regarding the ABC model. 186 college students were administered the Survey of Personal Beliefs and the State-Trait Anger Expression Inventory-Second Edition to measure irrational thinking and trait anger, respectively. Students who scored higher on Overall Irrational Thinking and Low Frustration Tolerance scored significantly higher on Trait Anger than did those who scored lower on Overall Irrational Thinking and Low Frustration Tolerance. This indicates support for the ABC model, especially Ellis's construct of irrational beliefs which is central to the model.

  17. Genome-wide identification, phylogenetic analysis, and expression profiles of ATP-binding cassette transporter genes in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).

    PubMed

    Xiao, Lin-Fan; Zhang, Wei; Jing, Tian-Xing; Zhang, Meng-Yi; Miao, Ze-Qing; Wei, Dan-Dan; Yuan, Guo-Rui; Wang, Jin-Jun

    2018-03-01

    The ATP-binding cassette (ABC) is the largest transporter gene family and the genes play key roles in xenobiotic resistance, metabolism, and development of all phyla. However, the specific functions of ABC gene families in insects is unclear. We report a genome-wide identification, phylogenetic, and transcriptional analysis of the ABC genes in the oriental fruit fly, Bactrocera dorsalis (Hendel). We identified a total of 47 ABC genes (BdABCs) from the transcriptomic and genomic databases of B. dorsalis and classified these genes into eight subfamilies (A-H), including 7 ABCAs, 7 ABCBs, 9 ABCCs, 2 ABCDs, 1 ABCE, 3 ABCFs, 15 ABCGs, and 3 ABCHs. Comparative phylogenetic analysis of the ABCs suggests an orthologous relationship between B. dorsalis and other insect species in which these genes have been related to pesticide resistance and essential biological processes. Comparison of transcriptome and relative expression patterns of BdABCs indicated diverse multifunctions within different B. dorsalis tissues. The expression of 4, 10, and 14 BdABCs from 18 BdABCs was significantly upregulated after exposure to LD 50 s of malathion, avermectin, and beta-cypermethrin, respectively. The maximum expression level of most BdABCs (including BdABCFs, BdABCGs, and BdABCHs) occurred at 48h post exposures, whereas BdABCEs peaked at 24h after treatment. Furthermore, RNA interference-mediated suppression of BdABCB7 resulted in increased toxicity of malathion against B. dorsalis. These data suggest that ABC transporter genes might play key roles in xenobiotic metabolism and biosynthesis in B. dorsalis. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. The ABC Model and its Applicability to Basal Angiosperms

    PubMed Central

    Soltis, Douglas E.; Chanderbali, André S.; Kim, Sangtae; Buzgo, Matyas; Soltis, Pamela S.

    2007-01-01

    Background Although the flower is the central feature of the angiosperms, little is known of its origin and subsequent diversification. The ABC model has long been the unifying paradigm for floral developmental genetics, but it is based on phylogenetically derived eudicot models. Synergistic research involving phylogenetics, classical developmental studies, genomics and developmental genetics has afforded valuable new insights into floral evolution in general, and the early flower in particular. Scope and Conclusions Genomic studies indicate that basal angiosperms, and by inference the earliest angiosperms, had a rich tool kit of floral genes. Homologues of the ABCE floral organ identity genes are also present in basal angiosperm lineages; however, C-, E- and particularly B-function genes are more broadly expressed in basal lineages. There is no single model of floral organ identity that applies to all angiosperms; there are multiple models that apply depending on the phylogenetic position and floral structure of the group in question. The classic ABC (or ABCE) model may work well for most eudicots. However, modifications are needed for basal eudicots and, the focus of this paper, basal angiosperms. We offer ‘fading borders’ as a testable hypothesis for the basal-most angiosperms and, by inference, perhaps some of the earliest (now extinct) angiosperms. PMID:17616563

  19. Phylodynamic Inference with Kernel ABC and Its Application to HIV Epidemiology.

    PubMed

    Poon, Art F Y

    2015-09-01

    The shapes of phylogenetic trees relating virus populations are determined by the adaptation of viruses within each host, and by the transmission of viruses among hosts. Phylodynamic inference attempts to reverse this flow of information, estimating parameters of these processes from the shape of a virus phylogeny reconstructed from a sample of genetic sequences from the epidemic. A key challenge to phylodynamic inference is quantifying the similarity between two trees in an efficient and comprehensive way. In this study, I demonstrate that a new distance measure, based on a subset tree kernel function from computational linguistics, confers a significant improvement over previous measures of tree shape for classifying trees generated under different epidemiological scenarios. Next, I incorporate this kernel-based distance measure into an approximate Bayesian computation (ABC) framework for phylodynamic inference. ABC bypasses the need for an analytical solution of model likelihood, as it only requires the ability to simulate data from the model. I validate this "kernel-ABC" method for phylodynamic inference by estimating parameters from data simulated under a simple epidemiological model. Results indicate that kernel-ABC attained greater accuracy for parameters associated with virus transmission than leading software on the same data sets. Finally, I apply the kernel-ABC framework to study a recent outbreak of a recombinant HIV subtype in China. Kernel-ABC provides a versatile framework for phylodynamic inference because it can fit a broader range of models than methods that rely on the computation of exact likelihoods. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  20. Abc Amino Acids: Design, Synthesis, and Properties of New Photoelastic Amino Acids

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Standaert, Robert F; Park, Dr Seung Bum

    2006-01-01

    Photoisomerizable amino acids provide a direct avenue to the experimental manipulation of bioactive polypeptides, potentially allowing real-time, remote control of biological systems and enabling useful applications in nanobiotechnology. Herein, we report a new class of photoisomerizable amino acids intended to cause pronounced expansion and contraction in the polypeptide backbone, i.e., to be photoelastic. These compounds, termed Abc amino acids, employ a photoisomerizable azobiphenyl chromophore to control the relative disposition of aminomethyl and carboxyl substituents. Molecular modeling of nine Abc isomers led to the identification of one with particularly attractive properties, including the ability to induce contractions up to 13A inmore » the backbone upon transa?cis photoisomerization. This isomer, designated mpAbc, has substituents at meta and para positions on the inner (azo-linked) and outer rings, respectively. An efficient synthesis of Fmoc-protected mpAbc was executed in which the biaryl components were formed via Suzuki couplings and the azo linkage was formed via amine/nitroso condensation; protected forms of three other Abc isomers were prepared similarly. A decapeptide incorporating mpAbc was synthesized by conventional solid-phase methods and displayed characteristic azobenzene photochemical behavior with optimal conversion to the cis isomer at 360 nm and a thermal cisa?trans half life of 100 min. at 80 AoC.« less

  1. Step 2: Know Your Diabetes ABCs

    MedlinePlus

    ... please turn JavaScript on. Feature: Type 2 Diabetes Step 2: Know Your Diabetes ABCs Past Issues / Fall ... 2 Diabetes" Articles Diabetes Is Serious But Manageable / Step 1: Learn About Diabetes / Step 2: Know Your ...

  2. The ABCs of Student Engagement

    ERIC Educational Resources Information Center

    Parsons, Seth A.; Nuland, Leila Richey; Parsons, Allison Ward

    2014-01-01

    Student engagement is an important consideration for teachers and administrators because it is explicitly associated with achievement. What the authors call the ABC's of engagement they outline as: Affective engagement, Behavioral engagement, and Cognitive engagement. They also present "Three Things Every Teacher Needs to Know about…

  3. Direct Spectroscopic Detection of ATP Turnover Reveals Mechanistic Divergence of ABC Exporters.

    PubMed

    Collauto, Alberto; Mishra, Smriti; Litvinov, Aleksei; Mchaourab, Hassane S; Goldfarb, Daniella

    2017-08-01

    We have applied high-field (W-band) pulse electron-nuclear double resonance (ENDOR) and electron-electron double resonance (ELDOR)-detected nuclear magnetic resonance (EDNMR) to characterize the coordination sphere of the Mn 2+ co-factor in the nucleotide binding sites (NBSs) of ABC transporters. MsbA and BmrCD are two efflux transporters hypothesized to represent divergent catalytic mechanisms. Our results reveal distinct coordination of Mn 2+ to ATP and transporter residues in the consensus and degenerate NBSs of BmrCD. In contrast, the coordination of Mn 2+ at the two NBSs of MsbA is similar, which provides a mechanistic rationale for its higher rate constant of ATP hydrolysis relative to BmrCD. Direct detection of vanadate ion, trapped in a high-energy post-hydrolysis intermediate, further supports the notion of asymmetric hydrolysis by the two NBSs of BmrCD. The integrated spectroscopic approach presented here, which link energy input to conformational dynamics, can be applied to a variety of systems powered by ATP turnover. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Implication of RuvABC and RecG in homologous recombination in Streptomyces ambofaciens.

    PubMed

    Hoff, Grégory; Bertrand, Claire; Piotrowski, Emilie; Thibessard, Annabelle; Leblond, Pierre

    2017-01-01

    Most bacterial organisms rely on homologous recombination to repair DNA double-strand breaks and for the post-replicative repair of DNA single-strand gaps. Homologous recombination can be divided into three steps: (i) a pre-synaptic step in which the DNA 3'-OH ends are processed, (ii) a recA-dependent synaptic step allowing the invasion of an intact copy and the formation of Holliday junctions, and (iii) a post-synaptic step consisting of migration and resolution of these junctions. Currently, little is known about factors involved in homologous recombination, especially for the post-synaptic step. In Escherichia coli, branch migration and resolution are performed by the RuvABC complex, but could also rely on the RecG helicase in a redundant manner. In this study, we show that recG and ruvABC are well-conserved among Streptomyces. ΔruvABC, ΔrecG and ΔruvABC ΔrecG mutant strains were constructed. ΔruvABC ΔrecG is only slightly affected by exposure to DNA damage (UV). We also show that conjugational recombination decreases in the absence of RuvABC and RecG, but that intra-chromosomal recombination is not affected. These data suggest that RuvABC and RecG are indeed involved in homologous recombination in Streptomyces ambofaciens and that alternative factors are able to take over Holliday junction in Streptomyces. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Candida Drug Resistance Protein 1, a Major Multidrug ATP Binding Cassette Transporter of Candida albicans, Translocates Fluorescent Phospholipids in a Reconstituted System†

    PubMed Central

    Shukla, Sudhanshu; Rai, Versha; Saini, Preeti; Banerjee, Dibyendu; Menon, Anant K.; Prasad, Rajendra

    2008-01-01

    Candida albicans drug resistance protein 1 (Cdr1p), an ATP-dependent drug efflux pump, contributes to multidrug resistance in Candida-infected immunocompromised patients. Previous cell-based assays suggested that Cdr1p also acts as a phospholipid translocator. To investigate this, we reconstituted purified Cdr1p into sealed membrane vesicles. Comparison of the ATPase activities of sealed and permeabilized proteoliposomes indicated that Cdr1p was asymmetrically reconstituted such that ~70% of the molecules had their ATP binding sites accessible to the extravesicular space. Fluorescent glycerophospholipids were incorporated into the outer leaflet of the proteoliposomes, and their transport into the inner leaflet was tracked with a quenching assay using membrane-impermeant dithionite. We observed ATP-dependent transport of the fluorescent lipids into the inner leaflet of the vesicles. With ~6 molecules of Cdr1p per vesicle on average, the half-time to reach the maximal extent of transport was ~15 min. Transport was reduced in vesicles reconstituted with Cdr1p variants with impaired ATPase activity and could be competed out to different levels by a molar excess of drugs such as fluconazole and miconazole that are known to be effluxed by Cdr1p. Transport was not affected by ampicillin, a compound that is not effluxed by Cdr1p. Our results suggest a direct link between the ability of Cdr1p to translocate fluorescent phospholipids and efflux drugs. We note that only a few members of the ABC superfamily of Candida have a well-defined role as drug exporters; thus, lipid translocation mediated by Cdr1p could reflect its cellular function. PMID:17924650

  6. Parents' Perspectives on Braille Literacy: Results from the ABC Braille Study

    ERIC Educational Resources Information Center

    Kamei-Hannan, Cheryl; Sacks, Sharon Zell

    2012-01-01

    Introduction: Parents who were the primary caretakers of children in the Alphabetic and Contracted Braille Study (ABC Braille Study) revealed their perspectives about braille literacy. Methods: A 30-item questionnaire was constructed by the ABC Braille research team, and researchers conducted telephone interviews with 31 parents who were the…

  7. Constitutive androstane receptor upregulates Abcb1 and Abcg2 at the blood-brain barrier after CITCO activation.

    PubMed

    Lemmen, Julia; Tozakidis, Iasson E P; Bele, Prachee; Galla, Hans-Joachim

    2013-03-21

    ATP-driven efflux transporters are considered to be the major hurdle in the treatment of central nervous system (CNS) diseases. Abcb1 (P-glycoprotein) and Abcg2 (breast cancer resistance protein/brain multidrug resistance protein) belong to the best known ABC-transporters. These ABC-transporters limit the permeability of the blood-brain barrier and protect the brain against toxic compounds in the blood but on the other hand they also reduce the efficacy of CNS pharmacotherapy. Even after 40 years of extensive research, the regulatory mechanisms of these efflux transporters are still not completely understood. To unravel the efflux transporter regulation, we analyzed the effect of the nuclear receptor CAR (constitutive androstane receptor) on the expression of Abcb1 and Abcg2 in primary cultures of porcine brain capillary endothelial cells (PBCEC). CAR is a xenobiotic-activated transcription factor, which is, like the other important nuclear receptor pregnane X receptor (PXR), highly expressed in barrier tissue and known to be a positive regulator of ABC-transporters. We demonstrate that activation of porcine CAR by the human CAR (hCAR) ligand CITCO (6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde) leads to an up-regulation of both transporters, whereas the mouse-specific CAR ligand TCPOBOP (1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene) had no effect on transporter expression. The stimulation of PBCEC with CITCO caused a significant up-regulation of both efflux-transporters on RNA-level, protein level and transport level. Furthermore the additional application of a CAR inhibitor significantly decreased the transporter expression to control niveau. In conclusion our data prove CAR activation only by the human ligand CITCO leading to an increased ABC-transporter expression and transport activity. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Enhanced gravi- and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1.

    PubMed

    Noh, Bosl; Bandyopadhyay, Anindita; Peer, Wendy Ann; Spalding, Edgar P; Murphy, Angus S

    2003-06-26

    Many aspects of plant growth and development are dependent on the flow of the hormone auxin down the plant from the growing shoot tip where it is synthesized. The direction of auxin transport in stems is believed to result from the basal localization within cells of the PIN1 membrane protein, which controls the efflux of the auxin anion. Mutations in two genes homologous to those encoding the P-glycoprotein ABC transporters that are especially abundant in multidrug-resistant tumour cells in animals were recently shown to block polar auxin transport in the hypocotyls of Arabidopsis seedlings. Here we show that the mdr mutants display faster and greater gravitropism and enhanced phototropism instead of the impaired curvature development expected in mutants lacking polar auxin transport. We find that these phenotypes result from a disruption of the normal accumulation of PIN1 protein along the basal end of hypocotyl cells associated with basipetal auxin flow. Lateral auxin conductance becomes relatively larger as a result, enhancing the growth differentials responsible for tropic responses.

  9. Early Observations of the Type Ia Supernova iPTF 16abc

    NASA Astrophysics Data System (ADS)

    Miller, Adam; iPTF Collaboration

    2018-01-01

    Early observations of Type Ia supernovae (SNe) provide a unique probe of their progenitor systems and explosion physics. Here, we report the intermediate Palomar Transient Factory (iPTF) discovery of an extraordinarily young SN Ia, iPTF 16abc. By fitting a power law to our early light curve, we infer that first light for the SN only occurred 0.15 +0.15-0.07 d before our first detection. In the ~24 hr after discovery, iPTF 16abc rose by ~2 mag, following a near-linear rise in flux for ~3 d. Strong C II absorption is detected in the early spectra of iPTF 16abc, before disappearing after ~7 d. Unlike the extensively-observed Type Ia SN 2011fe, the (B-V)_0 colors of iPTF 16abc are blue and nearly constant in the days after explosion. We show that our early observations of iPTF 16abc cannot be explained by either SN shock breakout and the associated, subsequent cooling, or the SN ejecta colliding with a stellar companion. Instead, we argue that the early characteristics of iPTF 16abc, including: (i) the rapid, near-linear rise, (ii) the non-evolving blue colors, and (iii) the strong absorption from ionized carbon, are the result of either vigorous mixing of radioactive-Ni in the SN ejecta, or ejecta interaction with diffuse material, or a combination of the two. In the next few years, dozens of very young normal SNe Ia will be discovered, and observations similar to those presented here will constrain the white dwarf explosion mechanism.

  10. The role of mass transport in protein crystallization.

    PubMed

    García-Ruiz, Juan Manuel; Otálora, Fermín; García-Caballero, Alfonso

    2016-02-01

    Mass transport takes place within the mesoscopic to macroscopic scale range and plays a key role in crystal growth that may affect the result of the crystallization experiment. The influence of mass transport is different depending on the crystallization technique employed, essentially because each technique reaches supersaturation in its own unique way. In the case of batch experiments, there are some complex phenomena that take place at the interface between solutions upon mixing. These transport instabilities may drastically affect the reproducibility of crystallization experiments, and different outcomes may be obtained depending on whether or not the drop is homogenized. In diffusion experiments with aqueous solutions, evaporation leads to fascinating transport phenomena. When a drop starts to evaporate, there is an increase in concentration near the interface between the drop and the air until a nucleation event eventually takes place. Upon growth, the weight of the floating crystal overcomes the surface tension and the crystal falls to the bottom of the drop. The very growth of the crystal then triggers convective flow and inhomogeneities in supersaturation values in the drop owing to buoyancy of the lighter concentration-depleted solution surrounding the crystal. Finally, the counter-diffusion technique works if, and only if, diffusive mass transport is assured. The technique relies on the propagation of a supersaturation wave that moves across the elongated protein chamber and is the result of the coupling of reaction (crystallization) and diffusion. The goal of this review is to convince protein crystal growers that in spite of the small volume of the typical protein crystallization setup, transport plays a key role in the crystal quality, size and phase in both screening and optimization experiments.

  11. Brassboard Astrometric Beam Combiner (ABC) Development for the Space Interferometry Mission (SIM)

    NASA Technical Reports Server (NTRS)

    Jeganathan, Muthu; Kuan, Gary; Rud, Mike; Lin, Sean; Sutherland, Kristen; Moore, James; An, Xin

    2008-01-01

    The Astrometric Beam Combiner (ABC) is a critical element of the Space Interferometry Mission (SIM) that performs three key functions: coherently combine starlight from two siderostats; individually detect starlight for angle tracking; and disperse and detect the interferometric fringes. In addition, the ABC contains: a stimulus, cornercubes and shutters for in-orbit calibration; several tip/tilt mirror mechanisms for in-orbit alignment; and internal metrology beam launcher for pathlength monitoring. The detailed design of the brassboard ABC (which has the form, fit and function of the flight unit) is complete, procurement of long-lead items is underway, and assembly and testing is expected to be completed in Spring 2009. In this paper, we present the key requirements for the ABC, details of the completed optical and mechanical design as well as plans for assembly and alignment.

  12. fbpABC gene cluster in Neisseria meningitidis is transcribed as an operon.

    PubMed

    Khun, H H; Deved, V; Wong, H; Lee, B C

    2000-12-01

    The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR.

  13. fbpABC Gene Cluster in Neisseria meningitidis Is Transcribed as an Operon

    PubMed Central

    Khun, Heng H.; Deved, Vinay; Wong, Howard; Lee, B. Craig

    2000-01-01

    The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with an fbpC-sequence-specific oligonucleotide indicates that fbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR. PMID:11083849

  14. Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells.

    PubMed

    Toussaint, Frédéric; Pierman, Baptiste; Bertin, Aurélie; Lévy, Daniel; Boutry, Marc

    2017-05-04

    Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia , which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min -1  mg -1 ) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

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

    PubMed Central

    Fredslund, Folmer; Vujičić Žagar, Andreja; Andersen, Thomas Lars; Svensson, Birte; Slotboom, Dirk Jan

    2016-01-01

    The molecular details and impact of oligosaccharide uptake by distinct human gut microbiota (HGM) are currently not well understood. Non-digestible dietary galacto- and gluco-α-(1,6)-oligosaccharides from legumes and starch, respectively, are preferentially fermented by mainly bifidobacteria and lactobacilli in the human gut. Here we show that the solute binding protein (BlG16BP) associated with an ATP binding cassette (ABC) transporter from the probiotic Bifidobacterium animalis subsp. lactis Bl-04 binds α-(1,6)-linked glucosides and galactosides of varying size, linkage, and monosaccharide composition with preference for the trisaccharides raffinose and panose. This preference is also reflected in the α-(1,6)-galactoside uptake profile of the bacterium. Structures of BlG16BP in complex with raffinose and panose revealed the basis for the remarkable ligand binding plasticity of BlG16BP, which recognizes the non-reducing α-(1,6)-diglycoside in its ligands. BlG16BP homologues occur predominantly in bifidobacteria and a few Firmicutes but lack in other HGMs. Among seven bifidobacterial taxa, only those possessing this transporter displayed growth on α-(1,6)-glycosides. Competition assays revealed that the dominant HGM commensal Bacteroides ovatus was out-competed by B. animalis subsp. lactis Bl-04 in mixed cultures growing on raffinose, the preferred ligand for the BlG16BP. By comparison, B. ovatus mono-cultures grew very efficiently on this trisaccharide. These findings suggest that the ABC-mediated uptake of raffinose provides an important competitive advantage, particularly against dominant Bacteroides that lack glycan-specific ABC-transporters. This novel insight highlights the role of glycan transport in defining the metabolic specialization of gut bacteria. PMID:27502277

  16. Transport proteins of parasitic protists and their role in nutrient salvage.

    PubMed

    Dean, Paul; Major, Peter; Nakjang, Sirintra; Hirt, Robert P; Embley, T Martin

    2014-01-01

    The loss of key biosynthetic pathways is a common feature of important parasitic protists, making them heavily dependent on scavenging nutrients from their hosts. This is often mediated by specialized transporter proteins that ensure the nutritional requirements of the parasite are met. Over the past decade, the completion of several parasite genome projects has facilitated the identification of parasite transporter proteins. This has been complemented by functional characterization of individual transporters along with investigations into their importance for parasite survival. In this review, we summarize the current knowledge on transporters from parasitic protists and highlight commonalities and differences in the transporter repertoires of different parasitic species, with particular focus on characterized transporters that act at the host-pathogen interface.

  17. Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana).

    PubMed

    Kanno, Akira; Saeki, Hiroshi; Kameya, Toshiaki; Saedler, Heinz; Theissen, Günter

    2003-07-01

    In higher eudicotyledonous angiosperms the floral organs are typically arranged in four different whorls, containing sepals, petals, stamens and carpels. According to the ABC model, the identity of these organs is specified by floral homeotic genes of class A, A+B, B+C and C, respectively. In contrast to the sepal and petal whorls of eudicots, the perianths of many plants from the Liliaceae family have two outer whorls of almost identical petaloid organs, called tepals. To explain the Liliaceae flower morphology, van Tunen et al. (1993) proposed a modified ABC model, exemplified with tulip. According to this model, class B genes are not only expressed in whorls 2 and 3, but also in whorl 1. Thus the organs of both whorls 1 and 2 express class A plus class B genes and, therefore, get the same petaloid identity. To test this modified ABC model we have cloned and characterized putative class B genes from tulip. Two DEF- and one GLO-like gene were identified, named TGDEFA, TGDEFB and TGGLO. Northern hybridization analysis showed that all of these genes are expressed in whorls 1, 2 and 3 (outer and inner tepals and stamens), thus corroborating the modified ABC model. In addition, these experiments demonstrated that TGGLO is also weakly expressed in carpels, leaves, stems and bracts. Gel retardation assays revealed that TGGLO alone binds to DNA as a homodimer. In contrast, TGDEFA and TGDEFB cannot homodimerize, but make heterodimers with PI. Homodimerization of GLO-like protein has also been reported for lily, suggesting that this phenomenon is conserved within Liliaceae plants or even monocot species.

  18. Interaction of oligomeric breast cancer resistant protein (BCRP) with adjudin: a male contraceptive with anti-cancer activity.

    PubMed

    Cheng, Yan Ho; Jenardhanan, Pranitha; Mathur, Premendu P; Qian, Xiaojing; Xia, Weiliang; Silvestrini, Bruno; Cheng, Chuen Yan

    2014-01-01

    Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein foun d in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis.

  19. Interaction of Oligomeric Breast Cancer Resistant Protein (BCRP) with Adjudin: A Male Contraceptive with Anti-Cancer Activity

    PubMed Central

    Cheng, Yan Ho; Jenardhanan, Pranitha; Mathur, Premendu P.; Qian, Xiaojing; Xia, Weiliang; Silvestrini, Bruno; Cheng, Chuen Yan

    2016-01-01

    Breast cancer resistant protein (BCRP, ABCG2) is an ATP-binding cassette (ABC) transporter, which together with two other ABC efflux drug pumps, namely P-glycoprotein (P-gp, ABCB1) and multidrug resistance-related protein 1 (MRP1, ABCC1) is the most important multidrug resistance protein found in eukaryotic cells including cells in the testis. However, unlike P-gp and MRP1, which are components of the Sertoli cell blood-testis barrier (BTB), BCRP is not expressed at the BTB in rodents and human testes. Instead, BCRP is expressed by peritubular myoid cells and endothelial cells of the lymphatic vessel in the tunica propria, residing outside the BTB. As such, the testis is equipped with two levels of defense against xenobiotics or drugs, preventing these harmful substances from entering the adluminal compartment to perturb meiosis and post-meiotic spermatid development: one at the level of the BTB conferred by P-gp and MRP1 and one at the tunica propria conferred by BCRP. The presence of drug transporters at the tunica propria as well as at the Sertoli cell BTB thus poses significant obstacles in developing non-hormonal contraceptives if these drugs (e.g., adjudin) exert their effects in germ cells behind the BTB, such as in the adluminal (apical) compartment of the seminiferous epithelium. Herein, we summarize recent findings pertinent to adjudin, a non-hormonal male contraceptive, and molecular interactions of adjudin with BCRP so that this information can be helpful to devise delivery strategies to evade BCRP in the tunica propria to improve its bioavailability in the testis. PMID:25620224

  20. Detection and functional characterization of Pgp1 (ABCB1) and MRP3 (ABCC3) efflux transporters in the PLHC-1 fish hepatoma cell line.

    PubMed

    Zaja, Roko; Klobucar, Roberta Sauerborn; Smital, Tvrtko

    2007-03-30

    The PLHC-1 hepatoma cell line derived from topminnow (Poeciliopsis lucida) is one of the most frequently used fish cell lines in aquatic ecotoxicology. These cells have been well characterized regarding the presence of phase I and phase II enzymes involved in the metabolism of xenobiotics. However, the presence of the ABC transport proteins possibly involved in the MultiXenobiotic Resistance (MXR) mechanism as phase III of cellular detoxification has never been described in the PLHC-1 cells. The main goal of this study was the detection and functional characterization of toxicologically relevant xenobiotic efflux transporters from ABCB and ABCC subfamily in the PLHC-1 cells. Using specific primer pairs two PCR products 1769 and 1023bp in length were successfully cloned and sequenced. Subsequent multiple alignment and phylogenetic analysis showed that these sequences share a high degree of homology with the P-glycoprotein (Pgp1; ABCB1) and the MRP3 (ABCC3). Functional experiments with fluorescent model substrates and specific inhibitors were used to verify that transport activities of Pgp- and MRP-related proteins are indeed present in PLHC-1 cells. Accumulation or efflux/retention rates of rhodamine 123, calcein-AM or monochlorbimane were time- and concentration-dependent. Cyclosporine A, MK571, verapamil, reversine 205, indomethacine and probenecid were used as specific inhibitors of Pgp1 and/or MRPs transport activities, resulting in a dose dependent inhibition of related transport activities in PLHC-1 cells. Similar to mammalian systems, the obtained IC(50) values were in the lower micromolar range. Taken together these data demonstrate that: (1) the PLHC-1 cells do express a functional MXR mechanism mediated by toxicologically relevant ABC efflux transporters; and (2) the presence of all three critical phases of cellular detoxification additionally affirms the PLHC-1 cells as a reliable in vitro model in aquatic toxicology.

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

    PubMed

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

    2015-10-01

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

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

    PubMed

    Sakamoto, K; Margolles, A; van Veen, H W; Konings, W N

    2001-09-01

    Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.

  3. ABC estimation of unit costs for emergency department services.

    PubMed

    Holmes, R L; Schroeder, R E

    1996-04-01

    Rapid evolution of the health care industry forces managers to make cost-effective decisions. Typical hospital cost accounting systems do not provide emergency department managers with the information needed, but emergency department settings are so complex and dynamic as to make the more accurate activity-based costing (ABC) system prohibitively expensive. Through judicious use of the available traditional cost accounting information and simple computer spreadsheets. managers may approximate the decision-guiding information that would result from the much more costly and time-consuming implementation of ABC.

  4. Structure determination of a sugar-binding protein from the phytopathogenic bacterium Xanthomonas citri

    PubMed Central

    Medrano, Francisco Javier; de Souza, Cristiane Santos; Romero, Antonio; Balan, Andrea

    2014-01-01

    The uptake of maltose and related sugars in Gram-negative bacteria is mediated by an ABC transporter encompassing a periplasmic component (the maltose-binding protein or MalE), a pore-forming membrane protein (MalF and MalG) and a membrane-associated ATPase (MalK). In the present study, the structure determination of the apo form of the putative maltose/trehalose-binding protein (Xac-MalE) from the citrus pathogen Xanthomonas citri in space group P6522 is described. The crystals contained two protein molecules in the asymmetric unit and diffracted to 2.8 Å resolution. Xac-MalE conserves the structural and functional features of sugar-binding proteins and a ligand-binding pocket with similar characteristics to eight different orthologues, including the residues for maltose and trehalose interaction. This is the first structure of a sugar-binding protein from a phytopathogenic bacterium, which is highly conserved in all species from the Xanthomonas genus. PMID:24817711

  5. Minireview: SLCO and ABC Transporters: A Role for Steroid Transport in Prostate Cancer Progression

    PubMed Central

    Cho, Eunpi; Montgomery, R. Bruce

    2014-01-01

    Androgens play a critical role in the development and progression of prostate cancer (PCa), and androgen deprivation therapy via surgical or medical castration is front-line therapy for patients with advanced PCa. However, intratumoral testosterone levels are elevated in metastases from patients with castration-resistant disease, and residual intratumoral androgens have been implicated in mediating ligand-dependent mechanisms of androgen receptor activation. The source of residual tissue androgens present despite castration has not been fully elucidated, but proposed mechanisms include uptake and conversion of adrenal androgens, such as dehdroepiandrosterone to testosterone and dihydrotestosterone, or de novo androgen synthesis from cholesterol or progesterone precursors. In this minireview, we discuss the emerging evidence that suggests a role for specific transporters in mediating transport of steroids into or out of prostate cells, thereby influencing intratumoral androgen levels and PCa development and progression. We focus on the solute carrier and ATP binding cassette gene families, which have the most published data for a role in PCa-related steroid transport, and review the potential impact of genetic variation on steroid transport activity and PCa outcomes. Continued assessment of transport activity in PCa models and human tumor tissue is needed to better delineate the different roles these transporters play in physiologic and neoplastic settings, and in order to determine whether targeting the uptake of steroid substrates by specific transporters may be a clinically feasible therapeutic strategy. PMID:25147980

  6. The ABCs of Activity-Based Costing: A Cost Containment and Reallocation Tool.

    ERIC Educational Resources Information Center

    Turk, Frederick J.

    1992-01-01

    This article describes activity-based costing (ABC) and how this tool may help management understand the costs of major activities and identify possible alternatives. Also discussed are the traditional costing systems used by higher education and ways of applying ABC to higher education. (GLR)

  7. Applying Activity Based Costing (ABC) Method to Calculate Cost Price in Hospital and Remedy Services

    PubMed Central

    Rajabi, A; Dabiri, A

    2012-01-01

    Background Activity Based Costing (ABC) is one of the new methods began appearing as a costing methodology in the 1990’s. It calculates cost price by determining the usage of resources. In this study, ABC method was used for calculating cost price of remedial services in hospitals. Methods: To apply ABC method, Shahid Faghihi Hospital was selected. First, hospital units were divided into three main departments: administrative, diagnostic, and hospitalized. Second, activity centers were defined by the activity analysis method. Third, costs of administrative activity centers were allocated into diagnostic and operational departments based on the cost driver. Finally, with regard to the usage of cost objectives from services of activity centers, the cost price of medical services was calculated. Results: The cost price from ABC method significantly differs from tariff method. In addition, high amount of indirect costs in the hospital indicates that capacities of resources are not used properly. Conclusion: Cost price of remedial services with tariff method is not properly calculated when compared with ABC method. ABC calculates cost price by applying suitable mechanisms but tariff method is based on the fixed price. In addition, ABC represents useful information about the amount and combination of cost price services. PMID:23113171

  8. Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

    PubMed Central

    Simpson, Brent W.; Owens, Tristan W.; Orabella, Matthew J.; Davis, Rebecca M.; May, Janine M.; Trauger, Sunia A.

    2016-01-01

    ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. PMID:27795402

  9. Identification of membrane-associated proteins with pathogenic potential expressed by Corynebacterium pseudotuberculosis grown in animal serum.

    PubMed

    Raynal, José Tadeu; Bastos, Bruno Lopes; Vilas-Boas, Priscilla Carolinne Bagano; Sousa, Thiago de Jesus; Costa-Silva, Marcos; de Sá, Maria da Conceição Aquino; Portela, Ricardo Wagner; Moura-Costa, Lília Ferreira; Azevedo, Vasco; Meyer, Roberto

    2018-01-25

    Previous works defining antigens that might be used as vaccine targets against Corynebacterium pseudotuberculosis, which is the causative agent of sheep and goat caseous lymphadenitis, have focused on secreted proteins produced in a chemically defined culture media. Considering that such antigens might not reflect the repertoire of proteins expressed during infection conditions, this experiment aimed to investigate the membrane-associated proteins with pathogenic potential expressed by C. pseudotuberculosis grown directly in animal serum. Its membrane-associated proteins have been extracted using an organic solvent enrichment methodology, followed by LC-MS/MS and bioinformatics analysis for protein identification and classification. The results revealed 22 membrane-associated proteins characterized as potentially pathogenic. An interaction network analysis indicated that the four potentially pathogenic proteins ciuA, fagA, OppA4 and OppCD were biologically connected within two distinct network pathways, which were both associated with the ABC Transporters KEGG pathway. These results suggest that C. pseudotuberculosis pathogenesis might be associated with the transport and uptake of nutrients; other seven identified potentially pathogenic membrane proteins also suggest that pathogenesis might involve events of bacterial resistance and adhesion. The proteins herein reported potentially reflect part of the protein repertoire expressed during real infection conditions and might be tested as vaccine antigens.

  10. Structural insight into lipopolysaccharide transport from the Gram-negative bacterial inner membrane to the outer membrane.

    PubMed

    Dong, Haohao; Tang, Xiaodi; Zhang, Zhengyu; Dong, Changjiang

    2017-11-01

    Lipopolysaccharide (LPS) is an important component of the outer membrane (OM) of Gram-negative bacteria, playing essential roles in protecting bacteria from harsh environments, in drug resistance and in pathogenesis. LPS is synthesized in the cytoplasm and translocated to the periplasmic side of the inner membrane (IM), where it matures. Seven lipopolysaccharide transport proteins, LptA-G, form a trans‑envelope complex that is responsible for LPS extraction from the IM and transporting it across the periplasm to the OM. The LptD/E of the complex transports LPS across the OM and inserts it into the outer leaflet of the OM. In this review we focus upon structural and mechanistic studies of LPS transport proteins, with a particular focus upon the LPS ABC transporter LptB 2 FG. This ATP binding cassette transporter complex consists of twelve transmembrane segments and has a unique mechanism whereby it extracts LPS from the periplasmic face of the IM through a pair of lateral gates and then powers trans‑periplasmic transport to the OM through a slide formed by either of the periplasmic domains of LptF or LptG, LptC, LptA and the N-terminal domain of LptD. The structural and functional studies of the seven lipopolysaccharide transport proteins provide a platform to explore the unusual mechanisms of LPS extraction, transport and insertion from the inner membrane to the outer membrane. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway.

    PubMed

    Jeong, Ji-Hye; Nam, Yeon-Ju; Kim, Seok-Yong; Kim, Eung-Gook; Jeong, Jooyoung; Kim, Hyong Kyu

    2007-09-01

    There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.

  12. Mechanism for the antibacterial action of epigallocatechin gallate (EGCg) on Bacillus subtilis.

    PubMed

    Nakayama, Motokazu; Shimatani, Kanami; Ozawa, Tadahiro; Shigemune, Naofumi; Tomiyama, Daisuke; Yui, Koji; Katsuki, Mao; Ikeda, Keisuke; Nonaka, Ai; Miyamoto, Takahisa

    2015-01-01

    Catechins are a class of polyphenols and have high anti-bacterial activity against various microorganisms. Here, we report the mechanism for antibacterial activity of epigallocatechin gallate (EGCg) against Gram-positive bacteria Bacillus subtilis, which is highly sensitive to EGCg. Transmission electron microscope analysis revealed that deposits containing EGCg were found throughout the cell envelope from the outermost surface to the outer surface of cytoplasmic membrane. Aggregating forms of proteins and EGCg were identified as spots that disappeared or showed markedly decreased intensity after the treatment with EGCg compared to the control by two-dimensional electrophoresis. Among the identified proteins included 4 cell surface proteins, such as oligopeptide ABC transporter binding lipoprotein, glucose phosphotransferase system transporter protein, phosphate ABC transporter substrate-binding protein, and penicillin-binding protein 5. Observations of glucose uptake of cells and cell shape B. subtilis after the treatment with EGCg suggested that EGCg inhibits the major functions of these proteins, leading to growth inhibition of B. subtilis.

  13. Molecular and cellular regulation of glucose transporter (GLUT) proteins in cancer.

    PubMed

    Macheda, Maria L; Rogers, Suzanne; Best, James D

    2005-03-01

    Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins, with overexpression of GLUT1 and/or GLUT3 a characteristic feature. Oncogenic transformation of cultured mammalian cells causes a rapid increase of glucose transport and GLUT1 expression via interaction with GLUT1 promoter enhancer elements. In human studies, high levels of GLUT1 expression in tumors have been associated with poor survival. Studies indicate that glucose transport in breast cancer is not fully explained by GLUT1 or GLUT3 expression, suggesting involvement of another glucose transporter. Recently, a novel glucose transporter protein, GLUT12, has been found in breast and prostate cancers. In human breast and prostate tumors and cultured cells, GLUT12 is located intracellularly and at the cell surface. Trafficking of GLUT12 to the plasma membrane could therefore contribute to glucose uptake. Several factors have been implicated in the regulation of glucose transporter expression in breast cancer. Hypoxia can increase GLUT1 levels and glucose uptake. Estradiol and epidermal growth factor, both of which can play a role in breast cancer cell growth, increase glucose consumption. Estradiol and epidermal growth factor also increase GLUT12 protein levels in cultured breast cancer cells. Targeting GLUT12 could provide novel methods for detection and treatment of breast and prostate cancer. 2004 Wiley-Liss, Inc.

  14. MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption

    PubMed Central

    Reznicek, Josef; Ceckova, Martina; Ptackova, Zuzana; Martinec, Ondrej; Tupova, Lenka; Cerveny, Lukas

    2017-01-01

    ABSTRACT Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo. Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir. PMID:28696229

  15. The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management.

    PubMed

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-09-03

    Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management.

  16. The novel ABC transporter ABCH1 is a potential target for RNAi-based insect pest control and resistance management

    PubMed Central

    Guo, Zhaojiang; Kang, Shi; Zhu, Xun; Xia, Jixing; Wu, Qingjun; Wang, Shaoli; Xie, Wen; Zhang, Youjun

    2015-01-01

    Insect pests cause serious crop damage and develop high-level resistance to chemical insecticides and Bacillus thuringiensis (Bt) insecticidal Cry toxins. A new promising approach for controlling them and overcoming this resistance is RNA interference (RNAi). The RNAi-based insect control strategy depends on the selection of suitable target genes. In this study, we cloned and characterized a novel ABC transporter gene PxABCH1 in diamondback moth, Plutella xylostella (L.). Phylogenetic analysis showed that PxABCH1 is closely related to ABCA and ABCG subfamily members. Spatial-temporal expression detection revealed that PxABCH1 was expressed in all tissues and developmental stages, and highest expressed in head and male adult. Midgut sequence variation and expression analyses of PxABCH1 in all the susceptible and Bt-resistant P. xylostella strains and the functional analysis by sublethal RNAi demonstrated that Cry1Ac resistance was independent of this gene. Silencing of PxABCH1 by a relatively high dose of dsRNA dramatically reduced its expression and resulted in larval and pupal lethal phenotypes in both susceptible and Cry1Ac-resistant P. xylostella strains. To our knowledge, this study provides the first insight into ABCH1 in lepidopterans and reveals it as an excellent target for RNAi-based insect pest control and resistance management. PMID:26333918

  17. mRMR-ABC: A Hybrid Gene Selection Algorithm for Cancer Classification Using Microarray Gene Expression Profiling

    PubMed Central

    Alshamlan, Hala; Badr, Ghada; Alohali, Yousef

    2015-01-01

    An artificial bee colony (ABC) is a relatively recent swarm intelligence optimization approach. In this paper, we propose the first attempt at applying ABC algorithm in analyzing a microarray gene expression profile. In addition, we propose an innovative feature selection algorithm, minimum redundancy maximum relevance (mRMR), and combine it with an ABC algorithm, mRMR-ABC, to select informative genes from microarray profile. The new approach is based on a support vector machine (SVM) algorithm to measure the classification accuracy for selected genes. We evaluate the performance of the proposed mRMR-ABC algorithm by conducting extensive experiments on six binary and multiclass gene expression microarray datasets. Furthermore, we compare our proposed mRMR-ABC algorithm with previously known techniques. We reimplemented two of these techniques for the sake of a fair comparison using the same parameters. These two techniques are mRMR when combined with a genetic algorithm (mRMR-GA) and mRMR when combined with a particle swarm optimization algorithm (mRMR-PSO). The experimental results prove that the proposed mRMR-ABC algorithm achieves accurate classification performance using small number of predictive genes when tested using both datasets and compared to previously suggested methods. This shows that mRMR-ABC is a promising approach for solving gene selection and cancer classification problems. PMID:25961028

  18. mRMR-ABC: A Hybrid Gene Selection Algorithm for Cancer Classification Using Microarray Gene Expression Profiling.

    PubMed

    Alshamlan, Hala; Badr, Ghada; Alohali, Yousef

    2015-01-01

    An artificial bee colony (ABC) is a relatively recent swarm intelligence optimization approach. In this paper, we propose the first attempt at applying ABC algorithm in analyzing a microarray gene expression profile. In addition, we propose an innovative feature selection algorithm, minimum redundancy maximum relevance (mRMR), and combine it with an ABC algorithm, mRMR-ABC, to select informative genes from microarray profile. The new approach is based on a support vector machine (SVM) algorithm to measure the classification accuracy for selected genes. We evaluate the performance of the proposed mRMR-ABC algorithm by conducting extensive experiments on six binary and multiclass gene expression microarray datasets. Furthermore, we compare our proposed mRMR-ABC algorithm with previously known techniques. We reimplemented two of these techniques for the sake of a fair comparison using the same parameters. These two techniques are mRMR when combined with a genetic algorithm (mRMR-GA) and mRMR when combined with a particle swarm optimization algorithm (mRMR-PSO). The experimental results prove that the proposed mRMR-ABC algorithm achieves accurate classification performance using small number of predictive genes when tested using both datasets and compared to previously suggested methods. This shows that mRMR-ABC is a promising approach for solving gene selection and cancer classification problems.

  19. Rab proteins: The key regulators of intracellular vesicle transport

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bhuin, Tanmay; Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied bymore » cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.« less

  20. Smart zwitterionic membranes with on/off behavior for protein transport.

    PubMed

    Su, Yanlei; Zheng, Lili; Li, Chao; Jiang, Zhongyi

    2008-09-25

    Poly(acrylonitrile) (PAN)-based zwitterionic membranes, composed of PAN and poly( N, N-dimethyl- N-methacryloxyethyl- N-(3-sulfopropyl) copolymer, are electrolyte-sensitive smart membranes. The hydrophilicity was increased and protein adsorption was remarkably decreased for the membranes in response to environmental stimuli. FTIR spectroscopic analysis directly provided molecular-level observation of the enhanced dissociation and hydration of zwitterionic sulfobetaine dipoles at higher electrolyte concentrations. The smart PAN-based zwitterionic membranes can close or open channels for protein transport under different NaCl concentrations. The electrolyte-sensitive switch of on/off behavior for protein transport is reversible.

  1. Computation of Thermal Transport in a Protein

    NASA Astrophysics Data System (ADS)

    Leitner, David M.

    2003-03-01

    Calculation of the coefficient of thermal conductivity and thermal diffusivity for a protein will be discussed. Thermal transport coefficients are obtained by computing the proteinÂ's normal modes, their lifetimes, the speed of sound and mean free path. We find the thermal diffusivity of myoglobin at 300 K to be 14 Å^2 /ps, the same as the value for water. The thermal conductivity at 300 K is calculated to be 2.0 mW/cm K in the absence of solvent and somewhat higher for the solvated protein, about one-third the value for water.

  2. Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

    PubMed

    Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

    2017-05-01

    The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pegos, Vanessa R.; Hey, Louis; LaMirande, Jacob

    Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP fromPolaromonasJS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently knownmore » PBPs. Here, it is shown that the PBP fromPolaromonasJS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP fromPolaromonasJS666 are reported.« less

  4. Position-dependent Effects of Polylysine on Sec Protein Transport*

    PubMed Central

    Liang, Fu-Cheng; Bageshwar, Umesh K.; Musser, Siegfried M.

    2012-01-01

    The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or “pause sites,” were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport. PMID:22367204

  5. Position-dependent effects of polylysine on Sec protein transport.

    PubMed

    Liang, Fu-Cheng; Bageshwar, Umesh K; Musser, Siegfried M

    2012-04-13

    The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.

  6. GPS-ABC radiated chamber testing overview and results : GPS-ABC Workshop VI : RTCA Washington, DC, March 30, 2017.

    DOT National Transportation Integrated Search

    2017-03-30

    This presentation, which was given during the GPS-ABC Workshop VI in Washington, DC on March 30, 2017 details the authors' radiated testing protocols and results. GPS receiver testing was carried out April 25-29, 2016 at the Army : Research Laborator...

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

    PubMed

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

    2014-10-06

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

  8. Role of Breast Cancer Resistance Protein (BCRP/ABCG2) in Cancer Drug Resistance

    PubMed Central

    Natarajan, Karthika; Xie, Yi; Baer, Maria R.; Ross, Douglas D.

    2012-01-01

    Since cloning of the ATP-binding cassette (ABC) family member breast cancer resistance protein (BCRP/ABCG2) and its characterization as a multidrug resistance efflux transporter in 1998, BCRP has been the subject of more than two thousand scholarly articles. In normal tissues, BCRP functions as a defense mechanism against toxins and xenobiotics, with expression in the gut, bile canaliculi, placenta, blood-testis and blood-brain barriers facilitating excretion and limiting absorption of potentially toxic substrate molecules, including many cancer chemotherapeutic drugs. BCRP also plays a key role in heme and folate homeostasis, which may help normal cells survive under conditions of hypoxia. BCRP expression appears to be a characteristic of certain normal tissue stem cells termed “side population cells,” which are identified on flow cytometric analysis by their ability to exclude Hoechst 33342, a BCRP substrate fluorescent dye. Hence, BCRP expression may contribute to the natural resistance and longevity of these normal stem cells. Malignant tissues can exploit the properties of BCRP to survive hypoxia and to evade exposure to chemotherapeutic drugs. Evidence is mounting that many cancers display subpopulations of stem cells that are responsible for tumor self-renewal. Such stem cells frequently manifest the “side population” phenotype characterized by expression of BCRP and other ABC transporters. Along with other factors, these transporters may contribute to the inherent resistance of these neoplasms and their failure to be cured. PMID:22248732

  9. Efflux proteins at the blood-brain barrier: review and bioinformatics analysis.

    PubMed

    Saidijam, Massoud; Karimi Dermani, Fatemeh; Sohrabi, Sareh; Patching, Simon G

    2018-05-01

    1. Efflux proteins at the blood-brain barrier provide a mechanism for export of waste products of normal metabolism from the brain and help to maintain brain homeostasis. They also prevent entry into the brain of a wide range of potentially harmful compounds such as drugs and xenobiotics. 2. Conversely, efflux proteins also hinder delivery of therapeutic drugs to the brain and central nervous system used to treat brain tumours and neurological disorders. For bypassing efflux proteins, a comprehensive understanding of their structures, functions and molecular mechanisms is necessary, along with new strategies and technologies for delivery of drugs across the blood-brain barrier. 3. We review efflux proteins at the blood-brain barrier, classified as either ATP-binding cassette (ABC) transporters (P-gp, BCRP, MRPs) or solute carrier (SLC) transporters (OATP1A2, OATP1A4, OATP1C1, OATP2B1, OAT3, EAATs, PMAT/hENT4 and MATE1). 4. This includes information about substrate and inhibitor specificity, structural organisation and mechanism, membrane localisation, regulation of expression and activity, effects of diseases and conditions and the principal technique used for in vivo analysis of efflux protein activity: positron emission tomography (PET). 5. We also performed analyses of evolutionary relationships, membrane topologies and amino acid compositions of the proteins, and linked these to structure and function.

  10. The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus

    PubMed Central

    Park, Jin Hwan; Jo, Youmi; Jang, Song Yee; Kwon, Haenaem; Irie, Yasuhiko; Parsek, Matthew R.; Kim, Myung Hee; Choi, Sang Ho

    2015-01-01

    A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3′,5′-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose. PMID:26406498

  11. A test of the ABC model underlying rational emotive behavior therapy.

    PubMed

    Ziegler, Daniel J; Leslie, Yvonne M

    2003-02-01

    The ABC model underlying Ellis's Rational Emotive Behavior Therapy predicts that people who think more irrationally should respond to daily stressors or hassles differently than do people who think less irrationally. This study tested this aspect of the ABC model. 192 college students were administered the Survey of Personal Beliefs and the Hassles Scale to measure irrational thinking and daily hassles, respectively. Students who scored higher on overall irrational thinking reported a significantly higher frequency of hassles than did those who scored lower on overall irrational thinking, while students who scored higher on awfulizing and low frustration tolerance reported a significantly greater intensity of hassles than did those who scored lower on awfulizing and low frustration tolerance. This indicates support for the ABC model, especially Ellis's construct of irrational beliefs central to this model.

  12. P-glycoprotein (Mdr1a/1b) and breast cancer resistance protein (Bcrp) decrease the uptake of hydrophobic alkyl triphenylphosphonium cations by the brain

    PubMed Central

    Porteous, Carolyn M.; Menon, David K.; Aigbirhio, Franklin I.; Smith, Robin A.J.; Murphy, Michael P.

    2013-01-01

    Background Mitochondrial dysfunction contributes to degenerative neurological disorders, consequently there is a need for mitochondria-targeted therapies that are effective within the brain. One approach to deliver pharmacophores is by conjugation to the lipophilic triphenylphosphonium (TPP) cation that accumulates in mitochondria driven by the membrane potential. While this approach has delivered TPP-conjugated compounds to the brain, the amounts taken up are lower than by other organs. Methods To discover why uptake of hydrophobic TPP compounds by the brain is relatively poor, we assessed the role of the P-glycoprotein (Mdr1a/b) and breast cancer resistance protein (Bcrp) ATP binding cassette (ABC) transporters, which drive the efflux of lipophilic compounds from the brain thereby restricting the uptake of lipophilic drugs. We used a triple transgenic mouse model lacking two isoforms of P-glycoprotein (Mdr1a/1b) and the Bcrp. Results There was a significant increase in the uptake into the brain of two hydrophobic TPP compounds, MitoQ and MitoF, in the triple transgenics following intra venous (IV) administration compared to control mice. Greater amounts of the hydrophobic TPP compounds were also retained in the liver of transgenic mice compared to controls. The uptake into the heart, white fat, muscle and kidneys was comparable between the transgenic mice and controls. Conclusion Efflux of hydrophobic TPP compounds by ABC transporters contributes to their lowered uptake into the brain and liver. General significance These findings suggest that strategies to bypass ABC transporters in the BBB will enhance delivery of mitochondria-targeted antioxidants, probes and pharmacophores to the brain. PMID:23454352

  13. Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance.

    PubMed

    Watanabe, M; Mizoguchi, H; Nishimura, A

    2000-01-01

    Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of deltapdr5 and deltasnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity - 1) x 1443] of the deltapdr5 and deltasnq2 strains rose faster than that of HL69. On the other hand, a deltayor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the deltayor1 slowed down compared with that of CTZ21. A deltayor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the deltapdr5 and deltasnq2 strains of CTZ21. In contrast, the deltayor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.

  14. Multidrug and toxin extrusion proteins as transporters of antimicrobial drugs.

    PubMed

    Nies, Anne T; Damme, Katja; Schaeffeler, Elke; Schwab, Matthias

    2012-12-01

    Antimicrobial drugs are essential in the treatment of infectious diseases. A better understanding of transport processes involved in drug disposition will improve the predictability of drug-drug interactions with consequences for drug response. Multidrug And Toxin Extrusion (MATE; SLC47A) proteins are efflux transporters mediating the excretion of several antimicrobial drugs as well as other organic compounds into bile and urine, thereby contributing to drug disposition. This review summarizes current knowledge of the structural and molecular features of human MATE transporters including their functional role in drug transport with a specific focus on antimicrobial drugs. The PubMed database was searched using the terms "MATE1," "MATE-2K," "MATE2," "SLC47A1," "SLC47A2," and "toxin extrusion protein" (up to June 2012). MATE proteins have been recognized as important transporters mediating the final excretion step of cationic drugs into bile and urine. These include the antiviral drugs acyclovir, amprenavir, and ganciclovir, the antibiotics cephalexin, cephradine and levofloxacin, as well as the antimalarial agents chloroquine and quinine. It is therefore important to enhance our understanding of the role of MATEs in drug extrusion with particular emphasis on the functional consequences of genetic variants on disposition of these antimicrobial drugs.

  15. The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum

    PubMed Central

    Martin, Rowena E; Henry, Roselani I; Abbey, Janice L; Clements, John D; Kirk, Kiaran

    2005-01-01

    Background The uptake of nutrients, expulsion of metabolic wastes and maintenance of ion homeostasis by the intraerythrocytic malaria parasite is mediated by membrane transport proteins. Proteins of this type are also implicated in the phenomenon of antimalarial drug resistance. However, the initial annotation of the genome of the human malaria parasite Plasmodium falciparum identified only a limited number of transporters, and no channels. In this study we have used a combination of bioinformatic approaches to identify and attribute putative functions to transporters and channels encoded by the malaria parasite, as well as comparing expression patterns for a subset of these. Results A computer program that searches a genome database on the basis of the hydropathy plots of the corresponding proteins was used to identify more than 100 transport proteins encoded by P. falciparum. These include all the transporters previously annotated as such, as well as a similar number of candidate transport proteins that had escaped detection. Detailed sequence analysis enabled the assignment of putative substrate specificities and/or transport mechanisms to all those putative transport proteins previously without. The newly-identified transport proteins include candidate transporters for a range of organic and inorganic nutrients (including sugars, amino acids, nucleosides and vitamins), and several putative ion channels. The stage-dependent expression of RNAs for 34 candidate transport proteins of particular interest are compared. Conclusion The malaria parasite possesses substantially more membrane transport proteins than was originally thought, and the analyses presented here provide a range of novel insights into the physiology of this important human pathogen. PMID:15774027

  16. Modeling Pressure-Driven Transport of Proteins through a Nanochannel

    PubMed Central

    Carr, Rogan; Comer, Jeffrey; Ginsberg, Mark D.; Aksimentiev, Aleksei

    2012-01-01

    Reducing the size of a nanofluidic channel not only creates new opportunities for high-precision manipulation of biological macromolecules, but also makes the performance of the entire nanofluidic system more susceptible to undesirable interactions between the transported biomolecules and the walls of the channel. In this manuscript, we report molecular dynamics simulations of a pressure-driven flow through a silica nanochannel that characterized, with atomic resolution, adsorption of a model protein to its surface. Although the simulated adsorption of the proteins was found to be nonspecific, it had a dramatic effect on the rate of the protein transport. To determine the relative strength of the protein–silica interactions in different adsorbed states, we simulated flow-induced desorption of the proteins from the silica surface. Our analysis of the protein conformations in the adsorbed states did not reveal any simple dependence of the adsorption strength on the size and composition of the protein–silica contact, suggesting that the heterogeneity of the silica surface may be a important factor. PMID:22611338

  17. Applying the Post-Modern Double ABC-X Model to Family Food Insecurity

    ERIC Educational Resources Information Center

    Hutson, Samantha; Anderson, Melinda; Swafford, Melinda

    2015-01-01

    This paper develops the argument that using the Double ABC-X model in family and consumer sciences (FCS) curricula is a way to educate nutrition and dietetics students regarding a family's perceptions of food insecurity. The Double ABC-X model incorporates ecological theory as a basis to explain family stress and the resulting adjustment and…

  18. Assembly and mechanism of a group II ECF transporter.

    PubMed

    Karpowich, Nathan K; Wang, Da-Neng

    2013-02-12

    Energy-coupling factor (ECF) transporters are a recently discovered family of primary active transporters for micronutrients and vitamins, such as biotin, thiamine, and riboflavin. Found exclusively in archaea and bacteria, including the human pathogens Listeria, Streptococcus, and Staphylococcus, ECF transporters may be the only means of vitamin acquisition in these organisms. The subunit composition of ECF transporters is similar to that of ATP binding cassette (ABC) importers, whereby both systems share two homologous ATPase subunits (A and A'), a high affinity substrate-binding subunit (S), and a transmembrane coupling subunit (T). However, the S subunit of ECF transporters is an integral membrane protein, and the transmembrane coupling subunits do not share an obvious sequence homology between the two transporter families. Moreover, the subunit stoichiometry of ECF transporters is controversial, and the detailed molecular interactions between subunits and the conformational changes during substrate translocation are unknown. We have characterized the ECF transporters from Thermotoga maritima and Streptococcus thermophilus. Our data suggests a subunit stoichiometry of 2S:2T:1A:1A' and that S subunits for different substrates can be incorporated into the same transporter complex simultaneously. In the first crystal structure of the A-A' heterodimer, each subunit contains a novel motif called the Q-helix that plays a key role in subunit coupling with the T subunits. Taken together, these findings suggest a mechanism for coupling ATP binding and hydrolysis to transmembrane transport by ECF transporters.

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

    PubMed

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

    2015-09-01

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

  20. Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192

    PubMed Central

    Keung, Hoi Yee; Li, Tsz Kai; Sham, Lok To; Cheung, Man Kit; Cheung, Peter Chi Keung

    2017-01-01

    ABSTRACT Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast (Saccharomyces cerevisiae) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter (malEFG1) and pullulanase (aapA) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically

  1. Mechanistic Study of Utilization of Water-Insoluble Saccharomyces cerevisiae Glucans by Bifidobacterium breve Strain JCM1192.

    PubMed

    Keung, Hoi Yee; Li, Tsz Kai; Sham, Lok To; Cheung, Man Kit; Cheung, Peter Chi Keung; Kwan, Hoi Shan

    2017-04-01

    Bifidobacteria exert beneficial effects on hosts and are extensively used as probiotics. However, due to the genetic inaccessibility of these bacteria, little is known about their mechanisms of carbohydrate utilization and regulation. Bifidobacterium breve strain JCM1192 can grow on water-insoluble yeast ( Saccharomyces cerevisiae ) cell wall glucans (YCWG), which were recently considered as potential prebiotics. According to the results of 1 H nuclear magnetic resonance (NMR) spectrometry, the YCWG were composed of highly branched (1→3,1→6)-β-glucans and (1→4,1→6)-α-glucans. Although the YCWG were composed of 78.3% β-glucans and 21.7% α-glucans, only α-glucans were consumed by the B. breve strain. The ABC transporter ( malEFG1 ) and pullulanase ( aapA ) genes were transcriptionally upregulated in the metabolism of insoluble yeast glucans, suggesting their potential involvement in the process. A nonsense mutation identified in the gene encoding an ABC transporter ATP-binding protein (MalK) led to growth failure of an ethyl methanesulfonate-generated mutant with yeast glucans. Coculture of the wild-type strain and the mutant showed that this protein was responsible for the import of yeast glucans or their breakdown products, rather than the export of α-glucan-catabolizing enzymes. Further characterization of the carbohydrate utilization of the mutant and three of its revertants indicated that this mutation was pleiotropic: the mutant could not grow with maltose, glycogen, dextrin, raffinose, cellobiose, melibiose, or turanose. We propose that insoluble yeast α-glucans are hydrolyzed by extracellular pullulanase into maltose and/or maltooligosaccharides, which are then transported into the cell by the ABC transport system composed of MalEFG1 and MalK. The mechanism elucidated here will facilitate the development of B. breve and water-insoluble yeast glucans as novel synbiotics. IMPORTANCE In general, Bifidobacterium strains are genetically intractable

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

    PubMed

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

    2014-01-01

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

  3. Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA

    PubMed Central

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

    2014-01-01

    Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli. PMID:25517996

  4. The ABC's of Learning in Infancy.

    ERIC Educational Resources Information Center

    Saunders, Minta M.

    Learning in infancy is based on activity, beginnings, and curiosity, the so-called ABC's. Earliest behavior consists of mass activity, the period from birth to 24 months of sensory-motor development which provides the foundation for all future learning. Adults must provide space, toys, and affectionate care to help infants proceed through…

  5. Our bodies are our own: resistance to ABC-based HIV-prevention programmes in northern Tanzanian conservation organisations.

    PubMed

    Reid-Hresko, John

    2014-01-01

    ABC-based HIV-prevention programmes have been widely employed in northern Tanzanian wildlife conservation settings in an attempt to (re)shape the sexual behaviours of conservation actors. Utilising findings from 66 semi-structured interviews conducted in 2009-2010, this paper examines ABC prevention as a form of Foucauldian governmentality--circulating technologies of power that mobilise disciplinary technologies and attempt to transform such efforts into technologies of the self--and explores how individuals understand and respond to attempts to govern their behaviour. ABC regimes attempt to rework subjectivity, positioning HIV-related behaviours within a risk-based neoliberal rationality. However, efforts to use ABC as a technology to govern populations and individual bodies are largely incommensurate with existing Tanzanian sociocultural formations, including economic and gendered inequalities, and local understandings of sexuality. The language research participants used to talk about ABC and the justifications they offered for non-compliance illuminate this discrepancy. Data reveal that the recipients of ABC campaigns are active producers of understandings that work for them in their lives, but may not produce the behavioural shifts envisioned by programme goals. These findings corroborate previous research, which questions the continued plausibility of ABC as a stand-alone HIV- prevention framework.

  6. Golgi Localized Barley MTP8 Proteins Facilitate Mn Transport

    PubMed Central

    Pedas, Pai; Schiller Stokholm, Michaela; Hegelund, Josefine Nymark; Ladegård, Anne Hald; Schjoerring, Jan Kofod; Husted, Søren

    2014-01-01

    Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species. PMID:25486417

  7. MDR1 and BCRP Transporter-Mediated Drug-Drug Interaction between Rilpivirine and Abacavir and Effect on Intestinal Absorption.

    PubMed

    Reznicek, Josef; Ceckova, Martina; Ptackova, Zuzana; Martinec, Ondrej; Tupova, Lenka; Cerveny, Lukas; Staud, Frantisek

    2017-09-01

    Rilpivirine (TMC278) is a highly potent nonnucleoside reverse transcriptase inhibitor (NNRTI) representing an effective component of combination antiretroviral therapy (cART) in the treatment of HIV-positive patients. Many antiretroviral drugs commonly used in cART are substrates of ATP-binding cassette (ABC) and/or solute carrier (SLC) drug transporters and, therefore, are prone to pharmacokinetic drug-drug interactions (DDIs). The aim of our study was to evaluate rilpivirine interactions with abacavir and lamivudine on selected ABC and SLC transporters in vitro and assess its importance for pharmacokinetics in vivo Using accumulation assays in MDCK cells overexpressing selected ABC or SLC drug transporters, we revealed rilpivirine as a potent inhibitor of MDR1 and BCRP, but not MRP2, OCT1, OCT2, or MATE1. Subsequent transport experiments across monolayers of MDCKII-MDR1, MDCKII-BCRP, and Caco-2 cells demonstrated that rilpivirine inhibits MDR1- and BCRP-mediated efflux of abacavir and increases its transmembrane transport. In vivo experiments in male Wistar rats confirmed inhibition of MDR1/BCRP in the small intestine, leading to a significant increase in oral bioavailability of abacavir. In conclusion, rilpivirine inhibits MDR1 and BCRP transporters and may affect pharmacokinetic behavior of concomitantly administered substrates of these transporters, such as abacavir. Copyright © 2017 American Society for Microbiology.

  8. Amino Acid Transporters and Release of Hydrophobic Amino Acids in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120.

    PubMed

    Pernil, Rafael; Picossi, Silvia; Herrero, Antonia; Flores, Enrique; Mariscal, Vicente

    2015-04-23

    Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium that can use inorganic compounds such as nitrate or ammonium as nitrogen sources. In the absence of combined nitrogen, it can fix N2 in differentiated cells called heterocysts. Anabaena also shows substantial activities of amino acid uptake, and three ABC-type transporters for amino acids have been previously characterized. Seven new loci encoding predicted amino acid transporters were identified in the Anabaena genomic sequence and inactivated. Two of them were involved in amino acid uptake. Locus alr2535-alr2541 encodes the elements of a hydrophobic amino acid ABC-type transporter that is mainly involved in the uptake of glycine. ORF all0342 encodes a putative transporter from the dicarboxylate/amino acid:cation symporter (DAACS) family whose inactivation resulted in an increased uptake of a broad range of amino acids. An assay to study amino acid release from Anabaena filaments to the external medium was set up. Net release of the alanine analogue α-aminoisobutyric acid (AIB) was observed when transport system N-I (a hydrophobic amino acid ABC-type transporter) was engaged in the uptake of a specific substrate. The rate of AIB release was directly proportional to the intracellular AIB concentration, suggesting leakage from the cells by diffusion.

  9. Structure of the transporter associated with antigen processing trapped by herpes simplex virus

    PubMed Central

    Oldham, Michael L; Grigorieff, Nikolaus; Chen, Jue

    2016-01-01

    The transporter associated with antigen processing (TAP) is an ATP-binding cassette (ABC) transporter essential to cellular immunity against viral infection. Some persistent viruses have evolved strategies to inhibit TAP so that they may go undetected by the immune system. The herpes simplex virus for example evades immune surveillance by blocking peptide transport with a small viral protein ICP47. In this study, we determined the structure of human TAP bound to ICP47 by electron cryo-microscopy (cryo-EM) to 4.0 Å. The structure shows that ICP47 traps TAP in an inactive conformation distinct from the normal transport cycle. The specificity and potency of ICP47 inhibition result from contacts between the tip of the helical hairpin and the apex of the transmembrane cavity. This work provides a clear molecular description of immune evasion by a persistent virus. It also establishes the molecular structure of TAP to facilitate mechanistic studies of the antigen presentation process. DOI: http://dx.doi.org/10.7554/eLife.21829.001 PMID:27935481

  10. The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

    PubMed Central

    Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

    2013-01-01

    Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

  11. Inner Blood-Retinal Barrier Dominantly Expresses Breast Cancer Resistance Protein: Comparative Quantitative Targeted Absolute Proteomics Study of CNS Barriers in Pig.

    PubMed

    Zhang, Zhengyu; Uchida, Yasuo; Hirano, Satoshi; Ando, Daisuke; Kubo, Yoshiyuki; Auriola, Seppo; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi; Urtti, Arto; Terasaki, Tetsuya; Tachikawa, Masanori

    2017-11-06

    The purpose of this study was to determine absolute protein expression levels of transporters at the porcine inner blood-retinal barrier (BRB) and to compare the transporter protein expression quantitatively among the inner BRB, outer BRB, blood-brain barrier (BBB), and blood-cerebrospinal fluid barrier (BCSFB). Crude membrane fractions of isolated retinal capillaries (inner BRB) and isolated retinal pigment epithelium (RPE, outer BRB) were prepared from porcine eyeballs, while plasma membrane fractions were prepared from isolated porcine brain capillaries (BBB) and isolated choroid plexus (BCSFB). Protein expression levels of 32 molecules, including 16 ATP-binding-cassette (ABC) transporters and 13 solute-carrier (SLC) transporters, were measured using a quantitative targeted absolute proteomic technique. At the inner BRB, five molecules were detected: breast cancer resistance protein (BCRP, ABCG2; 22.8 fmol/μg protein), multidrug resistance protein 1 (MDR1, ABCB1; 8.70 fmol/μg protein), monocarboxylate transporter 1 (MCT1, SLC16A1; 4.83 fmol/μg protein), glucose transporter 1 (GLUT1, SLC2A1; 168 fmol/μg protein), and sodium-potassium adenosine triphosphatase (Na + /K + -ATPase; 53.7 fmol/μg protein). Other proteins were under the limits of quantification. Expression of MCT1 was at least 17.6-, 11.0-, and 19.2-fold greater than those of MCT2, 3, and 4, respectively. The transporter protein expression at the inner BRB was most highly correlated with that at the BBB (R 2 = 0.8906), followed by outer BRB (R 2 = 0.7988) and BCSFB (R 2 = 0.4730). Sodium-dependent multivitamin transporter (SMVT, SLC5A6) and multidrug resistance-associated protein 1 (MRP1, ABCC1) were expressed at the outer BRB (0.378 and 1.03 fmol/μg protein, respectively) but were under the limit of quantification at the inner BRB. These findings may be helpful for understanding differential barrier function.

  12. astroABC : An Approximate Bayesian Computation Sequential Monte Carlo sampler for cosmological parameter estimation

    NASA Astrophysics Data System (ADS)

    Jennings, E.; Madigan, M.

    2017-04-01

    Given the complexity of modern cosmological parameter inference where we are faced with non-Gaussian data and noise, correlated systematics and multi-probe correlated datasets,the Approximate Bayesian Computation (ABC) method is a promising alternative to traditional Markov Chain Monte Carlo approaches in the case where the Likelihood is intractable or unknown. The ABC method is called "Likelihood free" as it avoids explicit evaluation of the Likelihood by using a forward model simulation of the data which can include systematics. We introduce astroABC, an open source ABC Sequential Monte Carlo (SMC) sampler for parameter estimation. A key challenge in astrophysics is the efficient use of large multi-probe datasets to constrain high dimensional, possibly correlated parameter spaces. With this in mind astroABC allows for massive parallelization using MPI, a framework that handles spawning of processes across multiple nodes. A key new feature of astroABC is the ability to create MPI groups with different communicators, one for the sampler and several others for the forward model simulation, which speeds up sampling time considerably. For smaller jobs the Python multiprocessing option is also available. Other key features of this new sampler include: a Sequential Monte Carlo sampler; a method for iteratively adapting tolerance levels; local covariance estimate using scikit-learn's KDTree; modules for specifying optimal covariance matrix for a component-wise or multivariate normal perturbation kernel and a weighted covariance metric; restart files output frequently so an interrupted sampling run can be resumed at any iteration; output and restart files are backed up at every iteration; user defined distance metric and simulation methods; a module for specifying heterogeneous parameter priors including non-standard prior PDFs; a module for specifying a constant, linear, log or exponential tolerance level; well-documented examples and sample scripts. This code is hosted

  13. Accelerator-based conversion (ABC) of weapons plutonium: Plant layout study and related design issues

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cowell, B.S.; Fontana, M.H.; Krakowski, R.A.

    1995-04-01

    In preparation for and in support of a detailed R and D Plan for the Accelerator-Based Conversion (ABC) of weapons plutonium, an ABC Plant Layout Study was conducted at the level of a pre-conceptual engineering design. The plant layout is based on an adaptation of the Molten-Salt Breeder Reactor (MSBR) detailed conceptual design that was completed in the early 1070s. Although the ABC Plant Layout Study included the Accelerator Equipment as an essential element, the engineering assessment focused primarily on the Target; Primary System (blanket and all systems containing plutonium-bearing fuel salt); the Heat-Removal System (secondary-coolant-salt and supercritical-steam systems); Chemicalmore » Processing; Operation and Maintenance; Containment and Safety; and Instrumentation and Control systems. Although constrained primarily to a reflection of an accelerator-driven (subcritical) variant of MSBR system, unique features and added flexibilities of the ABC suggest improved or alternative approaches to each of the above-listed subsystems; these, along with the key technical issues in need of resolution through a detailed R&D plan for ABC are described on the bases of the ``strawman`` or ``point-of-departure`` plant layout that resulted from this study.« less

  14. Inferring population history with DIY ABC: a user-friendly approach to approximate Bayesian computation.

    PubMed

    Cornuet, Jean-Marie; Santos, Filipe; Beaumont, Mark A; Robert, Christian P; Marin, Jean-Michel; Balding, David J; Guillemaud, Thomas; Estoup, Arnaud

    2008-12-01

    Genetic data obtained on population samples convey information about their evolutionary history. Inference methods can extract part of this information but they require sophisticated statistical techniques that have been made available to the biologist community (through computer programs) only for simple and standard situations typically involving a small number of samples. We propose here a computer program (DIY ABC) for inference based on approximate Bayesian computation (ABC), in which scenarios can be customized by the user to fit many complex situations involving any number of populations and samples. Such scenarios involve any combination of population divergences, admixtures and population size changes. DIY ABC can be used to compare competing scenarios, estimate parameters for one or more scenarios and compute bias and precision measures for a given scenario and known values of parameters (the current version applies to unlinked microsatellite data). This article describes key methods used in the program and provides its main features. The analysis of one simulated and one real dataset, both with complex evolutionary scenarios, illustrates the main possibilities of DIY ABC. The software DIY ABC is freely available at http://www.montpellier.inra.fr/CBGP/diyabc.

  15. Effect of body temperature on chondroitinase ABC's ability to cleave chondroitin sulfate glycosaminoglycans.

    PubMed

    Tester, Nicole J; Plaas, Anna H; Howland, Dena R

    2007-04-01

    Chondroitinase ABC (Ch'ase ABC) is a bacterial lyase that degrades chondroitin sulfate (CS), dermatan sulfate, and hyaluronan glycosaminoglycans (GAGs). This enzyme has received significant attention as a potential therapy for promoting central nervous system and peripheral nervous system repair based on its degradation of CS GAGs. Determination of the stability of Ch'ase ABC activity at temperatures equivalent to normal (37 degrees C) and elevated (39 degrees C) body temperatures is important for optimizing its clinical usage. We report here data obtained from examining enzymatic activity at these temperatures across nine lots of commercially available protease-free Ch'ase ABC. CS GAG degrading activity was assayed by using 1) immunohistochemical detection of unsaturated disaccharide stubs generated by digestion of proteoglycans in tissue sections and 2) fluorophore-assisted carbohydrate electrophoresis (FACE) and/or high-performance liquid chromatography (HPLC) to separate and quantify unsaturated disaccharide digestion products. Our results indicate that there is a significant effect of lot and time on enzymatic thermostability. Average enzymatic activity is significantly decreased at 1 and 3 days at 39 degrees C and 37 degrees C, respectively. Furthermore, the average activity seen after 1 day was significantly different between the two temperatures. Addition of bovine serum albumin as a stabilizer significantly preserved enzymatic activity at 1 day, but not 3 days, at 39 degrees C. These results show that the CS GAG degrading activity of Ch'ase ABC is significantly decreased with incubation at body temperature over time and that all lots do not show equal thermostability. These findings are important for the design and interpretation of experimental and potential clinical studies involving Ch'ase ABC. (c) 2007 Wiley-Liss, Inc.

  16. Beyond cellular detoxification: a plethora of physiological roles for MDR transporter homologs in plants

    PubMed Central

    Remy, Estelle; Duque, Paula

    2014-01-01

    Higher plants possess a multitude of Multiple Drug Resistance (MDR) transporter homologs that group into three distinct and ubiquitous families—the ATP-Binding Cassette (ABC) superfamily, the Major Facilitator Superfamily (MFS), and the Multidrug And Toxic compound Extrusion (MATE) family. As in other organisms, such as fungi, mammals, and bacteria, MDR transporters make a primary contribution to cellular detoxification processes in plants, mainly through the extrusion of toxic compounds from the cell or their sequestration in the central vacuole. This review aims at summarizing the currently available information on the in vivo roles of MDR transporters in plant systems. Taken together, these data clearly indicate that the biological functions of ABC, MFS, and MATE carriers are not restricted to xenobiotic and metal detoxification. Importantly, the activity of plant MDR transporters also mediates biotic stress resistance and is instrumental in numerous physiological processes essential for optimal plant growth and development, including the regulation of ion homeostasis and polar transport of the phytohormone auxin. PMID:24910617

  17. Aerosol and Cloud Properties during the Cloud Cheju ABC Plume -Asian Monsoon Experiment (CAPMEX) 2008: Linking between Ground-based and UAV Measurements

    NASA Astrophysics Data System (ADS)

    Kim, S.; Yoon, S.; Venkata Ramana, M.; Ramanathan, V.; Nguyen, H.; Park, S.; Kim, M.

    2009-12-01

    Cheju Atmospheric Brown Cloud (ABC) Plume-Monsoon Experiment (CAPMEX), comprehsensive ground-based measurements and a series of data-gathering flights by specially equipped autonomous unmanned aerial vehicles (AUAVs) for aerosol and cloud, had conducted at Jeju (formerly, Cheju), South Korea during August-September 2008, to improve our understanding of how the reduction of anthropogenic emissions in China (so-called “great shutdown” ) during and after the Summer Beijing Olympic Games 2008 effcts on the air quliaty and radiation budgets and how atmospheric brown clouds (ABCs) influences solar radiation budget off Asian continent. Large numbers of in-situ and remote sensing instruments at the Gosan ABC observatory and miniaturized instruments on the aircraft measure a range of properties such as the quantity of soot, size-segregated aerosol particle numbers, total particle numbers, size-segregated cloud droplet numbers (only AUAV), aerosol scattering properties (only ground), aerosol vertical distribution, column-integrated aerosol properties, and meteorological variables. By integrating ground-level and high-elevation AUAV measurements with NASA-satellite observations (e.g., MODIS, CALIPSO), we investigate the long range transport of aerosols, the impact of ABCs on clouds, and the role of biogenic and anthropogenic aerosols on cloud condensation nuclei (CCN). In this talk, we will present the results from CAPMEX focusing on: (1) the characteristics of aerosol optical, physical and chemical properties at Gosan observatory, (2) aerosol solar heating calculated from the ground-based micro-pulse lidar and AERONET sun/sky radiometer synergy, and comparison with direct measurements from UAV, and (3) aerosol-cloud interactions in conjunction with measurements by satellites and Gosan observatory.

  18. Ion transport across the biological membrane by computational protein design

    NASA Astrophysics Data System (ADS)

    Grigoryan, Gevorg

    The cellular membrane is impermeable to most of the chemicals the cell needs to take in or discard to survive. Therefore, transporters-a class of transmembrane proteins tasked with shuttling cargo chemicals in and out of the cell-are essential to all cellular life. From existing crystal structures, we know transporters to be complex machines, exquisitely tuned for specificity and controllability. But how could membrane-bound life have evolved if it needed such complex machines to exist first? To shed light onto this question, we considered the task of designing a transporter de novo. As our guiding principle, we took the ``alternating-access model''-a conceptual mechanism stating that transporters work by rocking between two conformations, each exposing the cargo-binding site to either the intra- or the extra-cellular environment. A computational design framework was developed to encode an anti-parallel four-helix bundle that rocked between two alternative states to orchestrate the movement of Zn(II) ions across the membrane. The ensemble nature of both states was accounted for using a free energy-based approach, and sequences were chosen based on predicted formation of the targeted topology in the membrane and bi-stability. A single sequence was prepared experimentally and shown to function as a Zn(II) transporter in lipid vesicles. Further, transport was specific to Zn(II) ions and several control peptides supported the underlying design principles. This included a mutant designed to retain all properties but with reduced rocking, which showed greatly depressed transport ability. These results suggest that early transporters could have evolved in the context of simple topologies, to be later tuned by evolution for improved properties and controllability. Our study also serves as an important advance in computational protein design, showing the feasibility of designing functional membrane proteins and of tuning conformational landscapes for desired function

  19. Transport of Proteins Dissolved in Organic Solvents Across Biomimetic Membranes

    NASA Astrophysics Data System (ADS)

    Bromberg, Lev E.; Klibanov, Alexander M.

    1995-02-01

    Using lipid-impregnated porous cellulose membranes as biomimetic barriers, we tested the hypothesis that to afford effective transmembrane transfer of proteins and nucleic acids, the vehicle solvent should be able to dissolve both the biopolymers and the lipids. While the majority of solvents dissolve one or the other, ethanol and methanol were found to dissolve both, especially if the protein had been lyophilized from an aqueous solution of a pH remote from the protein's isoelectric point. A number of proteins, as well as RNA and DNA, dissolved in these alcohols readily crossed the lipidized membranes, whereas the same biopolymers placed in nondissolving solvents (e.g., hexane and ethyl acetate) or in those unable to dissolve lipids (e.g., water and dimethyl sulfoxide) exhibited little transmembrane transport. The solubility of biopolymers in ethanol and methanol was further enhanced by complexation with detergents and poly(ethylene glycol); significant protein and nucleic acid transport through the lipidized membranes was observed from these solvents but not from water.

  20. PPAR-α, a lipid-sensing transcription factor, regulates blood–brain barrier efflux transporter expression

    PubMed Central

    More, Vijay R; Campos, Christopher R; Evans, Rebecca A; Oliver, Keith D; Chan, Gary NY; Miller, David S

    2016-01-01

    Lipid sensor peroxisome proliferator-activated receptor alpha (PPAR-α) is the master regulator of lipid metabolism. Dietary release of endogenous free fatty acids, fibrates, and certain persistent environmental pollutants, e.g. perfluoroalkyl fire-fighting foam components, are peroxisome proliferator-activated receptor alpha ligands. Here, we define a role for peroxisome proliferator-activated receptor alpha in regulating the expression of three ATP-driven drug efflux transporters at the rat and mouse blood–brain barriers: P-glycoprotein (Abcb1), breast cancer resistance protein (Bcrp/Abcg2), and multidrug resistance-associated protein 2 (Mrp2/Abcc2). Exposing isolated rat brain capillaries to linoleic acid, clofibrate, or PKAs increased the transport activity and protein expression of the three ABC transporters. These effects were blocked by the PPAR-α antagonist, GW6471. Dosing rats with 20 mg/kg or 200 mg/kg of clofibrate decreased the brain accumulation of the P-glycoprotein substrate, verapamil, by 50% (in situ brain perfusion; effects blocked by GW6471) and increased P-glycoprotein expression and activity in capillaries ex vivo. Fasting C57Bl/6 wild-type mice for 24 h increased both serum lipids and brain capillary P-glycoprotein transport activity. Fasting did not alter P-glycoprotein activity in PPAR-α knockout mice. These results indicate that hyperlipidemia, lipid-lowering fibrates and exposure to certain fire-fighting foam components activate blood–brain barrier peroxisome proliferator-activated receptor alpha, increase drug efflux transporter expression and reduce drug delivery to the brain. PMID:27193034

  1. The short version of the Activities-specific Balance Confidence (ABC) scale: its validity, reliability, and relationship to balance impairment and falls in older adults.

    PubMed

    Schepens, Stacey; Goldberg, Allon; Wallace, Melissa

    2010-01-01

    A shortened version of the ABC 16-item scale (ABC-16), the ABC-6, has been proposed as an alternative balance confidence measure. We investigated whether the ABC-6 is a valid and reliable measure of balance confidence and examined its relationship to balance impairment and falls in older adults. Thirty-five community-dwelling older adults completed the ABC-16, including the 6 questions of the ABC-6. They also completed the following clinical balance tests: unipedal stance time (UST), functional reach (FR), Timed Up and Go (TUG), and maximum step length (MSL). Participants reported 12-month falls history. Balance confidence on the ABC-6 was significantly lower than on the ABC-16, however scores were highly correlated. Fallers reported lower balance confidence than non-fallers as measured by the ABC-6 scale, but confidence did not differ between the groups with the ABC-16. The ABC-6 significantly correlated with all balance tests assessed and number of falls. The ABC-16 significantly correlated with all balance tests assessed, but not with number of falls. Test-retest reliability for the ABC-16 and ABC-6 was good to excellent. The ABC-6 is a valid and reliable measure of balance confidence in community-dwelling older adults, and shows stronger relationships to falls than does the ABC-16. The ABC-6 may be a more useful balance confidence assessment tool than the ABC-16. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

  2. Knowing the ABCs: A Comparative Effectiveness Study of Two Methods of Diabetes Education

    PubMed Central

    Naik, Aanand D.; Teal, Cayla R.; Rodriguez, Elisa; Haidet, Paul

    2011-01-01

    Objective To test an active-learning, empowerment approach to teaching patients about the “diabetes ABCs” (hemoglobin A1C, systolic blood pressure, and low density lipoprotein cholesterol). Methods 84 (97%) diabetic patients who participated in a randomized effectiveness trial of two clinic-based group educational methods and completed a post-intervention assessment. The empowerment arm participated in a group session that incorporated two educational innovations (a conceptual metaphor to foster understanding, and team-based learning methods to foster active learning). The traditional diabetes education arm received a didactic group session focused on self-management and educational materials about the diabetes ABCs. Participants in both arms received individual review of their current ABC values. Results A questionnaire evaluated knowledge, understanding, and recall of the diabetes ABCs was administered three months after enrollment in the study. At three months, participants in the empowerment group demonstrated greater understanding of the diabetes ABCs (P<.0001), greater knowledge of their own values (P<.0001), and greater knowledge of guideline-derived target goals for the ABCs compared with participants in the traditional arm (P<.0001). Conclusion An active-learning, empowerment-based approach applied to diabetes education can lead to greater understanding and knowledge retention. Practice Implications An empowerment approach to education can facilitate informed, activated patients and increase performance of self-management behaviors. PMID:21300516

  3. Structural basis of transport function in major facilitator superfamily protein from Trichoderma harzianum.

    PubMed

    Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2017-02-01

    Trichothecenes are the sesquiterpenes secreted by Trichoderma spp. residing in the rhizosphere. These compounds have been reported to act as plant growth promoters and bio-control agents. The structural knowledge for the transporter proteins of their efflux remained limited. In this study, three-dimensional structure of Thmfs1 protein, a trichothecene transporter from Trichoderma harzianum, was homology modelled and further Molecular Dynamics (MD) simulations were used to decipher its mechanism. Fourteen transmembrane helices of Thmfs1 protein are observed contributing to an inward-open conformation. The transport channel and ligand binding sites in Thmfs1 are identified based on heuristic, iterative algorithm and structural alignment with homologous proteins. MD simulations were performed to reveal the differential structural behaviour occurring in the ligand free and ligand bound forms. We found that two discrete trichothecene binding sites are located on either side of the central transport tunnel running from the cytoplasmic side to the extracellular side across the Thmfs1 protein. Detailed analysis of the MD trajectories showed an alternative access mechanism between N and C-terminal domains contributing to its function. These results also demonstrate that the transport of trichodermin occurs via hopping mechanism in which the substrate molecule jumps from one binding site to another lining the transport tunnel. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Inferring epidemiological parameters from phylogenies using regression-ABC: A comparative study

    PubMed Central

    Gascuel, Olivier

    2017-01-01

    Inferring epidemiological parameters such as the R0 from time-scaled phylogenies is a timely challenge. Most current approaches rely on likelihood functions, which raise specific issues that range from computing these functions to finding their maxima numerically. Here, we present a new regression-based Approximate Bayesian Computation (ABC) approach, which we base on a large variety of summary statistics intended to capture the information contained in the phylogeny and its corresponding lineage-through-time plot. The regression step involves the Least Absolute Shrinkage and Selection Operator (LASSO) method, which is a robust machine learning technique. It allows us to readily deal with the large number of summary statistics, while avoiding resorting to Markov Chain Monte Carlo (MCMC) techniques. To compare our approach to existing ones, we simulated target trees under a variety of epidemiological models and settings, and inferred parameters of interest using the same priors. We found that, for large phylogenies, the accuracy of our regression-ABC is comparable to that of likelihood-based approaches involving birth-death processes implemented in BEAST2. Our approach even outperformed these when inferring the host population size with a Susceptible-Infected-Removed epidemiological model. It also clearly outperformed a recent kernel-ABC approach when assuming a Susceptible-Infected epidemiological model with two host types. Lastly, by re-analyzing data from the early stages of the recent Ebola epidemic in Sierra Leone, we showed that regression-ABC provides more realistic estimates for the duration parameters (latency and infectiousness) than the likelihood-based method. Overall, ABC based on a large variety of summary statistics and a regression method able to perform variable selection and avoid overfitting is a promising approach to analyze large phylogenies. PMID:28263987

  5. Thermally activated charge transport in microbial protein nanowires

    PubMed Central

    Lampa-Pastirk, Sanela; Veazey, Joshua P.; Walsh, Kathleen A.; Feliciano, Gustavo T.; Steidl, Rebecca J.; Tessmer, Stuart H.; Reguera, Gemma

    2016-01-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors. PMID:27009596

  6. Thermally activated charge transport in microbial protein nanowires

    NASA Astrophysics Data System (ADS)

    Lampa-Pastirk, Sanela; Veazey, Joshua P.; Walsh, Kathleen A.; Feliciano, Gustavo T.; Steidl, Rebecca J.; Tessmer, Stuart H.; Reguera, Gemma

    2016-03-01

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors.

  7. Thermally activated charge transport in microbial protein nanowires.

    PubMed

    Lampa-Pastirk, Sanela; Veazey, Joshua P; Walsh, Kathleen A; Feliciano, Gustavo T; Steidl, Rebecca J; Tessmer, Stuart H; Reguera, Gemma

    2016-03-24

    The bacterium Geobacter sulfurreducens requires the expression of conductive protein filaments or pili to respire extracellular electron acceptors such as iron oxides and uranium and to wire electroactive biofilms, but the contribution of the protein fiber to charge transport has remained elusive. Here we demonstrate efficient long-range charge transport along individual pili purified free of metal and redox organic cofactors at rates high enough to satisfy the respiratory rates of the cell. Carrier characteristics were within the orders reported for organic semiconductors (mobility) and inorganic nanowires (concentration), and resistivity was within the lower ranges reported for moderately doped silicon nanowires. However, the pilus conductance and the carrier mobility decreased when one of the tyrosines of the predicted axial multistep hopping path was replaced with an alanine. Furthermore, low temperature scanning tunneling microscopy demonstrated the thermal dependence of the differential conductance at the low voltages that operate in biological systems. The results thus provide evidence for thermally activated multistep hopping as the mechanism that allows Geobacter pili to function as protein nanowires between the cell and extracellular electron acceptors.

  8. IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites.

    PubMed

    Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; Ratner, Anna; Palaniappan, Krishna; Szeto, Ernest; Huang, Jinghua; Reddy, T B K; Cimermančič, Peter; Fischbach, Michael A; Ivanova, Natalia N; Markowitz, Victor M; Kyrpides, Nikos C; Pati, Amrita

    2015-07-14

    In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of "big" genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in Alphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to

  9. Can the three pore model correctly describe peritoneal transport of protein?

    PubMed

    Waniewski, Jacek; Poleszczuk, Jan; Antosiewicz, Stefan; Baczynński, Daniel; Gałach, Magda; Pietribiasi, Mauro; Wanńkowicz, Zofia

    2014-01-01

    The three pore model (3PM) includes large pores for the description of protein leak to the peritoneal cavity during peritoneal dialysis. However, the reliability of this description has been not fully tested against clinical data yet. Peritoneal transport parameters were estimated using 3PM, extended 3p model (with estimation of fraction of large pores, ext3PM), ext3PM with modified size of pores and proteins (mext3PM), and simplified two pore (2PM, small and ultrasmall pores) models for 32 patients on peritoneal dialysis investigated using the sequential peritoneal equilibration test (consecutive peritoneal equilibration test [PET]: glucose 2.27%, 4 h, and miniPET: glucose 3.86%, 1 h). Urea, creatinine, glucose, sodium, phosphate, albumin, and IgM concentrations were measured in dialysis fluid and plasma. Ext3PM and mext3PM, with large pore fraction of about 0.14, provided a good description of fluid and small solute kinetics, but their predictions for albumin transport were less accurate. Two pore model precisely described the data on fluid and small solute transport. The 3p models could not describe the diffusive-convective transport of albumin as precisely as the transport of fluid, small solutes, and IgM. The 2p model (not applicable for proteins) was an efficient tool for modeling fluid and small solute transport.

  10. Computer Simulation of the Virulome of Bacillus anthracis Using Proteomics

    DTIC Science & Technology

    2006-07-31

    hypothetical protein gi|47526566 spermidine /putrescine ABC transporter, spermidine /putrescine-binding protein gi|47526625 oligoendopeptidase F, putative gi...glutamyl-trna(gln) amidotransferase, a subunit x gi|50196927 aspartate aminotransferase x gi|50196970 spermidine synthase x

  11. Mechanisms of Drug Resistance in Veterinary Oncology—A Review with an Emphasis on Canine Lymphoma

    PubMed Central

    Zandvliet, Maurice; Teske, Erik

    2015-01-01

    Drug resistance (DR) is the major limiting factor in the successful treatment of systemic neoplasia with cytotoxic chemotherapy. DR can be either intrinsic or acquired, and although the development and clinical implications are different, the underlying mechanisms are likely to be similar. Most causes for DR are pharmacodynamic in nature, result from adaptations within the tumor cell and include reduced drug uptake, increased drug efflux, changes in drug metabolism or drug target, increased capacity to repair drug-induced DNA damage or increased resistance to apoptosis. The role of active drug efflux transporters, and those of the ABC-transporter family in particular, have been studied extensively in human oncology and to a lesser extent in veterinary medicine. Methods reported to assess ABC-transporter status include detection of the actual protein (Western blot, immunohistochemistry), mRNA or ABC-transporter function. The three major ABC-transporters associated with DR in human oncology are ABCB1 or P-gp, ABCC1 or MRP1, and ABCG2 or BCRP, and have been demonstrated in canine cell lines, healthy dogs and dogs with cancer. Although this supports a causative role for these ABC-transporters in DR cytotoxic agents in the dog, the relative contribution to the clinical phenotype of DR in canine cancer remains an area of debate and requires further prospective studies. PMID:29061939

  12. Comparative vesicle proteomics reveals selective regulation of protein expression in chestnut blight fungus by a hypovirus.

    PubMed

    Wang, Jinzi; Wang, Fangzhen; Feng, Youjun; Mi, Ke; Chen, Qi; Shang, Jinjie; Chen, Baoshan

    2013-01-14

    The chestnut blight fungus (Cryphonectria parasitica) and hypovirus constitute a model system to study fungal pathogenesis and mycovirus-host interaction. Knowledge in this field has been gained largely from investigations at gene transcription level so far. Here we report a systematic analysis of the vesicle proteins of the host fungus with/without hypovirus infection. Thirty-three differentially expressed protein spots were identified in the purified vesicle protein samples by two-dimensional electrophoresis and mass spectrometry. Down-regulated proteins were mostly cargo proteins involved in primary metabolism and energy generation and up-regulated proteins were mostly vesicle associated proteins and ABC transporter. A virus-encoded protein p48 was found to have four forms with different molecular mass in vesicles from the virus-infected strain. While a few of the randomly selected differentially expressed proteins were in accordance with their transcription profiles, majority were not in agreement with their mRNA accumulation patterns, suggesting that an extensive post-transcriptional regulation may have occurred in the host fungus upon a hypovirus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. Inferring population history with DIY ABC: a user-friendly approach to approximate Bayesian computation

    PubMed Central

    Cornuet, Jean-Marie; Santos, Filipe; Beaumont, Mark A.; Robert, Christian P.; Marin, Jean-Michel; Balding, David J.; Guillemaud, Thomas; Estoup, Arnaud

    2008-01-01

    Summary: Genetic data obtained on population samples convey information about their evolutionary history. Inference methods can extract part of this information but they require sophisticated statistical techniques that have been made available to the biologist community (through computer programs) only for simple and standard situations typically involving a small number of samples. We propose here a computer program (DIY ABC) for inference based on approximate Bayesian computation (ABC), in which scenarios can be customized by the user to fit many complex situations involving any number of populations and samples. Such scenarios involve any combination of population divergences, admixtures and population size changes. DIY ABC can be used to compare competing scenarios, estimate parameters for one or more scenarios and compute bias and precision measures for a given scenario and known values of parameters (the current version applies to unlinked microsatellite data). This article describes key methods used in the program and provides its main features. The analysis of one simulated and one real dataset, both with complex evolutionary scenarios, illustrates the main possibilities of DIY ABC. Availability: The software DIY ABC is freely available at http://www.montpellier.inra.fr/CBGP/diyabc. Contact: j.cornuet@imperial.ac.uk Supplementary information: Supplementary data are also available at http://www.montpellier.inra.fr/CBGP/diyabc PMID:18842597

  14. Active and passive transport of drugs in the human placenta.

    PubMed

    Włoch, Stanisław; Pałasz, Artur; Kamiński, Marcin

    2009-10-01

    The human placenta, characterized by the processes of passive transport and facilitated diffusion, contains numerous active transport proteins, usually located in the microvilli of the syncytiotrophoblast or in the endothelium of the capillaries of the villi. These proteins use either the energy from ATP hydrolysis or other mechanisms resulting, among others, from the formation of the maternofetal ion gradient, which facilitates the transfer of various endogenous substances or xenobiotics across the body membranes. The proteins either trigger the efflux of these substances from the fetal tissues via the placenta into the maternal bloodstream, or conversely they accumulate them in the fetal tissues. Both the placenta and the fetus are equipped with independent systems of enzymes of 1st and 2nd phase of substrate metabolism, such as CYP450, glucuronyltransferase or sulphatase. An active therapy with a wide range of drugs, often at high toxicity levels, either shortly before or during pregnancy, has naturally posed a question concerning the degree of impermeability of the placental barrier and how effectively it can be crossed, including any possible negative embryotoxic or teratogenic consequences. Such hazards seem to be quite real, as many drugs are substrates for ABC transporters. Also the placenta itself, including its structure, is subject to vast transformations during pregnancy which may be observed as the thinning of the barrier separating the maternal blood from the fetal one, from 20-30 microm in the first trimester of gestation down to 2-4 microm in the third trimester of gestation.

  15. Phosphatidylglycerol directs binding and inhibitory action of EIIAGlc protein on the maltose transporter.

    PubMed

    Bao, Huan; Duong, Franck

    2013-08-16

    The signal-transducing protein EIIA(Glc) belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIA(Glc) is a negative regulator for several permeases, including the maltose transporter MalFGK2. How EIIA(Glc) is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIA(Glc) are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIA(Glc) binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIA(Glc) does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.

  16. ATP-binding cassette exporters: structure and mechanism with a focus on P-glycoprotein and MRP1.

    PubMed

    Arana, Maite Rocío; Altenberg, Guillermo

    2017-10-12

    The majority of proteins that belong to the ATP-binding cassette (ABC) superfamily are transporters that mediate the efflux of substrates from cells. These exporters include multidrug resistance proteins of the ABCB and ABCC subfamilies, such as P-glycoprotein (Pgp) and MRP1, respectively. These proteins are not only involved in the resistance of cancer to cytotoxic agents, but also in the protection from endo and xenobiotics, and the determination of drug pharmacokinetics, as well as in the pathophysiology of a variety of disorders. Here, we present a review of the information available on ABC exporters, with a focus on Pgp, MRP1 and related proteins. We describe tissue localization and function of these transporters in health and disease, and discuss the mechanisms of substrate transport. We also correlate recent structural information with the function of the exporters, and discuss details of their molecular mechanism with a focus on the nucleotide-binding domains. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Simulation-based estimation of mean and standard deviation for meta-analysis via Approximate Bayesian Computation (ABC).

    PubMed

    Kwon, Deukwoo; Reis, Isildinha M

    2015-08-12

    When conducting a meta-analysis of a continuous outcome, estimated means and standard deviations from the selected studies are required in order to obtain an overall estimate of the mean effect and its confidence interval. If these quantities are not directly reported in the publications, they must be estimated from other reported summary statistics, such as the median, the minimum, the maximum, and quartiles. We propose a simulation-based estimation approach using the Approximate Bayesian Computation (ABC) technique for estimating mean and standard deviation based on various sets of summary statistics found in published studies. We conduct a simulation study to compare the proposed ABC method with the existing methods of Hozo et al. (2005), Bland (2015), and Wan et al. (2014). In the estimation of the standard deviation, our ABC method performs better than the other methods when data are generated from skewed or heavy-tailed distributions. The corresponding average relative error (ARE) approaches zero as sample size increases. In data generated from the normal distribution, our ABC performs well. However, the Wan et al. method is best for estimating standard deviation under normal distribution. In the estimation of the mean, our ABC method is best regardless of assumed distribution. ABC is a flexible method for estimating the study-specific mean and standard deviation for meta-analysis, especially with underlying skewed or heavy-tailed distributions. The ABC method can be applied using other reported summary statistics such as the posterior mean and 95 % credible interval when Bayesian analysis has been employed.

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

    PubMed Central

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

    2014-01-01

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

  19. Validation of membrane vesicle-based breast cancer resistance protein and multidrug resistance protein 2 assays to assess drug transport and the potential for drug-drug interaction to support regulatory submissions.

    PubMed

    Elsby, Robert; Smith, Veronica; Fox, Lisa; Stresser, David; Butters, Caroline; Sharma, Pradeep; Surry, Dominic D

    2011-09-01

    Breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) can play a role in the absorption, distribution, metabolism, and excretion of drugs, impacting on the potential for drug-drug interactions. This study has characterized insect cell- and mammalian cell-derived ABC-transporter-expressing membrane vesicle test systems and validated methodologies for evaluation of candidate drugs as substrates or inhibitors of BCRP or MRP2. Concentration-dependent uptake of BCRP ([³H]oestrone 3-sulfate, [³H]methotrexate, [³H]rosuvastatin) and MRP2 ([³H]oestradiol 17β-glucuronide, [³H]pravastatin, carboxydichlorofluorescein) substrates, and inhibitory potencies (IC₅₀) of BCRP (sulfasalazine, novobiocin, fumitremorgin C) and MRP2 (benzbromarone, MK-571, terfenadine) inhibitors were determined. The apparent K(m) for probes [³H]oestrone 3-sulfate and [³H]oestradiol 17β-glucuronide was determined in insect cell vesicles to be 7.4 ± 1.7 and 105 ± 8.3 µM, respectively. All other substrates exhibited significant uptake ratios. Positive control inhibitors sulfasalazine and benzbromarone gave IC₅₀ values of 0.74 ± 0.18 and 36 ± 6.1 µM, respectively. All other inhibitors exhibited concentration-dependent inhibition. There was no significant difference in parameters generated between test systems. On the basis of the validation results, acceptance criteria to identify substrates/inhibitors of BCRP and MRP2 were determined for insect cell vesicles. The approach builds on earlier validations to support drug registration and extends from those cell-based systems to encompass assay formats using membrane vesicles.

  20. New Enhanced Artificial Bee Colony (JA-ABC5) Algorithm with Application for Reactive Power Optimization

    PubMed Central

    2015-01-01

    The standard artificial bee colony (ABC) algorithm involves exploration and exploitation processes which need to be balanced for enhanced performance. This paper proposes a new modified ABC algorithm named JA-ABC5 to enhance convergence speed and improve the ability to reach the global optimum by balancing exploration and exploitation processes. New stages have been proposed at the earlier stages of the algorithm to increase the exploitation process. Besides that, modified mutation equations have also been introduced in the employed and onlooker-bees phases to balance the two processes. The performance of JA-ABC5 has been analyzed on 27 commonly used benchmark functions and tested to optimize the reactive power optimization problem. The performance results have clearly shown that the newly proposed algorithm has outperformed other compared algorithms in terms of convergence speed and global optimum achievement. PMID:25879054

  1. New enhanced artificial bee colony (JA-ABC5) algorithm with application for reactive power optimization.

    PubMed

    Sulaiman, Noorazliza; Mohamad-Saleh, Junita; Abro, Abdul Ghani

    2015-01-01

    The standard artificial bee colony (ABC) algorithm involves exploration and exploitation processes which need to be balanced for enhanced performance. This paper proposes a new modified ABC algorithm named JA-ABC5 to enhance convergence speed and improve the ability to reach the global optimum by balancing exploration and exploitation processes. New stages have been proposed at the earlier stages of the algorithm to increase the exploitation process. Besides that, modified mutation equations have also been introduced in the employed and onlooker-bees phases to balance the two processes. The performance of JA-ABC5 has been analyzed on 27 commonly used benchmark functions and tested to optimize the reactive power optimization problem. The performance results have clearly shown that the newly proposed algorithm has outperformed other compared algorithms in terms of convergence speed and global optimum achievement.

  2. Expression and putative role of mitochondrial transport proteins in cancer.

    PubMed

    Lytovchenko, Oleksandr; Kunji, Edmund R S

    2017-08-01

    Cancer cells undergo major changes in energy and biosynthetic metabolism. One of them is the Warburg effect, in which pyruvate is used for fermentation rather for oxidative phosphorylation. Another major one is their increased reliance on glutamine, which helps to replenish the pool of Krebs cycle metabolites used for other purposes, such as amino acid or lipid biosynthesis. Mitochondria are central to these alterations, as the biochemical pathways linking these processes run through these organelles. Two membranes, an outer and inner membrane, surround mitochondria, the latter being impermeable to most organic compounds. Therefore, a large number of transport proteins are needed to link the biochemical pathways of the cytosol and mitochondrial matrix. Since the transport steps are relatively slow, it is expected that many of these transport steps are altered when cells become cancerous. In this review, changes in expression and regulation of these transport proteins are discussed as well as the role of the transported substrates. This article is part of a Special Issue entitled Mitochondria in Cancer, edited by Giuseppe Gasparre, Rodrigue Rossignol and Pierre Sonveaux. Copyright © 2017. Published by Elsevier B.V.

  3. Molecular mechanism of ligand recognition by membrane transport protein, Mhp1

    PubMed Central

    Simmons, Katie J; Jackson, Scott M; Brueckner, Florian; Patching, Simon G; Beckstein, Oliver; Ivanova, Ekaterina; Geng, Tian; Weyand, Simone; Drew, David; Lanigan, Joseph; Sharples, David J; Sansom, Mark SP; Iwata, So; Fishwick, Colin WG; Johnson, A Peter; Cameron, Alexander D; Henderson, Peter JF

    2014-01-01

    The hydantoin transporter Mhp1 is a sodium-coupled secondary active transport protein of the nucleobase-cation-symport family and a member of the widespread 5-helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site-directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5-substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5-substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5-(2-naphthylmethyl)-L-hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1. PMID:24952894

  4. Comparison between the Movement ABC-2 and the Zurich Neuromotor Assessment in Preschool Children.

    PubMed

    Kakebeeke, Tanja H; Knaier, Elisa; Köchli, Sabrina; Chaouch, Aziz; Rousson, Valentin; Kriemler, Susi; Jenni, Oskar G

    2016-12-01

    An established test instrument for the assessment of motor performance in children between 3 and 16 years is the Movement Assessment Battery for Children - Second Edition (M-ABC-2). The Zurich Neuromotor Assessment (ZNA) is also widely used for the evaluation of children's motor performance but has not been compared with the M-ABC-2 for children below five years for the purpose of convergent validity. Forty-seven children (26 boys, 21 girls) between three and five years of age were assessed using the M-ABC-2 and the ZNA3-5. Rank correlations between scores of different test components were calculated. Only low-to-moderate correlations were observed when separate components of these tests were compared (.31 to .68, p < .05), especially when involving the associated movements from the ZNA3-5 (-.05 to -.13, p > .05). However, the correlation between summary scores of the two tests was .77 (p < .001), and it increased to .84 when associated movements were excluded, which was comparable in magnitude to the test-retest reliability of the M-ABC-2, supporting convergent validity between the two tests. Although the ZNA3-5 and M-ABC-2 measure different aspects of motor behavior, the two instruments may thus measure essentially the same construct. © The Author(s) 2016.

  5. Protein folding optimization based on 3D off-lattice model via an improved artificial bee colony algorithm.

    PubMed

    Li, Bai; Lin, Mu; Liu, Qiao; Li, Ya; Zhou, Changjun

    2015-10-01

    Protein folding is a fundamental topic in molecular biology. Conventional experimental techniques for protein structure identification or protein folding recognition require strict laboratory requirements and heavy operating burdens, which have largely limited their applications. Alternatively, computer-aided techniques have been developed to optimize protein structures or to predict the protein folding process. In this paper, we utilize a 3D off-lattice model to describe the original protein folding scheme as a simplified energy-optimal numerical problem, where all types of amino acid residues are binarized into hydrophobic and hydrophilic ones. We apply a balance-evolution artificial bee colony (BE-ABC) algorithm as the minimization solver, which is featured by the adaptive adjustment of search intensity to cater for the varying needs during the entire optimization process. In this work, we establish a benchmark case set with 13 real protein sequences from the Protein Data Bank database and evaluate the convergence performance of BE-ABC algorithm through strict comparisons with several state-of-the-art ABC variants in short-term numerical experiments. Besides that, our obtained best-so-far protein structures are compared to the ones in comprehensive previous literature. This study also provides preliminary insights into how artificial intelligence techniques can be applied to reveal the dynamics of protein folding. Graphical Abstract Protein folding optimization using 3D off-lattice model and advanced optimization techniques.

  6. RECOVERY ACT - Thylakoid Assembly and Folded Protein Transport by the Tat Pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dabney-Smith, Carole

    Assembly of functional photosystems complete with necessary intrinsic (membrane-bound) and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. Our research focuses on one of those pathways, a unique and essential protein transport pathway found in the chloroplasts of plants, bacteria, and some archaebacteria, the Twin arginine translocation (Tat) system. The chloroplast Tat (cpTat) system is thought to be responsible for the proper location of ~50% of thylakoid lumen proteins, several of which are necessary for proper photosystem assembly, maintenance, and function. Specifically, cpTat systems are unique because they transport fully folded and assembledmore » proteins across ion tight membranes using only three membrane components, Tha4, Hcf106, and cpTatC, and the protonmotive force generated by photosynthesis. Despite the importance of the cpTat system in plants, the mechanism of transport of a folded precursor is not well known. Our long-term goal is to investigate the role protein transport systems have on organelle biogenesis, particularly the assembly of membrane protein complexes in thylakoids of chloroplasts. The objective of this proposal is to correlate structural changes in the membrane-bound cpTat component, Tha4, to the mechanism of translocation of folded-precursor substrates across the membrane bilayer by using a cysteine accessibility and crosslinking approach. Our central hypothesis is that the precursor passes through a proteinaceous pore of assembled Tha4 protomers that have undergone a conformational or topological change in response to transport. This research is predicated upon the observations that Tha4 exists in molar excess in the membrane relative to the other cpTat components; its regulated assembly to the precursor-bound receptor; and our data showing oligomerization of Tha4 into very large complexes in response to transport. Our rationale for these studies is that understanding cp

  7. Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

    PubMed Central

    Walsh, Dustin R.; Nolin, Thomas D.

    2015-01-01

    Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

  8. The actin cytoskeleton may control the polar distribution of an auxin transport protein

    NASA Technical Reports Server (NTRS)

    Muday, G. K.; Hu, S.; Brady, S. R.; Davies, E. (Principal Investigator)

    2000-01-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  9. The actin cytoskeleton may control the polar distribution of an auxin transport protein.

    PubMed

    Muday, G K; Hu, S; Brady, S R

    2000-06-01

    The gravitropic bending of plants has long been linked to the changes in the transport of the plant hormone auxin. To understand the mechanism by which gravity alters auxin movement, it is critical to know how polar auxin transport is initially established. In shoots, polar auxin transport is basipetal (i.e., from the shoot apex toward the base). It is driven by the basal localization of the auxin efflux carrier complex. One mechanism for localizing this efflux carrier complex to the basal membrane may be through attachment to the actin cytoskeleton. The efflux carrier protein complex is believed to consist of several polypeptides, including a regulatory subunit that binds auxin transport inhibitors, such as naphthylphthalamic acid (NPA). Several lines of experimentation have been used to determine if the NPA binding protein interacts with actin filaments. The NPA binding protein has been shown to partition with the actin cytoskeleton during detergent extraction. Agents that specifically alter the polymerization state of the actin cytoskeleton change the amount of NPA binding protein and actin recovered in these cytoskeletal pellets. Actin-affinity columns were prepared with polymers of actin purified from zucchini hypocotyl tissue. NPA binding activity was eluted in a single peak from the actin filament column. Cytochalasin D, which fragments the actin cytoskeleton, was shown to reduce polar auxin transport in zucchini hypocotyls. The interaction of the NPA binding protein with the actin cytoskeleton may localize it in one plane of the plasma membrane, and thereby control the polarity of auxin transport.

  10. The Role of Activity Based Costing (ABC) in Educational Support Services: A White Paper.

    ERIC Educational Resources Information Center

    Edds, Daniel B.

    Many front-line managers who are assuming more financial responsibility for their organizations find traditional cost accounting inadequate for their needs and are turning to Activity Based Costing (ABC). ABC is not a financial reporting system to serve the needs of regulatory agencies, but a tool that tracks costs from the general ledger…

  11. Sublingual immunization with the phosphate-binding-protein (PstS) reduces oral colonization by Streptococcus mutans.

    PubMed

    Ferreira, E L; Batista, M T; Cavalcante, R C M; Pegos, V R; Passos, H M; Silva, D A; Balan, A; Ferreira, L C S; Ferreira, R C C

    2016-10-01

    Bacterial ATP-binding cassette (ABC) transporters play a crucial role in the physiology and pathogenicity of different bacterial species. Components of ABC transporters have also been tested as target antigens for the development of vaccines against different bacterial species, such as those belonging to the Streptococcus genus. Streptococcus mutans is the etiological agent of dental caries, and previous studies have demonstrated that deletion of the gene encoding PstS, the substrate-binding component of the phosphate uptake system (Pst), reduced the adherence of the bacteria to abiotic surfaces. In the current study, we generated a recombinant form of the S. mutans PstS protein (rPstS) with preserved structural features, and we evaluated the induction of antibody responses in mice after sublingual mucosal immunization with a formulation containing the recombinant protein and an adjuvant derived from the heat-labile toxin from enterotoxigenic Escherichia coli strains. Mice immunized with rPstS exhibited systemic and secreted antibody responses, measured by the number of immunoglobulin A-secreting cells in draining lymph nodes. Serum antibodies raised in mice immunized with rPstS interfered with the adhesion of bacteria to the oral cavity of naive mice challenged with S. mutans. Similarly, mice actively immunized with rPstS were partially protected from oral colonization after challenge with the S. mutans NG8 strain. Therefore, our results indicate that S. mutans PstS is a potential target antigen capable of inducing specific and protective antibody responses after sublingual administration. Overall, these observations raise interesting perspectives for the development of vaccines to prevent dental caries. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus.

    PubMed

    Alves, Ingrid R; Lima-Noronha, Marco A; Silva, Larissa G; Fernández-Silva, Frank S; Freitas, Aline Luiza D; Marques, Marilis V; Galhardo, Rodrigo S

    2017-11-01

    imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. IMG-ABC. A knowledge base to fuel discovery of biosynthetic gene clusters and novel secondary metabolites

    DOE PAGES

    Hadjithomas, Michalis; Chen, I-Min Amy; Chu, Ken; ...

    2015-07-14

    In the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG’s comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve asmore » the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC’s focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time in lphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules. IMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG’s extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC

  14. Measurement of mitochondrial Ca2+ transport mediated by three transport proteins: VDAC1, the Na+/Ca2+ exchanger, and the Ca2+ uniporter.

    PubMed

    Ben-Hail, Danya; Palty, Raz; Shoshan-Barmatz, Varda

    2014-02-01

    Ca(2+) is a ubiquitous cellular signal, with changes in intracellular Ca(2+) concentration not only stimulating a number of intercellular events but also triggering cell death pathways, including apoptosis. Mitochondrial Ca(2+) uptake and release play pivotal roles in cellular physiology by regulating intracellular Ca(2+) signaling, energy metabolism and cell death. Ca(2+) transport across the inner and outer mitochondrial membranes is mediated by several proteins, including channels, antiporters, and a uniporter. In this article, we present the background to several methods now established for assaying mitochondrial Ca(2+) transport activity across both mitochondrial membranes. The first of these is Ca(2+) transport mediated by the outer mitochondrial protein, the voltage-dependent anion-selective channel protein 1 (VDAC1, also known as porin 1), both as a purified protein reconstituted into a planar lipid bilayer (PLB) or into liposomes and as a mitochondrial membrane-embedded protein. The second method involves isolated mitochondria for assaying the activity of an inner mitochondrial membrane transport protein, the mitochondrial Ca(2+) uniporter (MCU) that transports Ca(2+) and is powered by the steep mitochondrial membrane potential. In the event of Ca(2+) overload, this leads to opening of the mitochondrial permeability transition pore (MPTP) and cell death. The third method describes how Na(+)-dependent mitochondrial Ca(2+) efflux mediated by mitochondrial NCLX, a member of the Na(+)/Ca(2+) exchanger superfamily, can be assayed in digitonin-permeabilized HEK-293 cells. The Ca(2+)-transport assays can be performed under various conditions and in combination with inhibitors, allowing detailed characterization of the transport activity of interest.

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

    PubMed Central

    Hedditch, Ellen L.; Gao, Bo; Russell, Amanda J.; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E.; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T.; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W.; Ekici, Arif B.; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K.; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P.; Berchuck, Andrew; Goode, Ellen; Bowtell, David D.; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D.; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J.

    2014-01-01

    Background ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. Methods The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA–mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan–Meier analysis and log-rank tests. All statistical tests were two-sided. Results Associations with outcome were observed with ABC transporters of the “A” subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e−6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Conclusions Expression of ABCA transporters was associated with poor

  16. ABCA transporter gene expression and poor outcome in epithelial ovarian cancer.

    PubMed

    Hedditch, Ellen L; Gao, Bo; Russell, Amanda J; Lu, Yi; Emmanuel, Catherine; Beesley, Jonathan; Johnatty, Sharon E; Chen, Xiaoqing; Harnett, Paul; George, Joshy; Williams, Rebekka T; Flemming, Claudia; Lambrechts, Diether; Despierre, Evelyn; Lambrechts, Sandrina; Vergote, Ignace; Karlan, Beth; Lester, Jenny; Orsulic, Sandra; Walsh, Christine; Fasching, Peter; Beckmann, Matthias W; Ekici, Arif B; Hein, Alexander; Matsuo, Keitaro; Hosono, Satoyo; Nakanishi, Toru; Yatabe, Yasushi; Pejovic, Tanja; Bean, Yukie; Heitz, Florian; Harter, Philipp; du Bois, Andreas; Schwaab, Ira; Hogdall, Estrid; Kjaer, Susan K; Jensen, Allan; Hogdall, Claus; Lundvall, Lene; Engelholm, Svend Aage; Brown, Bob; Flanagan, James; Metcalf, Michelle D; Siddiqui, Nadeem; Sellers, Thomas; Fridley, Brooke; Cunningham, Julie; Schildkraut, Joellen; Iversen, Ed; Weber, Rachel P; Berchuck, Andrew; Goode, Ellen; Bowtell, David D; Chenevix-Trench, Georgia; deFazio, Anna; Norris, Murray D; MacGregor, Stuart; Haber, Michelle; Henderson, Michelle J

    2014-07-01

    ATP-binding cassette (ABC) transporters play various roles in cancer biology and drug resistance, but their association with outcomes in serous epithelial ovarian cancer (EOC) is unknown. The relationship between clinical outcomes and ABC transporter gene expression in two independent cohorts of high-grade serous EOC tumors was assessed with real-time quantitative polymerase chain reaction, analysis of expression microarray data, and immunohistochemistry. Associations between clinical outcomes and ABCA transporter gene single nucleotide polymorphisms were tested in a genome-wide association study. Impact of short interfering RNA-mediated gene suppression was determined by colony forming and migration assays. Association with survival was assessed with Kaplan-Meier analysis and log-rank tests. All statistical tests were two-sided. Associations with outcome were observed with ABC transporters of the "A" subfamily, but not with multidrug transporters. High-level expression of ABCA1, ABCA6, ABCA8, and ABCA9 in primary tumors was statistically significantly associated with reduced survival in serous ovarian cancer patients. Low levels of ABCA5 and the C-allele of rs536009 were associated with shorter overall survival (hazard ratio for death = 1.50; 95% confidence interval [CI] =1.26 to 1.79; P = 6.5e-6). The combined expression pattern of ABCA1, ABCA5, and either ABCA8 or ABCA9 was associated with particularly poor outcome (mean overall survival in group with adverse ABCA1, ABCA5 and ABCA9 gene expression = 33.2 months, 95% CI = 26.4 to 40.1; vs 55.3 months in the group with favorable ABCA gene expression, 95% CI = 49.8 to 60.8; P = .001), independently of tumor stage or surgical debulking status. Suppression of cholesterol transporter ABCA1 inhibited ovarian cancer cell growth and migration in vitro, and statin treatment reduced ovarian cancer cell migration. Expression of ABCA transporters was associated with poor outcome in serous ovarian cancer, implicating lipid

  17. Genome Sequence Alterations Detected upon Passage of Burkholderia mallei ATCC 23344 in Culture and in Mammalian Hosts

    DTIC Science & Technology

    2006-09-05

    NA NA no yes BMAA1128 ABC Transporter 3 GGGAAACGCGAAAC 6 5 yes no BMAA1873 Hypothetical protein 4 No (-C) NA NA no yes BMAA1868 Aconitate hydratase 5...no yes BMAA1868 Aconitate hydratase 3 GTGCTGTC 21 22 no yes BMAA0375 Transcriptional regulator Human Blood 1 TTGGCGC 111 109 no no BMAA1866 Conserved...NA NA no yes BMAA1128 ABC transporter 5 No (-C) NA NA no yes BMAA1868 Aconitate hydratase NA: Not applicable.Page 3 of 11 (page number not for

  18. Identification of a Third Osmoprotectant Transport System, the OsmU System, in Salmonella enterica

    PubMed Central

    Frossard, Stephen M.; Khan, Aftab A.; Warrick, Eric C.; Gately, Jonathan M.; Hanson, Andrew D.; Oldham, Michael L.; Sanders, David Avram

    2012-01-01

    The growth of Salmonella enterica serovar Typhimurium mutants lacking the ProP and ProU osmoprotectant transport systems is stimulated by glycine betaine in high-osmolarity media, suggesting that this organism has an additional osmoprotectant transport system. Bioinformatic analysis revealed that the genome of this organism contains a hitherto-unidentified operon, designated osmU, consisting of four genes whose products show high similarity to ABC-type transport systems for osmoprotectants in other bacteria. The osmU operon was inactivated by a site-directed deletion, which abolished the ability of glycine betaine to alleviate the inhibitory effect of high osmolarity and eliminated the accumulation of [14C]glycine betaine and [14C]choline-O-sulfate in high-osmolarity media in a strain lacking the ProP and ProU systems. Although the OsmU system can take up glycine betaine and choline-O-sulfate, these two osmoprotectants are recognized at low affinity by this transporter, suggesting that there might be more efficient substrates that are yet to be discovered. The transcription of osmU is induced 23-fold by osmotic stress (0.3 M NaCl). The osmU operon is present in the genomes of a number of Enterobacteriaceae, and orthologs of the OsmU system can be recognized in a wide variety of Bacteria and Archaea. The structure of the periplasmic binding protein component of this transporter, OsmX, was modeled on the crystallographic structure of the glycine betaine-binding protein ProX of Archaeoglobus fulgidus; the resultant model indicated that the amino acids that constitute substrate-binding site, including an “aromatic cage” made up of four tyrosines, are conserved between these two proteins. PMID:22609924

  19. Transport of soluble proteins through the Golgi occurs by diffusion via continuities across cisternae

    PubMed Central

    Beznoussenko, Galina V; Parashuraman, Seetharaman; Rizzo, Riccardo; Polishchuk, Roman; Martella, Oliviano; Di Giandomenico, Daniele; Fusella, Aurora; Spaar, Alexander; Sallese, Michele; Capestrano, Maria Grazia; Pavelka, Margit; Vos, Matthijn R; Rikers, Yuri GM; Helms, Volkhard; Mironov, Alexandre A; Luini, Alberto

    2014-01-01

    The mechanism of transport through the Golgi complex is not completely understood, insofar as no single transport mechanism appears to account for all of the observations. Here, we compare the transport of soluble secretory proteins (albumin and α1-antitrypsin) with that of supramolecular cargoes (e.g., procollagen) that are proposed to traverse the Golgi by compartment progression–maturation. We show that these soluble proteins traverse the Golgi much faster than procollagen while moving through the same stack. Moreover, we present kinetic and morphological observations that indicate that albumin transport occurs by diffusion via intercisternal continuities. These data provide evidence for a transport mechanism that applies to a major class of secretory proteins and indicate the co-existence of multiple intra-Golgi trafficking modes. DOI: http://dx.doi.org/10.7554/eLife.02009.001 PMID:24867214

  20. Calculus ABCs: A Gateway for Freshman Calculus

    ERIC Educational Resources Information Center

    Fulton, Scott R.

    2003-01-01

    This paper describes a gateway testing program designed to ensure that students acquire basic skills in freshman calculus. Students must demonstrate they have mastered standards for "Absolutely Basic Competency"--the Calculus ABCs--in order to pass the course with a grade of C or better. We describe the background, standards, and testing program.…