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Sample records for abc membrane transporters

  1. Control of Plasma Membrane Permeability by ABC Transporters.

    PubMed

    Khakhina, Svetlana; Johnson, Soraya S; Manoharlal, Raman; Russo, Sarah B; Blugeon, Corinne; Lemoine, Sophie; Sunshine, Anna B; Dunham, Maitreya J; Cowart, L Ashley; Devaux, Frédéric; Moye-Rowley, W Scott

    2015-05-01

    ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2. PMID:25724885

  2. ABC transporters: bacterial exporters.

    PubMed Central

    Fath, M J; Kolter, R

    1993-01-01

    The ABC transporters (also called traffic ATPases) make up a large superfamily of proteins which share a common function and a common ATP-binding domain. ABC transporters are classified into three major groups: bacterial importers (the periplasmic permeases), eukaryotic transporters, and bacterial exporters. We present a comprehensive review of the bacterial ABC exporter group, which currently includes over 40 systems. The bacterial ABC exporter systems are functionally subdivided on the basis of the type of substrate that each translocates. We describe three main groups: protein exporters, peptide exporters, and systems that transport nonprotein substrates. Prototype exporters from each group are described in detail to illustrate our current understanding of this protein family. The prototype systems include the alpha-hemolysin, colicin V, and capsular polysaccharide exporters from Escherichia coli, the protease exporter from Erwinia chrysanthemi, and the glucan exporters from Agrobacterium tumefaciens and Rhizobium meliloti. Phylogenetic analysis of the ATP-binding domains from 29 bacterial ABC exporters indicates that the bacterial ABC exporters can be divided into two primary branches. One branch contains the transport systems where the ATP-binding domain and the membrane-spanning domain are present on the same polypeptide, and the other branch contains the systems where these domains are found on separate polypeptides. Differences in substrate specificity do not correlate with evolutionary relatedness. A complete survey of the known and putative bacterial ABC exporters is included at the end of the review. PMID:8302219

  3. ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria.

    PubMed

    Narita, Shin-ichiro

    2011-01-01

    The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane. The transport systems for lipoproteins have been characterized in detail. An ATP-binding cassette (ABC) transporter, LolCDE, initiates sorting by mediating the detachment of lipoproteins from the inner membrane to form a water-soluble lipoprotein-LolA complex in the periplasm. Lipoproteins are then transferred to LolB at the outer membrane and are incorporated into the lipid bilayer. A model analogous to the Lol system has been suggested for the transport of LPS, where an ABC transporter, LptBFG, mediates the detachment of LPS from the inner membrane. Recent developments in the functional characterization of ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria are discussed. PMID:21670534

  4. Plant ABC Transporters

    PubMed Central

    Kang, Joohyun; Park, Jiyoung; Choi, Hyunju; Burla, Bo; Kretzschmar, Tobias; Lee, Youngsook; Martinoia, Enrico

    2011-01-01

    ABC transporters constitute one of the largest protein families found in all living organisms. ABC transporters are driven by ATP hydrolysis and can act as exporters as well as importers. The plant genome encodes for more than 100 ABC transporters, largely exceeding that of other organisms. In Arabidopsis, only 22 out of 130 have been functionally analyzed. They are localized in most membranes of a plant cell such as the plasma membrane, the tonoplast, chloroplasts, mitochondria and peroxisomes and fulfill a multitude of functions. Originally identified as transporters involved in detoxification processes, they have later been shown to be required for organ growth, plant nutrition, plant development, response to abiotic stresses, pathogen resistance and the interaction of the plant with its environment. To fulfill these roles they exhibit different substrate specifies by e.g. depositing surface lipids, accumulating phytate in seeds, and transporting the phytohormones auxin and abscisic acid. The aim of this review is to give an insight into the functions of plant ABC transporters and to show their importance for plant development and survival. PMID:22303277

  5. ABC transporters in CSCs membranes as a novel target for treating tumor relapse

    PubMed Central

    Zinzi, Laura; Contino, Marialessandra; Cantore, Mariangela; Capparelli, Elena; Leopoldo, Marcello; Colabufo, Nicola A.

    2014-01-01

    CSCs are responsible for the high rate of recurrence and chemoresistance of different types of cancer. The current antineoplastic agents able to inhibit bulk replicating cancer cells and radiation treatment are not efficacious toward CSCs since this subpopulation has several intrinsic mechanisms of resistance. Among these mechanisms, the expression of ATP-Binding Cassette (ABC) transporters family and the activation of different signaling pathways (such as Wnt/β-catenin signaling, Hedgehog, Notch, Akt/PKB) are reported. Therefore, considering ABC transporters expression on CSCs membranes, compounds able to modulate MDR could induce cytotoxicity in these cells disclosing an exciting and alternative strategy for targeting CSCs in tumor therapy. The next challenge in the cure of cancer relapse may be a multimodal strategy, an approach where specific CSCs targeting drugs exert simultaneously the ability to circumvent tumor drug resistance (ABC transporters modulation) and cytotoxic activity toward CSCs and the corresponding differentiated tumor cells. The efficacy of suggested multimodal strategy could be probed by using several scaffolds active toward MDR pumps on CSCs isolated by tumor specimens. PMID:25071581

  6. ABC transporters: The power to change

    PubMed Central

    Rees, Douglas C.; Johnson, Eric; Lewinson, Oded

    2010-01-01

    ATP binding cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins that are responsible for the ATP powered translocation of many substrates across membranes. The highly conserved ABC domains provide the nucleotide dependent engine that drives transport. By contrast, the transmembrane domains that create the translocation pathway are more variable. Recent structural advances with prokaryotic ABC transporters have provided a qualitative molecular framework for deciphering the transport cycle; an important goal is to develop quantitative models that detail the kinetic and molecular mechanisms by which ABC transporters couple the binding and hydrolysis of ATP to substrate translocation. PMID:19234479

  7. Mapping the functional yeast ABC transporter interactome.

    PubMed

    Snider, Jamie; Hanif, Asad; Lee, Mid Eum; Jin, Ke; Yu, Analyn R; Graham, Chris; Chuk, Matthew; Damjanovic, Dunja; Wierzbicka, Marta; Tang, Priscilla; Balderes, Dina; Wong, Victoria; Jessulat, Matthew; Darowski, Katelyn D; San Luis, Bryan-Joseph; Shevelev, Igor; Sturley, Stephen L; Boone, Charles; Greenblatt, Jack F; Zhang, Zhaolei; Paumi, Christian M; Babu, Mohan; Park, Hay-Oak; Michaelis, Susan; Stagljar, Igor

    2013-09-01

    ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters. PMID:23831759

  8. Bioinformatic survey of ABC transporters in dermatophytes.

    PubMed

    Gadzalski, Marek; Ciesielska, Anita; Stączek, Paweł

    2016-01-15

    ATP binding cassette (ABC) transporters constitute a very large and ubiquitous superfamily of membrane proteins. They are responsible for ATP hydrolysis driven translocation of countless substrates. Being a very old and diverse group of proteins present in all organisms they share a common feature, which is the presence of an evolutionary conservative nucleotide binding domain (NBD)--the engine that drives the transport. Another common domain is a transmembrane domain (TMD) which consists of several membrane-spanning helices. This part of protein is substrate-specific, thus it is much more variable. ABC transporters are known for driving drug efflux in many pathogens and cancer cells, therefore they are the subject of extensive studies. There are many examples of conferring a drug resistance phenotype in fungal pathogens by ABC transporters, however, little is known about these proteins in dermatophytes--a group of fungi causing superficial mycoses. So far only a single ABC transporter has been extensively studied in this group of pathogens. We analyzed available genomic sequences of seven dermatophyte species in order to provide an insight into dermatophyte ABC protein inventory. Phylogenetic studies of ABC transporter genes and their products were conducted and included ABC transporters of other fungi. Our results show that each dermatophyte genome studied possesses a great variety of ABC transporter genes. Detailed analysis of selected genes and their products indicates that relatively recent duplication of ABC transporter genes could lead to novel substrate specificity. PMID:26524502

  9. Comparison of Cytotoxicity and Inhibition of Membrane ABC Transporters Induced by MWCNTs with Different Length and Functional Groups.

    PubMed

    Yu, Jing; Liu, Su; Wu, Bing; Shen, Zhuoyan; Cherr, Gary N; Zhang, Xu-Xiang; Li, Mei

    2016-04-01

    Experimental studies indicate that multiwalled carbon nanotubes (MWCNTs) have the potential to induce cytotoxicity. However, the reports are often inconsistent and even contradictory. Additionally, adverse effects of MWCNTs at low concentration are not well understood. In this study, we systemically compared adverse effects of six MWCNTs including pristine MWCNTs, hydroxyl-MWCNTs and carboxyl-MWCNTs of two different lengths (0.5-2 μm and 10-30 μm) on human hepatoma cell line HepG2. Results showed that MWCNTs induced cytotoxicity by increasing reactive oxygen species (ROS) generation and damaging cell function. Pristine short MWCNTs induced higher cytotoxicity than pristine long MWCNTs. Functionalization increased cytotoxicity of long MWCNTs, but reduced cytotoxicity of short MWCNTs. Further, our results indicated that the six MWCNTs, at nontoxic concentration, might not be environmentally safe as they inhibited ABC transporters' efflux capabilities. This inhibition was observed even at very low concentrations, which were 40-1000 times lower than their effective concentrations on cytotoxicity. The inhibition of ABC transporters significantly increased cytotoxicity of arsenic, a known substrate of ABC transporters, indicating a chemosensitizing effect of MWCNTs. Plasma membrane damage was likely the mechanism by which the six MWCNTs inhibited ABC transporter activity. This study provides insight into risk assessments of low levels of MWCNTs in the environment. PMID:26943274

  10. The ABC transporters in Candidatus Liberibacter asiaticus.

    PubMed

    Li, Wenlin; Cong, Qian; Pei, Jimin; Kinch, Lisa N; Grishin, Nick V

    2012-11-01

    Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram-negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter-related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In-depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid-like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. PMID:22807026

  11. The ABC transporters in Candidatus Liberibacter asiaticus

    PubMed Central

    Li, Wenlin; Cong, Qian; Pei, Jimin; Kinch, Lisa N; Grishin, Nick V

    2012-01-01

    Candidatus Liberibacter asiaticus (Ca. L. asiaticus) is a Gram-negative bacterium and the pathogen of Citrus Greening disease (Huanglongbing, HLB). As a parasitic bacterium, Ca. L. asiaticus harbors ABC transporters that play important roles in exchanging chemical compounds between Ca. L. asiaticus and its host. Here, we analyzed all the ABC transporter-related proteins in Ca. L. asiaticus. We identified 14 ABC transporter systems and predicted their structures and substrate specificities. In-depth sequence and structure analysis including multiple sequence alignment, phylogenetic tree reconstruction, and structure comparison further support their function predictions. Our study shows that this bacterium could use these ABC transporters to import metabolites (amino acids and phosphates) and enzyme cofactors (choline, thiamine, iron, manganese, and zinc), resist to organic solvent, heavy metal, and lipid-like drugs, maintain the composition of the outer membrane (OM), and secrete virulence factors. Although the features of most ABC systems could be deduced from the abundant experimental data on their orthologs, we reported several novel observations within ABC system proteins. Moreover, we identified seven nontransport ABC systems that are likely involved in virulence gene expression regulation, transposon excision regulation, and DNA repair. Our analysis reveals several candidates for further studies to understand and control the disease, including the type I virulence factor secretion system and its substrate that are likely related to Ca. L. asiaticus pathogenicity and the ABC transporter systems responsible for bacterial OM biosynthesis that are good drug targets. PMID:22807026

  12. ABC transporters and neuroblastoma.

    PubMed

    Yu, Denise M T; Huynh, Tony; Truong, Alan M; Haber, Michelle; Norris, Murray D

    2015-01-01

    Neuroblastoma is the most common cancer of infancy and accounts for 15% of all pediatric oncology deaths. Survival rates of high-risk neuroblastoma remain less than 50%, with amplification of the MYCN oncogene the most important aberration associated with poor outcome. Direct transcriptional targets of MYCN include a number of ATP-binding cassette (ABC) transporters, of which ABCC1 (MRP1), ABCC3 (MRP3), and ABCC4 (MRP4) are the best characterized. These three transporter genes have been shown to be strongly prognostic of neuroblastoma outcome in primary untreated neuroblastoma. In addition to their ability to efflux a number of chemotherapeutic drugs, evidence suggests that these transporters also contribute to neuroblastoma outcome independent of any role in cytotoxic drug efflux. Endogenous substrates of ABCC1 and ABCC4 that may be potential candidates affecting neuroblastoma biology include molecules such as prostaglandins and leukotrienes. These bioactive lipid mediators have the ability to influence biological processes contributing to cancer initiation and progression, such as angiogenesis, cell signaling, inflammation, proliferation, and migration and invasion. ABCC1 and ABCC4 are thus potential targets for therapeutic suppression in high-risk neuroblastoma, and recently developed small-molecule inhibitors may be an effective strategy in treating aggressive forms of this cancer, as well as other cancers that express high levels of these transporters. PMID:25640269

  13. Structural insights into ABC transporter mechanism

    SciTech Connect

    Oldham, Michael L.; Davidson, Amy L.; Chen, Jue

    2010-07-27

    ATP-binding cassette (ABC) transporters utilize the energy from ATP hydrolysis to transport substances across the membrane. In recent years, crystal structures of several ABC transporters have become available. These structures show that both importers and exporters oscillate between two conformations: an inward-facing conformation with the substrate translocation pathway open to the cytoplasm and an outward-facing conformation with the translocation pathway facing the opposite side of the membrane. In this review, conformational differences found in the structures of homologous ABC transporters are analyzed to understand how alternating-access is achieved. It appears that rigid-body rotations of the transmembrane subunits, coinciding with the opening and closing of the nucleotide-binding subunits, couples ATP hydrolysis to substrate translocation.

  14. Defining key roles for auxiliary proteins in an ABC transporter that maintains bacterial outer membrane lipid asymmetry

    PubMed Central

    Thong, Shuhua; Ercan, Bilge; Torta, Federico; Fong, Zhen Yang; Wong, Hui Yi Alvina; Wenk, Markus R; Chng, Shu-Sin

    2016-01-01

    In Gram-negative bacteria, lipid asymmetry is critical for the function of the outer membrane (OM) as a selective permeability barrier, but how it is established and maintained is poorly understood. Here, we characterize a non-canonical ATP-binding cassette (ABC) transporter in Escherichia coli that provides energy for maintaining OM lipid asymmetry via the transport of aberrantly localized phospholipids (PLs) from the OM to the inner membrane (IM). We establish that the transporter comprises canonical components, MlaF and MlaE, and auxiliary proteins, MlaD and MlaB, of previously unknown functions. We further demonstrate that MlaD forms extremely stable hexamers within the complex, functions in substrate binding with strong affinity for PLs, and modulates ATP hydrolytic activity. In addition, MlaB plays critical roles in both the assembly and activity of the transporter. Our work provides mechanistic insights into how the MlaFEDB complex participates in ensuring active retrograde PL transport to maintain OM lipid asymmetry. DOI: http://dx.doi.org/10.7554/eLife.19042.001 PMID:27529189

  15. Membrane topology and functional importance of the periplasmic region of ABC transporter LolCDE.

    PubMed

    Yasuda, Masaki; Iguchi-Yokoyama, Asako; Matsuyama, Shin-ichi; Tokuda, Hajime; Narita, Shin-ichiro

    2009-10-01

    The LolCDE complex is an ATP-binding cassette transporter that mediates the release of newly synthesized lipoproteins from the cytoplasmic membrane of gram-negative bacteria, which results in the initiation of outer-membrane sorting of lipoproteins through the Lol pathway. LolCDE is composed of one copy each of membrane subunits LolC and LolE, and two copies of nucleotide-binding subunit LolD. In this study, we examined the membrane topology of LolC and LolE by PhoA fusion analysis. Both LolC and LolE were found to have four transmembrane segments with a large periplasmic loop exposed to the periplasm. Despite similarities in sequence and topology, the accessibility of a sulfhydryl reagent to Cys introduced into the periplasmic loop suggested that the structure of the periplasmic region differs between LolC and LolE. Inhibition of the release of lipoproteins by the sulfhydryl reagent supported a previous proposal that LolC and LolE have distinct functions. PMID:19809197

  16. Identification and functional characterization of Penicillium marneffei pleiotropic drug resistance transporters ABC1 and ABC2.

    PubMed

    Panapruksachat, Siribun; Iwatani, Shun; Oura, Takahiro; Vanittanakom, Nongnuch; Chindamporn, Ariya; Niimi, Kyoko; Niimi, Masakazu; Lamping, Erwin; Cannon, Richard D; Kajiwara, Susumu

    2016-07-01

    Penicilliosis caused by the dimorphic fungus Penicillium marneffei is an endemic, AIDS-defining illness and, after tuberculosis and cryptococcosis, the third most common opportunistic infection of AIDS patients in tropical Southeast Asia. Untreated, patients have poor prognosis; however, primary amphotericin B treatment followed by prolonged itraconazole prophylaxis is effective. To identify ATP-binding cassette (ABC) transporters that may play a role in potential multidrug resistance of P. marneffei, we identified and classified all 46 P. marneffei ABC transporters from the genome sequence. PmABC1 and PmABC2 were most similar to the archetype Candida albicans multidrug efflux pump gene CDR1. P. marneffei Abc1p (PmAbc1p) was functionally expressed in Saccharomyces cerevisiae, although at rather low levels, and correctly localized to the plasma membrane, causing cells to be fourfold to eightfold more resistant to azoles and many other xenobiotics than untransformed cells. P. marneffei Abc2p (PmAbc2p) was expressed at similarly low levels, but it had no efflux activity and did not properly localize to the plasma membrane. Interestingly, PmAbc1p mislocalized and lost its transport activity when cells were shifted to 37 °C. We conclude that expression of PmAbc1p in S. cerevisiae confers resistance to several xenobiotics indicating that PmAbc1p may be a multidrug efflux pump. PMID:26782644

  17. 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

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

    PubMed Central

    2009-01-01

    There exist four fundamentally different classes of membrane-bound transport proteins: ion channels; transporters; aquaporins; and ATP-powered pumps. ATP-binding cassette (ABC) transporters are an example of ATP-dependent pumps. ABC transporters are ubiquitous membrane-bound proteins, present in all prokaryotes, as well as plants, fungi, yeast and animals. These pumps can move substrates in (influx) or out (efflux) of cells. In mammals, ABC transporters are expressed predominantly in the liver, intestine, blood-brain barrier, blood-testis barrier, placenta and kidney. ABC proteins transport a number of endogenous substrates, including inorganic anions, metal ions, peptides, amino acids, sugars and a large number of hydrophobic compounds and metabolites across the plasma membrane, and also across intracellular membranes. The human genome contains 49 ABC genes, arranged in eight subfamilies and named via divergent evolution. That ABC genes are important is underscored by the fact that mutations in at least I I of these genes are already known to cause severe inherited diseases (eg cystic fibrosis and X-linked adrenoleukodystrophy [X-ALD]). ABC transporters also participate in the movement of most drugs and their metabolites across cell surface and cellular organelle membranes; thus, defects in these genes can be important in terms of cancer therapy, pharmacokinetics and innumerable pharmacogenetic disorders. PMID:19403462

  19. Sub-cellular localisation of the white/scarlet ABC transporter to pigment granule membranes within the compound eye of Drosophila melanogaster.

    PubMed

    Mackenzie, S M; Howells, A J; Cox, G B; Ewart, G D

    2000-01-01

    The white, scarlet, and brown genes of Drosophila melanogaster encode ABC transporters involved with the uptake and storage of metabolic precursors to the red and brown eye colour pigments. It has generally been assumed that these proteins are localised in the plasma membrane and transport precursor molecules from the heamolymph into the eye pigment cells. However, the immuno-electron microscopy experiments in this study reveal that the White and Scarlet proteins are located in the membranes of pigment granules within pigment cells and retinula cells of the compound eye. No evidence of their presence in the plasma membrane was observed. This result suggests that, rather than tranporting tryptophan into the cell across the plasma membrane, the White/Scarlet complex transports a metabolic intermediate (such as 3-hydroxy kynurenine) from the cytoplasm into the pigment granules. Other functional implications of this new finding are discussed. PMID:11294610

  20. Peroxisomal ABC transporters: functions and mechanism

    PubMed Central

    Baker, Alison; Carrier, David J.; Schaedler, Theresia; Waterham, Hans R.; van Roermund, Carlo W.; Theodoulou, Frederica L.

    2015-01-01

    Peroxisomes are arguably the most biochemically versatile of all eukaryotic organelles. Their metabolic functions vary between different organisms, between different tissue types of the same organism and even between different developmental stages or in response to changed environmental conditions. New functions for peroxisomes are still being discovered and their importance is underscored by the severe phenotypes that can arise as a result of peroxisome dysfunction. The β-oxidation pathway is central to peroxisomal metabolism, but the substrates processed are very diverse, reflecting the diversity of peroxisomes across species. Substrates for β-oxidation enter peroxisomes via ATP-binding cassette (ABC) transporters of subfamily D; (ABCD) and are activated by specific acyl CoA synthetases for further metabolism. Humans have three peroxisomal ABCD family members, which are half transporters that homodimerize and have distinct but partially overlapping substrate specificity; Saccharomyces cerevisiae has two half transporters that heterodimerize and plants have a single peroxisomal ABC transporter that is a fused heterodimer and which appears to be the single entry point into peroxisomes for a very wide variety of β-oxidation substrates. Our studies suggest that the Arabidopsis peroxisomal ABC transporter AtABCD1 accepts acyl CoA substrates, cleaves them before or during transport followed by reactivation by peroxisomal synthetases. We propose that this is a general mechanism to provide specificity to this class of transporters and by which amphipathic compounds are moved across peroxisome membranes. PMID:26517910

  1. The ABCs of membrane transporters in health and disease (SLC series): Introduction☆☆☆

    PubMed Central

    Hediger, Matthias A.; Clémençon, Benjamin; Burrier, Robert E.; Bruford, Elspeth A.

    2013-01-01

    The field of transport biology has steadily grown over the past decade and is now recognized as playing an important role in manifestation and treatment of disease. The SLC (solute carrier) gene series has grown to now include 52 families and 395 transporter genes in the human genome. A list of these genes can be found at the HUGO Gene Nomenclature Committee (HGNC) website (see www.genenames.org/genefamilies/SLC). This special issue features mini-reviews for each of these SLC families written by the experts in each field. The existing online resource for solute carriers, the Bioparadigms SLC Tables (www.bioparadigms.org), has been updated and significantly extended with additional information and cross-links to other relevant databases, and the nomenclature used in this database has been validated and approved by the HGNC. In addition, the Bioparadigms SLC Tables functionality has been improved to allow easier access by the scientific community. This introduction includes: an overview of all known SLC and “non-SLC” transporter genes; a list of transporters of water soluble vitamins; a summary of recent progress in the structure determination of transporters (including GLUT1/SLC2A1); roles of transporters in human diseases and roles in drug approval and pharmaceutical perspectives. PMID:23506860

  2. Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits.

    PubMed

    Mourez, M; Hofnung, M; Dassa, E

    1997-06-01

    The cytoplasmic membrane proteins of bacterial binding protein-dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA---G---------I-LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose-negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane-bound, as in wild-type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose-negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain. PMID:9214624

  3. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

    PubMed

    Lin, Hong Ting; Bavro, Vassiliy N; Barrera, Nelson P; Frankish, Helen M; Velamakanni, Saroj; van Veen, Hendrik W; Robinson, Carol V; Borges-Walmsley, M Inês; Walmsley, Adrian R

    2009-01-01

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled. PMID:18955484

  4. MacB ABC Transporter Is a Dimer Whose ATPase Activity and Macrolide-binding Capacity Are Regulated by the Membrane Fusion Protein MacA*S⃞

    PubMed Central

    Lin, Hong Ting; Bavro, Vassiliy N.; Barrera, Nelson P.; Frankish, Helen M.; Velamakanni, Saroj; van Veen, Hendrik W.; Robinson, Carol V.; Borges-Walmsley, M. Inês; Walmsley, Adrian R.

    2009-01-01

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled. PMID:18955484

  5. Cloning of two novel ABC transporters mapping on human chromosome 9

    SciTech Connect

    Luciani, M.F.; Savary, S.; Chimini, G. ); Denizot, F. ); Mattei, M.G. )

    1994-05-01

    The family of ATP binding cassette (ABC) transporters or traffic ATPases is composed of several membrane-associated proteins that transport a great variety of solutes across cellular membranes. Two novel mammalian members of the family, ABC1 and ABC2, have been identified by a PCR-based approach. They belong to a group of traffic ATPases encoded as a single multifunctional protein, such as CFTR, STE 6, and P-glycoproteins. Their peculiar structural features and close relationship to ABC transporters involved in nodulation suggest that ABC1 and ABC2 define a novel subgroup of mammalian traffic ATPases. 51 refs., 7 figs.

  6. Regulation of ABC Transporter Function Via Phosphorylation by Protein Kinases

    PubMed Central

    Stolarczyk, Elzbieta I.; Reiling, Cassandra J.; Paumi, Christian M.

    2011-01-01

    ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases [1]. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation. PMID:21118091

  7. The ABC transporter gene family of Daphnia pulex

    PubMed Central

    Sturm, Armin; Cunningham, Phil; Dean, Michael

    2009-01-01

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

  8. THE ABC TRANSPORTER, AbcB3, MEDIATES cAMP EXPORT IN D. DISCOIDEUM DEVELOPMENT

    PubMed Central

    Miranda, Edward Roshan; Nam, Edward A.; Kuspa, Adam; Shaulsky, Gad

    2014-01-01

    Extracellular cAMP functions as a primary ligand for cell surface cAMP receptors throughout Dictyostelium discoideum development, controlling chemotaxis and morphogenesis. The developmental consequences of cAMP signaling and the metabolism of cAMP have been studied in great detail, but it has been unclear how cells export cAMP across the plasma membrane. Here we show pharmacologically and genetically that ABC transporters mediate cAMP export. Using an evolutionary-developmental biology approach, we identified several candidate abc genes and characterized one of them, abcB3, in more detail. Genetic and biochemical evidence suggest that AbcB3 is a component of the cAMP export mechanism in D. discoideum development. PMID:25448698

  9. ABC transporter research: going strong 40 years on

    PubMed Central

    Theodoulou, Frederica L.; Kerr, Ian D.

    2015-01-01

    In most organisms, ABC transporters constitute one of the largest families of membrane proteins. In humans, their functions are diverse and underpin numerous key physiological processes, as well as being causative factors in a number of clinically relevant pathologies. Advances in our understanding of these diseases have come about through combinations of genetic and protein biochemical investigations of these transporters and the power of in vitro and in vivo investigations is helping to develop genotype–phenotype understanding. However, the importance of ABC transporter research goes far beyond human biology; microbial ABC transporters are of great interest in terms of understanding virulence and drug resistance and industrial biotechnology researchers are exploring the potential of prokaryotic ABC exporters to increase the capacity of synthetic biology systems. Plant ABC transporters play important roles in transport of hormones, xenobiotics, metals and secondary metabolites, pathogen responses and numerous aspects of development, all of which are important in the global food security area. For 3 days in Chester, this Biochemical Society Focused Meeting brought together researchers with diverse experimental approaches and with different fundamental questions, all of which are linked by the commonality of ABC transporters. PMID:26517919

  10. In vitro interaction between components of the inner membrane complex of the maltose ABC transporter of Escherichia coli: modulation by ATP.

    PubMed

    Mourez, M; Jéhanno, M; Schneider, E; Dassa, E

    1998-10-01

    Interactions between domains of ATP-binding cassette (ABC) transporters are of great functional importance and yet are poorly understood. To gain further knowledge of these protein-protein interactions, we studied the inner membrane complex of the maltose transporter of Escherichia coli. We focused on interactions between the nucleotide-binding protein, MalK, and the transmembrane proteins, MalF and MalG. We incubated purified MalK with inverted membrane vesicles containing MalF and MalG. MalK bound specifically to MalF and MalG and reconstituted a functional complex. We used this approach and limited proteolysis with trypsin to show that binding and hydrolysis of ATP, inducing conformational changes in MalK, modulate its interaction with MalF and MalG. MalK in the reconstituted complex was less sensitive to protease added from the cytoplasmic side of the membrane, and one proteolytic cleavage site located in the middle of a putative helical domain of MalK was protected. These results suggest that the putative helical domain of the nucleotide-binding domains is involved, through its conformational changes, in the coupling between the transmembrane domains and ATP binding/hydrolysis at the nucleotide-binding domains. PMID:9791180

  11. Identification of ABC transporters in Sarcoptes scabiei.

    PubMed

    Mounsey, K E; Holt, D C; McCarthy, J; Walton, S F

    2006-06-01

    We have identified and partially sequenced 8 ABC transporters from an EST dataset of Sarcoptes scabiei var. hominis, the causative agent of scabies. Analysis confirmed that most of the known ABC subfamilies are represented in the EST dataset including several members of the multidrug resistance protein subfamily (ABC-C). Although P-glycoprotein (ABC-B) sequences were not found in the EST dataset, a partial P-glycoprotein sequence was subsequently obtained using a degenerate PCR strategy and library screening. Thus a total of 9 potential S. scabiei ABC transporters representing the subfamilies A, B, C, E, F and H have been identified. Ivermectin is currently used in the treatment of hyper-infested (crusted) scabies, and has also been identified as a potentially effective acaricide for mass treatment programmes in scabies-endemic communities. The observation of clinical and in vitro ivermectin resistance in 2 crusted scabies patients who received multiple treatments has raised serious concerns regarding the sustainability of such programmes. One possible mechanism for ivermectin resistance is through ABC transporters such as P-glycoprotein. This work forms an important foundation for further studies to elucidate the potential role of ABC transporters in ivermectin resistance of S. scabiei. PMID:16454864

  12. Yeast ABC transporters in lipid trafficking.

    PubMed

    Prasad, Rajendra; Khandelwal, Nitesh Kumar; Banerjee, Atanu

    2016-08-01

    Throughout its evolution, the ATP-binding cassette (ABC) transporter superfamily has experienced a rapid expansion in its substrate repertoire and functions. Of the diverse functions that these pumps offer, their drug transport properties have attracted considerable attention primarily owing to their clinical significance. Despite this fact, emerging evidence suggests that physiological substrates of transporters also affect the overall functioning of an organism. Lipids, as substrates of ABC transporters, constitute one feature found in all representative groups of the living kingdom. Due to the importance of lipid species in the cellular physiology of an organism, their proper distribution within cells is crucial. This fact is well exemplified by the vast number of medical conditions that have been caused as a result of perturbations in ABC transporter-mediated lipid transport in higher organisms. In yeasts, apart from providing transport functions, ABC transporters also coordinate regulatory networks with lipids. This review focuses on yeast ABC transporters involved in the transport of lipids and briefly discusses the integration of their regulatory network with that of the lipid species. PMID:27259587

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

    PubMed

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

    2005-03-21

    ATP binding cassette (ABC) proteins of both eukaryotic and prokaryotic origins are implicated in the transport of lipids. In humans, members of the ABC protein families A, B, C, D and G are mutated in a number of lipid transport and metabolism disorders, such as Tangier disease, Stargardt syndrome, progressive familial intrahepatic cholestasis, pseudoxanthoma elasticum, adrenoleukodystrophy or sitosterolemia. Studies employing transfection, overexpression, reconstitution, deletion and inhibition indicate the transbilayer transport of endogenous lipids and their analogs by some of these proteins, modulating lipid transbilayer asymmetry. Other proteins appear to be involved in the exposure of specific lipids on the exoplasmic leaflet, allowing their uptake by acceptors and further transport to specific sites. Additionally, lipid transport by ABC proteins is currently being studied in non-human eukaryotes, e.g. in sea urchin, trypanosomatides, arabidopsis and yeast, as well as in prokaryotes such as Escherichia coli and Lactococcus lactis. Here, we review current information about the (putative) role of both pro- and eukaryotic ABC proteins in the various phenomena associated with lipid transport. Besides providing a better understanding of phenomena like lipid metabolism, circulation, multidrug resistance, hormonal processes, fertilization, vision and signalling, studies on pro- and eukaryotic ABC proteins might eventually enable us to put a name on some of the proteins mediating transbilayer lipid transport in various membranes of cells and organelles. It must be emphasized, however, that there are still many uncertainties concerning the functions and mechanisms of ABC proteins interacting with lipids. In particular, further purification and reconstitution experiments with an unambiguous role of ATP hydrolysis are needed to demonstrate a clear involvement of ABC proteins in lipid transbilayer asymmetry. PMID:15749056

  14. Fungal ABC transporter deletion and localization analysis.

    PubMed

    Kovalchuk, Andriy; Weber, Stefan S; Nijland, Jeroen G; Bovenberg, Roel A L; Driessen, Arnold J M

    2012-01-01

    Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological function. Among different approaches available, the localization analysis based on the expression of GFP fusions is commonly used as a relatively fast and cost-efficient method that allows visualization of proteins of interest in both live and fixed cells. In addition, inactivation of transporter genes is an important tool to resolve their specific function. Here we provide a detailed protocol for the deletion and localization analysis of ABC transporters in the filamentous fungus Penicillium chrysogenum. It includes construction of expression plasmids, their transformation into fungal strains, cultivation of transformants, microscopy analysis, as well as additional protocols on staining of fungal cells with organelle-specific dyes like Hoechst 33342, MitoTracker DeepRed, and FM4-64. PMID:22183644

  15. A Drosophila ABC transporter regulates lifespan.

    PubMed

    Huang, He; Lu-Bo, Ying; Haddad, Gabriel G

    2014-12-01

    MRP4 (multidrug resistance-associated protein 4) is a member of the MRP/ABCC subfamily of ATP-binding cassette (ABC) transporters that are essential for many cellular processes requiring the transport of substrates across cell membranes. Although MRP4 has been implicated as a detoxification protein by transport of structurally diverse endogenous and xenobiotic compounds, including antivirus and anticancer drugs, that usually induce oxidative stress in cells, its in vivo biological function remains unknown. In this study, we investigate the biological functions of a Drosophila homolog of human MRP4, dMRP4. We show that dMRP4 expression is elevated in response to oxidative stress (paraquat, hydrogen peroxide and hyperoxia) in Drosophila. Flies lacking dMRP4 have a shortened lifespan under both oxidative and normal conditions. Overexpression of dMRP4, on the other hand, is sufficient to increase oxidative stress resistance and extend lifespan. By genetic manipulations, we demonstrate that dMRP4 is required for JNK (c-Jun NH2-terminal kinase) activation during paraquat challenge and for basal transcription of some JNK target genes under normal condition. We show that impaired JNK signaling is an important cause for major defects associated with dMRP4 mutations, suggesting that dMRP4 regulates lifespan by modulating the expression of a set of genes related to both oxidative resistance and aging, at least in part, through JNK signaling. PMID:25474322

  16. The ABCs of Candida albicans Multidrug Transporter Cdr1

    PubMed Central

    Banerjee, Atanu; Khandelwal, Nitesh Kumar; Dhamgaye, Sanjiveeni

    2015-01-01

    In the light of multidrug resistance (MDR) among pathogenic microbes and cancer cells, membrane transporters have gained profound clinical significance. Chemotherapeutic failure, by far, has been attributed mainly to the robust and diverse array of these proteins, which are omnipresent in every stratum of the living world. Candida albicans, one of the major fungal pathogens affecting immunocompromised patients, also develops MDR during the course of chemotherapy. The pivotal membrane transporters that C. albicans has exploited as one of the strategies to develop MDR belongs to either the ATP binding cassette (ABC) or the major facilitator superfamily (MFS) class of proteins. The ABC transporter Candida drug resistance 1 protein (Cdr1p) is a major player among these transporters that enables the pathogen to outplay the battery of antifungals encountered by it. The promiscuous Cdr1 protein fulfills the quintessential need of a model to study molecular mechanisms of multidrug transporter regulation and structure-function analyses of asymmetric ABC transporters. In this review, we cover the highlights of two decades of research on Cdr1p that has provided a platform to study its structure-function relationships and regulatory circuitry for a better understanding of MDR not only in yeast but also in other organisms. PMID:26407965

  17. The ABCs of Candida albicans Multidrug Transporter Cdr1.

    PubMed

    Prasad, Rajendra; Banerjee, Atanu; Khandelwal, Nitesh Kumar; Dhamgaye, Sanjiveeni

    2015-12-01

    In the light of multidrug resistance (MDR) among pathogenic microbes and cancer cells, membrane transporters have gained profound clinical significance. Chemotherapeutic failure, by far, has been attributed mainly to the robust and diverse array of these proteins, which are omnipresent in every stratum of the living world. Candida albicans, one of the major fungal pathogens affecting immunocompromised patients, also develops MDR during the course of chemotherapy. The pivotal membrane transporters that C. albicans has exploited as one of the strategies to develop MDR belongs to either the ATP binding cassette (ABC) or the major facilitator superfamily (MFS) class of proteins. The ABC transporter Candida drug resistance 1 protein (Cdr1p) is a major player among these transporters that enables the pathogen to outplay the battery of antifungals encountered by it. The promiscuous Cdr1 protein fulfills the quintessential need of a model to study molecular mechanisms of multidrug transporter regulation and structure-function analyses of asymmetric ABC transporters. In this review, we cover the highlights of two decades of research on Cdr1p that has provided a platform to study its structure-function relationships and regulatory circuitry for a better understanding of MDR not only in yeast but also in other organisms. PMID:26407965

  18. ABC transporters in fish species: a review

    PubMed Central

    Ferreira, Marta; Costa, Joana; Reis-Henriques, Maria A.

    2014-01-01

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

  19. Insights into how nucleotide-binding domains power ABC transport.

    PubMed

    Newstead, Simon; Fowler, Philip W; Bilton, Paul; Carpenter, Elisabeth P; Sadler, Peter J; Campopiano, Dominic J; Sansom, Mark S P; Iwata, So

    2009-09-01

    The mechanism by which nucleotide-binding domains (NBDs) of ABC transporters power the transport of substrates across cell membranes is currently unclear. Here we report the crystal structure of an NBD, FbpC, from the Neisseria gonorrhoeae ferric iron uptake transporter with an unusual and substantial domain swap in the C-terminal regulatory domain. This entanglement suggests that FbpC is unable to open to the same extent as the homologous protein MalK. Using molecular dynamics we demonstrate that this is not the case: both NBDs open rapidly once ATP is removed. We conclude from this result that the closed structures of FbpC and MalK have higher free energies than their respective open states. This result has important implications for our understanding of the mechanism of power generation in ABC transporters, because the unwinding of this free energy ensures that the opening of these two NBDs is also powered. PMID:19748342

  20. Multidrug resistance ABC transporter structure predictions by homology modeling approaches.

    PubMed

    Honorat, Mylène; Falson, Pierre; Terreux, Raphael; Di Pietro, Attilio; Dumontet, Charles; Payen, Léa

    2011-03-01

    Human multidrug resistance ABC transporters are ubiquitous membrane proteins responsible for the efflux of multiple, endogenous or exogenous, compounds out of the cells, and therefore they are involved in multi-drug resistance phenotype (MDR). They thus deeply impact the pharmacokinetic parameters and toxicity properties of drugs. A great pressure to develop inhibitors of these pumps is carried out, by either ligand-based drug design or (more ideally) structure-based drug design. In that goal, many biochemical studies have been carried out to characterize their transport functions, and many efforts have been spent to get high-resolution structures. Currently, beside the 3D-structures of bacterial ABC transporters Sav1866 and MsbA, only the mouse ABCB1 complete structure has been published at high-resolution, illustrating the tremendous difficulty in getting such information, taking into account that the human genome accounts for 48 ABC transporters encoding genes. Homology modeling is consequently a reasonable approach to overcome this obstacle. The present review describes, in the first part, the different approaches which have been published to set up human ABC pump 3D-homology models allowing the localization of binding sites for drug candidates, and the identification of critical residues therein. In a second part, the review proposes a more accurate strategy and practical keys to use such biological tools for initiating structure-based drug design. PMID:21470105

  1. An ABC transporter and an outer membrane lipoprotein participate in posttranslational activation of type VI secretion in Pseudomonas aeruginosa.

    PubMed

    Casabona, Maria G; Silverman, Julie M; Sall, Khady M; Boyer, Frédéric; Couté, Yohann; Poirel, Jessica; Grunwald, Didier; Mougous, Joseph D; Elsen, Sylvie; Attree, Ina

    2013-02-01

    Pseudomonas aeruginosa is capable of injecting protein toxins into other bacterial cells through one of its three type VI secretion systems (T6SSs). The activity of this T6SS is tightly regulated on the posttranslational level by phosphorylation-dependent and -independent pathways. The phosphorylation-dependent pathway consists of a Threonine kinase/phosphatase pair (PpkA/PppA) that acts on a forkhead domain-containing protein, Fha1, and a periplasmic protein, TagR, that positively regulates PpkA. In the present work, we biochemically and functionally characterize three additional proteins of the phosphorylation-dependent regulatory cascade that controls T6S activation: TagT, TagS and TagQ. We show that similar to TagR, these proteins act upstream of the PpkA/PppA checkpoint and influence phosphorylation of Fha1 and, apparatus assembly and effector export. Localization studies demonstrate that TagQ is an outer membrane lipoprotein and TagR is associated with the outer membrane. Consistent with their homology to lipoprotein outer membrane localization (Lol) components, TagT and TagS form a stable inner membrane complex with ATPase activity. However, we find that outer membrane association of T6SS lipoproteins TagQ and TssJ1, and TagR, is unaltered in a ΔtagTS background. Notably, we found that TagQ is indispensible for anchoring of TagR to the outer membrane fraction. As T6S-dependent fitness of P. aeruginosa requires TagT, S, R and Q, we conclude that these proteins likely participate in a trans-membrane signalling pathway that promotes H1-T6SS activity under optimal environmental conditions. PMID:22765374

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

    PubMed

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

    2012-01-01

    Alzheimer's disease (AD) is considered the "disease of the twenty-first century." With a 10-fold increase in global incidence over the past 100 years, AD is now reaching epidemic proportions and by all projections, AD patient numbers will continue to rise. Despite intense research efforts, AD remains a mystery and effective therapies are still unavailable. This represents an unmet need resulting in clinical, social, and economic problems. Over the last decade, a new AD research focus has emerged: ATP-binding cassette (ABC) transporters. In this article, we provide an overview of the ABC transporters ABCA1, ABCA2, P-glycoprotein (ABCB1), MRP1 (ABCC1), and BCRP (ABCG2), all of which are expressed in the brain and have been implicated in AD. We summarize recent findings on the role of these five transporters in AD, and discuss their potential to serve as therapeutic targets. PMID:22675311

  3. Apical ABC transporters and cancer chemotherapeutic drug disposition.

    PubMed

    Durmus, Selvi; Hendrikx, Jeroen J M A; Schinkel, Alfred H

    2015-01-01

    ATP-binding cassette (ABC) transporters are transmembrane efflux transporters that mediate cellular extrusion of a broad range of substrates ranging from amino acids, lipids, and ions to xenobiotics including many anticancer drugs. ABCB1 (P-GP) and ABCG2 (BCRP) are the most extensively studied apical ABC drug efflux transporters. They are highly expressed in apical membranes of many pharmacokinetically relevant tissues such as epithelial cells of the small intestine and endothelial cells of the blood capillaries in brain and testis, and in the placental maternal-fetal barrier. In these tissues, they have a protective function as they efflux their substrates back to the intestinal lumen or blood and thus restrict the intestinal uptake and tissue disposition of many compounds. This presents a major challenge for the use of many (anticancer) drugs, as most currently used anticancer drugs are substrates of these transporters. Herein, we review the latest findings on the role of apical ABC transporters in the disposition of anticancer drugs. We discuss that many new, rationally designed anticancer drugs are substrates of these transporters and that their oral availability and/or brain disposition are affected by this interaction. We also summarize studies that investigate the improvement of oral availability and brain disposition of many cytotoxic (e.g., taxanes) and rationally designed (e.g., tyrosine kinase inhibitor) anticancer drugs, using chemical inhibitors of these transporters. These findings provide a better understanding of the importance of apical ABC transporters in chemotherapy and may therefore advance translation of promising preclinical insights and approaches to clinical studies. PMID:25640265

  4. The ABC gene family in arthropods: comparative genomics and role in insecticide transport and resistance.

    PubMed

    Dermauw, Wannes; Van Leeuwen, Thomas

    2014-02-01

    About a 100 years ago, the Drosophila white mutant marked the birth of Drosophila genetics. The white gene turned out to encode the first well studied ABC transporter in arthropods. The ABC gene family is now recognized as one of the largest transporter families in all kingdoms of life. The majority of ABC proteins function as primary-active transporters that bind and hydrolyze ATP while transporting a large diversity of substrates across lipid membranes. Although extremely well studied in vertebrates for their role in drug resistance, less is known about the role of this family in the transport of endogenous and exogenous substances in arthropods. The ABC families of five insect species, a crustacean and a chelicerate have been annotated in some detail. We conducted a thorough phylogenetic analysis of the seven arthropod and human ABC protein subfamilies, to infer orthologous relationships that might suggest conserved function. Most orthologous relationships were found in the ABCB half transporter, ABCD, ABCE and ABCF subfamilies, but specific expansions within species and lineages are frequently observed and discussed. We next surveyed the role of ABC transporters in the transport of xenobiotics/plant allelochemicals and their involvement in insecticide resistance. The involvement of ABC transporters in xenobiotic resistance in arthropods is historically not well documented, but an increasing number of studies using unbiased differential gene expression analysis now points to their importance. We give an overview of methods that can be used to link ABC transporters to resistance. ABC proteins have also recently been implicated in the mode of action and resistance to Bt toxins in Lepidoptera. Given the enormous interest in Bt toxicology in transgenic crops, such findings will provide an impetus to further reveal the role of ABC transporters in arthropods. PMID:24291285

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

    PubMed

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

    2014-12-01

    Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5'-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum. PMID:25506302

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

    PubMed Central

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

    2014-01-01

    Fungi tolerate exposure to various abiotic stresses, including cytotoxic compounds and fungicides, via their ATP-driven efflux pumps belonging to ATP-binding cassette (ABC) transporters. To clarify the molecular basis of interaction between the fungus and various abiotic stresses including fungicides, we constructed a cDNA library from germinated conidia of Colletotrichum acutatum, a major anthracnose pathogen of pepper (Capsicum annum L.). Over 1,000 cDNA clones were sequenced, of which single clone exhibited significant nucleotide sequence homology to ABC transporter genes. We isolated three fosmid clones containing the C. acutatum ABC1 (CaABC1) gene in full-length from genomic DNA library screening. The CaABC1 gene consists of 4,059 bp transcript, predicting a 1,353-aa protein. The gene contains the typical ABC signature and Walker A and B motifs. The 5′-flanking region contains a CAAT motif, a TATA box, and a Kozak region. Phylogenetic and structural analysis suggested that the CaABC1 is a typical ABC transporter gene highly conserved in various fungal species, as well as in Chromista, Metazoans, and Viridiplantae. We also found that CaABC1 was up-regulated during conidiation and a minimal medium condition. Moreover, CaABC1 was induced in iprobenfos, kresoxim-methyl, thiophanate-methyl, and hygromycin B. These results demonstrate that CaABC1 is necessary for conidiation, abiotic stress, and various fungicide resistances. These results will provide the basis for further study on the function of ABC transporter genes in C. acutatum. PMID:25506302

  7. Plant ABC Transporters Enable Many Unique Aspects of a Terrestrial Plant's Lifestyle.

    PubMed

    Hwang, Jae-Ung; Song, Won-Yong; Hong, Daewoong; Ko, Donghwi; Yamaoka, Yasuyo; Jang, Sunghoon; Yim, Sojeong; Lee, Eunjung; Khare, Deepa; Kim, Kyungyoon; Palmgren, Michael; Yoon, Hwan Su; Martinoia, Enrico; Lee, Youngsook

    2016-03-01

    Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us to propose that diverse ABC transporters enabled many unique and essential aspects of a terrestrial plant's lifestyle, by transporting various compounds across specific membranes of the plant. PMID:26902186

  8. A reciprocating twin-channel model for ABC transporters.

    PubMed

    Jones, Peter M; George, Anthony M

    2014-08-01

    ABC transporters comprise a large, diverse, and ubiquitous superfamily of membrane active transporters. Their core architecture is a dimer of dimers, comprising two transmembrane (TM) domains that bind substrate, and two ATP-binding cassettes, which use the cell's energy currency to couple substrate translocation to ATP hydrolysis. Despite the availability of over a dozen resolved structures and a wealth of biochemical and biophysical data, this field is bedeviled by controversy and long-standing mechanistic questions remain unresolved. The prevailing paradigm for the ABC transport mechanism is the Switch Model, in which the ATP-binding cassettes dimerize upon binding two ATP molecules, and thence dissociate upon sequential ATP hydrolysis. This cycle of nucleotide-binding domain (NBD) dimerization and dissociation is coupled to a switch between inward- or outward facing conformations of a single TM channel; this alternating access enables substrate binding on one face of the membrane and its release at the other. Notwithstanding widespread acceptance of the Switch Model, there is substantial evidence that the NBDs do not separate very much, if at all, and thus physical separation of the ATP cassettes observed in crystallographic structures may be an artefact. An alternative Constant Contact Model has been proposed, in which ATP hydrolysis occurs alternately at the two ATP-binding sites, with one of the sites remaining closed and containing occluded nucleotide at all times. In this model, the cassettes remain in contact and the active sites swing open in an alternately seesawing motion. Whilst the concept of NBD association/dissociation in the Switch Model is naturally compatible with a single alternating-access channel, the asymmetric functioning proposed by the Constant Contact model suggests an alternating or reciprocating function in the TMDs. Here, a new model for the function of ABC transporters is proposed in which the sequence of ATP binding, hydrolysis, and

  9. How heterogeneous is the involvement of ABC transporters against insecticides?

    PubMed

    Porretta, Daniele; Epis, Sara; Mastrantonio, Valentina; Ferrari, Marco; Bellini, Romeo; Favia, Guido; Urbanelli, Sandra

    2016-05-01

    Understanding the molecular mechanisms underlying cellular defense against xenobiotic compounds is a main research issue in medical and veterinary entomology, as insecticide/acaricide resistance is a major threat in the control of arthropods. ABC transporters are recognized as a component of the detoxifying mechanism in arthropods. We investigated the possible involvement of ABC transporters in defense to the organophosphate insecticide temephos in the malarial vector Anopheles stephensi. We performed bioassays on larvae of An. stephensi, using insecticide alone and in combination with ABC-transporter inhibitors, to assess synergism between these compounds. Next, we investigated the expression profiles of six ABC transporter genes in larvae exposed to temephos. Surprisingly, neither bioassays nor gene expression analyses provided any evidence for a major role of ABC transporters in defense against temephos in An. stephensi. We thus decided to review existing literature to generate a record of other studies that failed to reveal a role for ABC transporters against particular insecticides/acaricides. A review of the scientific literature led to the recovery of 569 papers about ABC transporters; among these, 50 involved arthropods, and 10 reported negative results. Our study on An. stephensi and accompanying literature review highlight the heterogeneity that exists in ABC transporter involvement in defense/resistance mechanisms in arthropods. PMID:26855383

  10. The Predicted ABC Transporter AbcEDCBA Is Required for Type IV Secretion System Expression and Lysosomal Evasion by Brucella ovis

    PubMed Central

    Silva, Teane M. A.; Mol, Juliana P. S.; Winter, Maria G.; Atluri, Vidya; Xavier, Mariana N.; Pires, Simone F.; Paixão, Tatiane A.; Andrade, Hélida M.; Santos, Renato L.; Tsolis, Renee M.

    2014-01-01

    Brucella ovis is a major cause of reproductive failure in rams and it is one of the few well-described Brucella species that is not zoonotic. Previous work showed that a B. ovis mutant lacking a species-specific ABC transporterabcBA) was attenuated in mice and was unable to survive in macrophages. The aim of this study was to evaluate the role of this ABC transporter during intracellular survival of B. ovis. In HeLa cells, B. ovis WT was able to survive and replicate at later time point (48 hpi), whereas an ΔabcBA mutant was attenuated at 24 hpi. The reduced survival of the ΔabcBA mutant was associated with a decreased ability to exclude the lysosomal marker LAMP1 from its vacuolar membrane, suggesting a failure to establish a replicative niche. The ΔabcBA mutant showed a reduced abundance of the Type IV secretion system (T4SS) proteins VirB8 and VirB11 in both rich and acid media, when compared to WT B. ovis. However, mRNA levels of virB1, virB8, hutC, and vjbR were similar in both strains. These results support the notion that the ABC transporter encoded by abcEDCBA or its transported substrate acts at a post-transcriptional level to promote the optimal expression of the B. ovis T4SS within infected host cells. PMID:25474545

  11. Defining the blanks – Pharmacochaperoning of SLC6 transporters and ABC transporters?

    PubMed Central

    Chiba, Peter; Freissmuth, Michael; Stockner, Thomas

    2014-01-01

    SLC6 family members and ABC transporters represent two extremes: SLC6 transporters are confined to the membrane proper and only expose small segments to the hydrophilic milieu. In ABC transporters the hydrophobic core is connected to a large intracellular (eponymous) ATP binding domain that is comprised of two discontiguous repeats. Accordingly, their folding problem is fundamentally different. This can be gauged from mutations that impair the folding of the encoded protein and give rise to clinically relevant disease phenotypes: in SLC6 transporters, these cluster at the protein–lipid interface on the membrane exposed surface. Mutations in ABC-transporters map to the interface between nucleotide binding domains and the coupling helices, which provide the connection to the hydrophobic core. Folding of these mutated ABC-transporters can be corrected with ligands/substrates that bind to the hydrophobic core. This highlights a pivotal role of the coupling helices in the folding trajectory. In contrast, insights into pharmacochaperoning of SLC6 transporters are limited to monoamine transporters – in particular the serotonin transporter (SERT) – because of their rich pharmacology. Only ligands that stabilize the inward facing conformation act as effective pharmacochaperones. This indicates that the folding trajectory of SERT proceeds via the inward facing conformation. Mutations that impair folding of SLC6 family members can be transmitted as dominant or recessive alleles. The dominant phenotype of the mutation can be rationalized, because SLC6 transporters are exported in oligomeric form from the endoplasmic reticulum (ER). Recessive transmission requires shielding of the unaffected gene product from the mutated transporter in the ER. This can be accounted for by a chaperone-COPII (coatomer protein II) exchange model, where proteinaceous ER-resident chaperones engage various intermediates prior to formation of the oligomeric state and subsequent export from the

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

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

    2005-01-01

    ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (mal)function in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD) have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases. PMID:15967026

  14. 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

  15. Active transmembrane drug transport in microgravity: a validation study using an ABC transporter model

    PubMed Central

    Vaquer, Sergi; Cuyàs, Elisabet; Rabadán, Arnau; González, Albert; Fenollosa, Felip; de la Torre, Rafael

    2014-01-01

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

  16. 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. PMID:27164440

  17. An ABC Transporter Plays a Developmental Aggregation Role in Myxococcus xanthus

    PubMed Central

    Ward, Mandy J.; Mok, Kenny C.; Astling, David P.; Lew, Helen; Zusman, David R.

    1998-01-01

    Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle. Autochemotaxis, a process whereby cells release a self-generated signaling molecule, may be the principal mechanism facilitating directed motility in both the vegetative swarming and developmental aggregation stages of this life cycle. The process requires the Frz signal transduction system, including FrzZ, a protein which is composed of two domains, both showing homology to the enteric chemotaxis response regulator CheY. The first domain of FrzZ (FrzZ1), when expressed as bait in the yeast two-hybrid system and screened against a library, was shown to potentially interact with the C-terminal portion of a protein encoding an ATP-binding cassette (AbcA). The activation domain-AbcA fusion protein did not interact with the second domain of FrzZ (FrzZ2) or with two other M. xanthus response regulator-containing proteins presented as bait, suggesting that the FrzZ1-AbcA interaction may be specific. Cloning and sequencing of the upstream region of the abcA gene showed the ATP-binding cassette to be linked to a large hydrophobic, potentially membrane-spanning domain. This domain organization is characteristic of a subgroup of ABC transporters which perform export functions. Cloning and sequencing downstream of abcA indicated that the ABC transporter is at the start of an operon containing three open reading frames. An insertion mutation in the abcA gene resulted in cells displaying the frizzy aggregation phenotype, providing additional evidence that FrzZ and AbcA may be part of the same signal transduction pathway. Cells with mutations in genes downstream of abcA showed no developmental defects. Analysis of the proposed exporter role of AbcA in cell mixing experiments showed that the ABC transporter mutant could be rescued by extracellular complementation. We speculate that the AbcA protein may be involved in the export of a molecule required for the autochemotactic process. PMID:9791121

  18. ABC multidrug transporters in schistosomes and other parasitic flatworms

    PubMed Central

    Greenberg, Robert M.

    2013-01-01

    Schistosomiasis, a neglected tropical disease affecting hundreds of millions, is caused by parasitic flatworms of the genus Schistosoma. Treatment and control of schistosomiasis relies almost exclusively on a single drug, praziquantel (PZQ), a dangerous situation for a disease of this magnitude. Though PZQ is highly effective overall, it has drawbacks, and reports of worms showing PZQ resistance, either induced in the laboratory or isolated from the field, are disconcerting. Multidrug transporters underlie multidrug resistance (MDR), a phenomenon in which resistance to a single drug is accompanied by unexpected cross-resistance to several structurally unrelated compounds. Some of the best studied multidrug transporters are members of the ancient and very large ATP-binding cassette (ABC) superfamily of efflux transporters. ABC multidrug transporters such as P-glycoprotein (Pgp; ABCB1) are also associated with drug resistance in parasites, including helminths such as schistosomes. In addition to their association with drug resistance, however, ABC transporters also function in a wide variety of physiological processes in metazoans. In this review, we examine recent studies that help define the role of schistosome ABC transporters in regulating drug susceptibility, and in normal schistosome physiology, including reproduction and excretory activity. We postulate that schistosome ABC transporters could be useful targets for compounds that enhance the effectiveness of current therapeutics as well as for agents that act as antischistosomals on their own. PMID:23474413

  19. Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis.

    PubMed

    Goldman, B S; Beckman, D L; Bali, A; Monika, E M; Gabbert, K K; Kranz, R G

    1997-05-16

    The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis. The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components. Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes. To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated. We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex. The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region. In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins. Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters. PMID:9175857

  20. Adaptive Evolution of Thermotoga maritima Reveals Plasticity of the ABC Transporter Network

    PubMed Central

    Latif, Haythem; Sahin, Merve; Tarasova, Janna; Tarasova, Yekaterina; Portnoy, Vasiliy A.; Nogales, Juan

    2015-01-01

    Thermotoga maritima is a hyperthermophilic anaerobe that utilizes a vast network of ABC transporters to efficiently metabolize a variety of carbon sources to produce hydrogen. For unknown reasons, this organism does not metabolize glucose as readily as it does glucose di- and polysaccharides. The leading hypothesis implicates the thermolability of glucose at the physiological temperatures at which T. maritima lives. After a 25-day laboratory evolution, phenotypes were observed with growth rates up to 1.4 times higher than and glucose utilization rates exceeding 50% those of the wild type. Genome resequencing revealed mutations in evolved cultures related to glucose-responsive ABC transporters. The native glucose ABC transporter, GluEFK, has more abundant transcripts either as a result of gene duplication-amplification or through mutations to the operator sequence regulating this operon. Conversely, BglEFGKL, a transporter of beta-glucosides, is substantially downregulated due to a nonsense mutation to the solute binding protein or due to a deletion of the upstream promoter. Analysis of the ABC2 uptake porter families for carbohydrate and peptide transport revealed that the solute binding protein, often among the transcripts detected at the highest levels, is predominantly downregulated in the evolved cultures, while the membrane-spanning domain and nucleotide binding components are less varied. Similar trends were observed in evolved strains grown on glycerol, a substrate that is not dependent on ABC transporters. Therefore, improved growth on glucose is achieved through mutations favoring GluEFK expression over BglEFGKL, and in lieu of carbon catabolite repression, the ABC transporter network is modulated to achieve improved growth fitness. PMID:26048924

  1. Overcoming ABC transporter-mediated multidrug resistance: Molecular mechanisms and novel therapeutic drug strategies.

    PubMed

    Li, Wen; Zhang, Han; Assaraf, Yehuda G; Zhao, Kun; Xu, Xiaojun; Xie, Jinbing; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-07-01

    Multidrug resistance is a key determinant of cancer chemotherapy failure. One of the major causes of multidrug resistance is the enhanced efflux of drugs by membrane ABC transporters. Targeting ABC transporters projects a promising approach to eliminating or suppressing drug resistance in cancer treatment. To reveal the functional mechanisms of ABC transporters in drug resistance, extensive studies have been conducted from identifying drug binding sites to elucidating structural dynamics. In this review article, we examined the recent crystal structures of ABC proteins to depict the functionally important structural elements, such as domains, conserved motifs, and critical amino acids that are involved in ATP-binding and drug efflux. We inspected the drug-binding sites on ABC proteins and the molecular mechanisms of various substrate interactions with the drug binding pocket. While our continuous battle against drug resistance is far from over, new approaches and technologies have emerged to push forward our frontier. Most recent developments in anti-MDR strategies include P-gp inhibitors, RNA-interference, nano-medicines, and delivering combination strategies. With the advent of the 'Omics' era - genomics, epigenomics, transcriptomics, proteomics, and metabolomics - these disciplines play an important role in fighting the battle against chemoresistance by further unraveling the molecular mechanisms of drug resistance and shed light on medical therapies that specifically target MDR. PMID:27449595

  2. Identification of a new biocontrol gene in Trichoderma atroviride: the role of an ABC transporter membrane pump in the interaction with different plant-pathogenic fungi.

    PubMed

    Ruocco, Michelina; Lanzuise, Stefania; Vinale, Francesco; Marra, Roberta; Turrà, David; Woo, Sheridan Lois; Lorito, Matteo

    2009-03-01

    Successful biocontrol interactions often require that the beneficial microbes involved are resistant or tolerant to a variety of toxicants, including antibiotics produced by themselves or phytopathogens, plant antimicrobial compounds, and synthetic chemicals or contaminants. The ability of Trichoderma spp., the most widely applied biocontrol fungi, to withstand different chemical stresses, including those associated with mycoparasitism, is well known. In this work, we identified an ATP-binding cassette transporter cell membrane pump as an important component of the above indicated resistance mechanisms that appears to be supported by an extensive and powerful cell detoxification system. The encoding gene, named Taabc2, was cloned from a strain of Trichoderma atroviride and characterized. Its expression was found to be upregulated in the presence of pathogen-secreted metabolites, specific mycotoxins and some fungicides, and in conditions that stimulate the production in Trichoderma spp. of antagonism-related factors (toxins and enzymes). The key role of this gene in antagonism and biocontrol was demonstrated by the characterization of the obtained deletion mutants. They suffered an increased susceptibility to inhibitory compounds either secreted by pathogenic fungi or possibly produced by the biocontrol microbe itself and lost, partially or entirely, the ability to protect tomato plants from Pythium ultimum and Rhizoctonia solani attack. PMID:19245323

  3. Conformational coupling of the nucleotide-binding and the transmembrane domains in ABC transporters.

    PubMed

    Wen, Po-Chao; Tajkhorshid, Emad

    2011-08-01

    Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree

  4. Regulation of ABC transporters blood-brain barrier: the good, the bad, and the ugly.

    PubMed

    Miller, David S

    2015-01-01

    The brain capillary endothelial cells that constitute the blood-brain barrier express multiple ABC transport proteins on the luminal, blood-facing, plasma membrane. These transporters function as ATP-driven efflux pumps for xenobiotics and endogenous metabolites. High expression of these ABC transporters at the barrier is a major obstacle to the delivery of therapeutics, including chemotherapeutics, to the CNS. Here, I review the signals that alter ABC transporter expression and transport function with an emphasis on P-glycoprotein, Mrp2, and breast cancer resistance protein (BCRP), the efflux transporters for which we have the most detailed picture of regulation. Recent work shows that transporter protein expression can be upregulated in response to inflammatory and oxidative stress, therapeutic drugs, diet, and persistent environmental pollutants; as a consequence, drug delivery to the brain is reduced (potentially bad and ugly). In contrast, basal transport activity of P-glycoprotein and BCRP can be reduced through complex signaling pathways that involve events in and on the brain capillary endothelial cells. Targeting these signaling events provides opportunities to rapidly and reversibly increase brain accumulation of drugs that are substrates for the transporters (potentially good). The clinical usefulness of targeting signaling to reduce efflux transporter activity and improve drug delivery to the CNS remains to be established. PMID:25640266

  5. Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7).

    PubMed

    Bai, Yonghong; Li, Min; Hwang, Tzyh-Chang

    2011-11-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel. PMID:22042986

  6. Structural basis for the channel function of a degraded ABC transporter, CFTR (ABCC7)

    PubMed Central

    Bai, Yonghong

    2011-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily, but little is known about how this ion channel that harbors an uninterrupted ion permeation pathway evolves from a transporter that works by alternately exposing its substrate conduit to the two sides of the membrane. Here, we assessed reactivity of intracellularly applied thiol-specific probes with cysteine residues substituted into the 12th transmembrane segment (TM12) of CFTR. Our experimental data showing high reaction rates of substituted cysteines toward the probes, strong blocker protection of cysteines against reaction, and reaction-induced alterations in channel conductance support the idea that TM12 of CFTR contributes to the lining of the ion permeation pathway. Together with previous work, these findings raise the possibility that pore-lining elements of CFTR involve structural components resembling those that form the substrate translocation pathway of ABC transporters. In addition, comparison of reaction rates in the open and closed states of the CFTR channel leads us to propose that upon channel opening, the wide cytoplasmic vestibule tightens and the pore-lining TM12 rotates along its helical axis. This simple model for gating conformational changes in the inner pore domain of CFTR argues that the gating transition of CFTR and the transport cycle of ABC proteins share analogous conformational changes. Collectively, our data corroborate the popular hypothesis that degradation of the cytoplasmic-side gate turned an ABC transporter into the CFTR channel. PMID:22042986

  7. Membrane Transport

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The selective movement and redistribution of ions and small organic molecules is essential for plant growth and cellular homeostasis. Because of this, plants have evolved numerous proteins that facilitate the transport of minerals, sugars, metabolites, and other compounds through the limiting membra...

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

    PubMed Central

    Shipp, Lauren E.; Hamdoun, Amro

    2012-01-01

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

  9. Tonoplast-localized Abc2 Transporter Mediates Phytochelatin Accumulation in Vacuoles and Confers Cadmium Tolerance*

    PubMed Central

    Mendoza-Cózatl, David G.; Zhai, Zhiyang; Jobe, Timothy O.; Akmakjian, Garo Z.; Song, Won-Yong; Limbo, Oliver; Russell, Matthew R.; Kozlovskyy, Volodymyr I.; Martinoia, Enrico; Vatamaniuk, Olena K.; Russell, Paul; Schroeder, Julian I.

    2010-01-01

    Phytochelatins mediate tolerance to heavy metals in plants and some fungi by sequestering phytochelatin-metal complexes into vacuoles. To date, only Schizosaccharomyces pombe Hmt1 has been described as a phytochelatin transporter and attempts to identify orthologous phytochelatin transporters in plants and other organisms have failed. Furthermore, recent data indicate that the hmt1 mutant accumulates significant phytochelatin levels in vacuoles, suggesting that unidentified phytochelatin transporters exist in fungi. Here, we show that deletion of all vacuolar ABC transporters abolishes phytochelatin accumulation in S. pombe vacuoles and abrogates 35S-PC2 uptake into S. pombe microsomal vesicles. Systematic analysis of the entire S. pombe ABC transporter family identified Abc2 as a full-size ABC transporter (ABCC-type) that mediates phytochelatin transport into vacuoles. The S. pombe abc1 abc2 abc3 abc4 hmt1 quintuple and abc2 hmt1 double mutant show no detectable phytochelatins in vacuoles. Abc2 expression restores phytochelatin accumulation into vacuoles and suppresses the cadmium sensitivity of the abc quintuple mutant. A novel, unexpected, function of Hmt1 in GS-conjugate transport is also shown. In contrast to Hmt1, Abc2 orthologs are widely distributed among kingdoms and are proposed as the long-sought vacuolar phytochelatin transporters in plants and other organisms. PMID:20937798

  10. Cellodextrin and Laminaribiose ABC Transporters in Clostridium thermocellum▿

    PubMed Central

    Nataf, Yakir; Yaron, Sima; Stahl, Frank; Lamed, Raphael; Bayer, Edward A.; Scheper, Thomas-Helmut; Sonenshein, Abraham L.; Shoham, Yuval

    2009-01-01

    Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only β-1,3 and β-1,4 glucans and prefers long cellodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins (G2 to G5), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose (G3), while another protein, Lbp (laminaribiose-binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins (G3 to G5) are assimilated faster than cellobiose. PMID:18952792

  11. ABC transporter AtABCG25 is involved in abscisic acid transport and responses

    PubMed Central

    Kuromori, Takashi; Miyaji, Takaaki; Yabuuchi, Hikaru; Shimizu, Hidetada; Sugimoto, Eriko; Kamiya, Asako; Moriyama, Yoshinori; Shinozaki, Kazuo

    2010-01-01

    Abscisic acid (ABA) is one of the most important phytohormones involved in abiotic stress responses, seed maturation, germination, and senescence. ABA is predominantly produced in vascular tissues and exerts hormonal responses in various cells, including guard cells. Although ABA responses require extrusion of ABA from ABA-producing cells in an intercellular ABA signaling pathway, the transport mechanisms of ABA through the plasma membrane remain unknown. Here we isolated an ATP-binding cassette (ABC) transporter gene, AtABCG25, from Arabidopsis by genetically screening for ABA sensitivity. AtABCG25 was expressed mainly in vascular tissues. The fluorescent protein-fused AtABCG25 was localized at the plasma membrane in plant cells. In membrane vesicles derived from AtABCG25-expressing insect cells, AtABCG25 exhibited ATP-dependent ABA transport. The AtABCG25-overexpressing plants showed higher leaf temperatures, implying an influence on stomatal regulation. These results strongly suggest that AtABCG25 is an exporter of ABA and is involved in the intercellular ABA signaling pathway. The presence of the ABA transport mechanism sheds light on the active control of multicellular ABA responses to environmental stresses among plant cells. PMID:20133881

  12. Fungal ABC transporters and microbial interactions in natural environments.

    PubMed

    Schoonbeek, Henk-jan; Raaijmakers, Jos M; De Waard, Maarten A

    2002-11-01

    In natural environments, microorganisms are exposed to a wide variety of antibiotic compounds produced by competing organisms. Target organisms have evolved various mechanisms of natural resistance to these metabolites. In this study, the role of ATP-binding cassette (ABC) transporters in interactions between the plant-pathogenic fungus Botrytis cinerea and antibiotic-producing Pseudomonas bacteria was investigated in detail. We discovered that 2,4-diacetylphloroglucinol, phenazine-1-carboxylic acid and phenazine-1-carboxamide (PCN), broad-spectrum antibiotics produced by Pseudomonas spp., induced expression of several ABC transporter genes in B. cinerea. Phenazines strongly induced expression of BcatrB, and deltaBcatrB mutants were significantly more sensitive to these antibiotics than their parental strain. Treatment of B. cinerea germlings with PCN strongly affected the accumulation of [14C]fludioxonil, a phenylpyrrole fungicide known to be transported by BcatrB, indicating that phenazines also are transported by BcatrB. Pseudomonas strains producing phenazines displayed a stronger antagonistic activity in vitro toward ABcatrB mutants than to the parental B. cinerea strain. On tomato leaves, phenazine-producing Pseudomonas strains were significantly more effective in reducing gray mold symptoms incited by a ABcatrB mutant than by the parental strain. We conclude that the ABC transporter BcatrB provides protection to B. cinerea in phenazine-mediated interactions with Pseudomonas spp. Collectively, these results indicate that fungal ABC transporters can play an important role in antibiotic-mediated interactions between bacteria and fungi in plant-associated environments. The implications of these findings for the implementation and sustainability of crop protection by antagonistic microorganisms are discussed. PMID:12423022

  13. Active transporters as enzymes: an energetic framework applied to major facilitator superfamily and ABC importer systems.

    PubMed

    Shilton, Brian H

    2015-04-15

    Active membrane transporters are dynamic molecular machines that catalyse transport across a membrane by coupling solute movement to a source of energy such as ATP or a secondary ion gradient. A central question for many active transporters concerns the mechanism by which transport is coupled to a source of energy. The transport process and associated energetic coupling involve conformational changes in the transporter. For efficient transport, the conformational changes must be tightly regulated and they must link energy use to movement of the substrate across the membrane. The present review discusses active transport using the well-established energetic framework for enzyme-mediated catalysis. In particular, membrane transport systems can be viewed as ensembles consisting of low-energy and high-energy conformations. The transport process involves binding interactions that selectively stabilize the higher energy conformations, and in this way promote conformational changes in the system that are coupled to decreases in free energy and substrate translocation. The major facilitator superfamily of secondary active transporters is used to illustrate these ideas, which are then be expanded to primary active transport mediated by ABC (ATP-binding cassette) import systems, with a focus on the well-studied maltose transporter. PMID:25837849

  14. 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

  15. Know your ABCs: Characterization and gene expression dynamics of ABC transporters in the polyphagous herbivore Helicoverpa armigera.

    PubMed

    Bretschneider, Anne; Heckel, David G; Vogel, Heiko

    2016-05-01

    Polyphagous insect herbivores are adapted to many different secondary metabolites of their host plants. However, little is known about the role of ATP-binding cassette (ABC) transporters, a multigene family involved in detoxification processes. To study the larval response of the generalist Helicoverpa armigera (Lepidoptera) and the putative role of ABC transporters, we performed developmental assays on artificial diet supplemented with secondary metabolites from host plants (atropine-scopolamine, nicotine and tomatine) and non-host plants (taxol) in combination with a replicated RNAseq experiment. A maximum likelihood phylogeny identified the subfamily affiliations of the ABC transporter sequences. Larval performance was equal on the atropine-scopolamine diet and the tomatine diet. For the latter we could identify a treatment-specific upregulation of five ABC transporters in the gut. No significant developmental difference was detected between larvae fed on nicotine or taxol. This was also mirrored in the upregulation of five ABC transporters when fed on either of the two diets. The highest number of differentially expressed genes was recorded in the gut samples in response to feeding on secondary metabolites. Our results are consistent with the expectation of a general detoxification response in a polyphagous herbivore. This is the first study to characterize the multigene family of ABC transporters and identify gene expression changes across different developmental stages and tissues, as well as the impact of secondary metabolites in the agricultural pest H. armigera. PMID:26951878

  16. 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

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

    PubMed

    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

  18. 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. PMID:19244140

  19. Correction: Learning from each other: ABC transporter regulation by protein phosphorylation in plant and mammalian systems.

    PubMed

    Aryal, Bibek; Laurent, Christophe; Geisler, Markus

    2016-04-15

    The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABCB subfamily display very high substrate specificity compared with their mammalian counterparts that are often associated with multidrug resistance (MDR) phenomena. In this review we highlight prominent functions of plant and mammalian ABC transporters and summarize our knowledge on their post-transcriptional regulation with a focus on protein phosphorylation. A deeper comparison of regulatory events of human cystic fibrosis transmembrane conductance regulator (CFTR) and ABCB1 from the model plantArabidopsisreveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins (FKBPs) that chaperon both transporters to the plasma membrane in an action that seems to involve Hsp90. Further both transporters are phosphorylated at regulatory domains that connect both nucleotide-binding folds. Taken together it appears that ABC transporters exhibit an evolutionary conserved but complex regulation by protein phosphorylation, which apparently is, at least in some cases, tightly connected with protein-protein interactions (PPI). PMID:27068986

  20. A Bacterial-Type ABC Transporter Is Involved in Aluminum Tolerance in Rice[W

    PubMed Central

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

    2009-01-01

    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. PMID:19244140

  1. Schistosome ABC multidrug transporters: From pharmacology to physiology

    PubMed Central

    Greenberg, Robert M.

    2014-01-01

    Praziquantel (PZQ) is essentially the only drug currently available for treatment and control of schistosomiasis, a disease affecting hundreds of millions worldwide. Though highly effective overall, PZQ has limitations, most notably its significant lack of activity against immature schistosomes. Furthermore, the availability of only a single drug for a disease of this magnitude makes reports of PZQ-resistant isolates particularly troubling. ATP-binding cassette (ABC) multidrug transporters such as P-glycoprotein (Pgp; ABCB1) are efflux transporters that underlie multidrug resistance (MDR); changes in their expression or structure are also associated with drug resistance in parasites, including helminths. This review will discuss the role these transporters might play in modulating schistosome susceptibility to PZQ, and the implications for developing new or repurposed treatments that enhance the efficacy of PZQ. However, in addition to influencing drug susceptibility, ABC transporters play important roles in several critical physiological functions such as excretion and maintenance of permeability barriers. They also transport signaling molecules with high affinity, and several lines of evidence implicate mammalian transporters in a diverse array of physiological functions, including regulation of immune responses. Like their mammalian counterparts, schistosome ABC transporters appear to be involved in functions critical to the parasite, including excretory activity and reproduction, and we hypothesize that they underlie at least some aspects of parasite–host interactions. Thus, in addition to their potential as targets for enhancers of PZQ susceptibility, these transporters might also serve as candidate targets for agents that disrupt the parasite life cycle and act as antischistosomals on their own. PMID:25516841

  2. Environment sensing and response mediated by ABC transporters

    SciTech Connect

    Giuliani, Sarah E; Frank, Ashley M; Corgliano, Danielle M; Siefert, Catherine; Hauser, Loren John; Collart, Frank

    2011-01-01

    Abstract Background: Transporter proteins are one of an organism s primary interfaces with the environment. The expressed set of transporters mediates cellular metabolic capabilities and influences signal transduction pathways and regulatory networks. The functional annotation of most transporters is currently limited to general classification into families. The development of capabilities to map ligands with specific transporters would improve our knowledge of the function of these proteins, improve the annotation of related genomes, and facilitate predictions for their role in cellular responses to environmental changes. Results: To improve the utility of the functional annotation for ABC transporters, we expressed and purified the set of solute binding proteins from Rhodopseudomonas palustris and characterized their ligand-binding specificity. Our approach utilized ligand libraries consisting of environmental and cellular metabolic compounds, and fluorescence thermal shift based high throughput ligand binding screens. This process resulted in the identification of specific binding ligands for approximately 64% of the purified and screened proteins. The collection of binding ligands is representative of common functionalities associated with many bacterial organisms as well as specific capabilities linked to the ecological niche occupied by R. palustris. Conclusion: The functional screen identified specific ligands that bound to ABC transporter periplasmic binding subunits from R. palustris. These assignments provide unique insight for the metabolic capabilities of this organism and are consistent with the ecological niche of strain isolation. This functional insight can be used to improve the annotation of related organisms and provides a route to evaluate the evolution of this important and diverse group of transporter proteins.

  3. How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

    PubMed

    Theodoulou, Frederica L; Carrier, David J; Schaedler, Theresia A; Baldwin, Stephen A; Baker, Alison

    2016-06-15

    Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data. PMID:27284041

  4. How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?

    PubMed Central

    Theodoulou, Frederica L.; Carrier, David J.; Schaedler, Theresia A.; Baldwin, Stephen A.; Baker, Alison

    2016-01-01

    Import of β-oxidation substrates into peroxisomes is mediated by ATP binding cassette (ABC) transporters belonging to subfamily D. In order to enter the β-oxidation pathway, fatty acids are activated by conversion to fatty acyl-CoA esters, a reaction which is catalysed by acyl-CoA synthetases (ACSs). Here, we present evidence for an unusual transport mechanism, in which fatty acyl-CoA substrates are accepted by ABC subclass D protein (ABCD) transporters, cleaved by the transporters during transit across the lipid bilayer to release CoA, and ultimately re-esterified in the peroxisome lumen by ACSs which interact with the transporter. We propose that this solves the biophysical problem of moving an amphipathic molecule across the peroxisomal membrane, since the intrinsic thioesterase activity of the transporter permits separate membrane translocation pathways for the hydrophobic fatty acid moiety and the polar CoA moiety. The cleavage/re-esterification mechanism also has the potential to control entry of disparate substrates into the β-oxidation pathway when coupled with distinct peroxisomal ACSs. A different solution to the movement of amphipathic molecules across a lipid bilayer is deployed by the bacterial lipid-linked oligosaccharide (LLO) flippase, PglK, in which the hydrophilic head group and the hydrophobic polyprenyl tail of the substrate are proposed to have distinct translocation pathways but are not chemically separated during transport. We discuss a speculative alternating access model for ABCD proteins based on the mammalian ABC transporter associated with antigen processing (TAP) and compare it to the novel mechanism suggested by the recent PglK crystal structures and biochemical data. PMID:27284041

  5. An ABC Transporter Mutation Is Correlated with Insect Resistance to Bacillus thuringiensis Cry1Ac Toxin

    PubMed Central

    Gahan, Linda J.; Pauchet, Yannick; Vogel, Heiko; Heckel, David G.

    2010-01-01

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt–expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field. PMID:21187898

  6. An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin.

    PubMed

    Gahan, Linda J; Pauchet, Yannick; Vogel, Heiko; Heckel, David G

    2010-12-01

    Transgenic crops producing insecticidal toxins from Bacillus thuringiensis (Bt) are commercially successful in reducing pest damage, yet knowledge of resistance mechanisms that threaten their sustainability is incomplete. Insect resistance to the pore-forming Cry1Ac toxin is correlated with the loss of high-affinity, irreversible binding to the mid-gut membrane, but the genetic factors responsible for this change have been elusive. Mutations in a 12-cadherin-domain protein confer some Cry1Ac resistance but do not block this toxin binding in in vitro assays. We sought to identify mutations in other genes that might be responsible for the loss of binding. We employed a map-based cloning approach using a series of backcrosses with 1,060 progeny to identify a resistance gene in the cotton pest Heliothis virescens that segregated independently from the cadherin mutation. We found an inactivating mutation of the ABC transporter ABCC2 that is genetically linked to Cry1Ac resistance and is correlated with loss of Cry1Ac binding to membrane vesicles. ABC proteins are integral membrane proteins with many functions, including export of toxic molecules from the cell, but have not been implicated in the mode of action of Bt toxins before. The reduction in toxin binding due to the inactivating mutation suggests that ABCC2 is involved in membrane integration of the toxin pore. Our findings suggest that ABC proteins may play a key role in the mode of action of Bt toxins and that ABC protein mutations can confer high levels of resistance that could threaten the continued utilization of Bt-expressing crops. However, such mutations may impose a physiological cost on resistant insects, by reducing export of other toxins such as plant secondary compounds from the cell. This weakness could be exploited to manage this mechanism of Bt resistance in the field. PMID:21187898

  7. Identification and characterization of a Streptococcus pyogenes ABC transporter with multiple specificity for metal cations.

    PubMed

    Janulczyk, R; Pallon, J; Björck, L

    1999-11-01

    Metal ions are crucial trace elements for bacteria infecting the human host. The LraI (lipoprotein receptor-associated antigen I) transporter in Streptococcus spp. belongs to the superfamily of ABC transporters. The transporter consists of a lipoprotein, an ATP-binding protein and a hydrophobic integral membrane protein. Here, we describe a new member of the LraI family in the important human pathogen Streptococcus pyogenes. The system was identified in silico by analysis of the S. pyogenes Genome Sequencing Project. The S. pyogenes operon exhibits an atypical organization compared with equivalents in other Streptococcus spp. The presence and atypical organization of the operon was verified in a number of S. pyogenes strains of different serotypes. Transcriptional analysis of the LraI operon demonstrates a polycistronic transcription attenuated by a stable stem-loop structure, which allows the lipoprotein to be expressed in larger quantities than the other two components. The localization of the native lipoprotein at the bacterial surface was shown by proteolytic digestion of S. pyogenes bacteria and NH2-terminal sequencing of a released lipoprotein fragment. Recombinant lipoprotein was expressed as a GST fusion protein, and studies of molecular interactions with metal radioisotopes demonstrated that the protein has affinity for Zn(II), Fe(III) and Cu(II). Zn(II) and Cu(II) were found to compete for the same binding site, whereas Fe(III) uses a second site. Also, proton-induced X-ray analysis of lipoprotein samples identified iron, copper and zinc. Finally, a mutant strain lacking a functional mtsABC operon was generated and showed reduced uptake of 55Fe and 65Zn compared with the wild-type strain. The operon encoding this novel ABC transporter with multiple specificity for metal cations is designated mtsABC, for metal transporter of Streptococcus. PMID:10564500

  8. Release of Entropic Spring Reveals Conformational Coupling Mechanism in the ABC Transporter BtuCD-F.

    PubMed

    Prieß, Marten; Schäfer, Lars V

    2016-06-01

    Substrate translocation by ATP-binding cassette (ABC) transporters involves coupling of ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) to conformational changes in the transmembrane domains. We used molecular dynamics simulations to investigate the atomic-level mechanism of conformational coupling in the ABC transporter BtuCD-F, which imports vitamin B12 across the inner membrane of Escherichia coli. Our simulations show how an engineered disulfide bond across the NBD dimer interface reduces conformational fluctuations and hence configurational entropy. As a result, the disulfide bond is under substantial mechanical stress. Releasing this entropic spring, as is the case in the wild-type transporter, combined with analyzing the pairwise forces between individual residues, unravels the coupling mechanism. The identified pathways along which force is propagated from the NBDs via the coupling helix to the transmembrane domains are composed of highly conserved residues, underlining their functional relevance. This study not only reveals the details of conformational coupling in BtuCD-F, it also provides a promising approach to other long-range conformational couplings, e.g., in ABC exporters or other ATP-driven molecular machines. PMID:27276259

  9. [Structural and Pharmacological Studies of an ABC Multidrug Transporter].

    PubMed

    Yamaguchi, Tomohiro

    2016-01-01

    A drug's effectiveness against a disease depends not only on its interaction with receptors but also its pharmacokinetics (absorption, distribution, metabolism, and extrusion; ADME). ATP binding cassette (ABC) multidrug transporters are important proteins that influence the ADME properties of a drug, especially the ABC transporter subfamily B member 1 (ABCB1). Elucidation of the molecular mechanisms of ABCB1 will contribute to our understanding of the molecular basis of ADME. Human ABCB1 is expressed in many organelles, and exports various substrates from cells using energy generated by its ATP hydrolase (ATPase) activity. The ATPase activity depends on the concentration of the transport substrates, and the characteristic behavior of the substrate-dependent ATPase activity can be related to the molecular mechanism of ABCB1. Recently, we have revealed the molecular mechanisms of a eukaryotic ABCB1 homolog, CmABCB1, based on structural and functional studies. In this review, I discuss the relationship between key structural features and the behavior of transport substrate-dependent ATPase activity of CmABCB1, including its role in determining the molecular basis of ADME. PMID:26831793

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

    PubMed Central

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

    1995-01-01

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

  11. [Role of ABC efflux transporters in the oral bioavailability and drug-induced intestinal toxicity].

    PubMed

    Yokooji, Tomoharu

    2013-01-01

    The gastrointestinal tract is the organ that absorbs nutrients and water from foods and drinks. This organ is often exposed to various harmful xenobiotics, and therefore possesses various detoxification/barrier systems, including metabolizing enzymes and efflux transporters. Intestinal epithelial cells express ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein, multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein, in addition to various solute carrier (SLC) influx transporters. These transporters are expressed site- and membrane-specifically in enterocytes, which affects the bioavailability of ingested substrate drugs. Expression and/or function of transporters can be modulated by various compounds, including therapeutic drugs, herbal products, some foods, and by disease states. The modulation of transporters could cause unexpectedly higher or lower blood concentrations, marked inter- and intra-individual variations in pharmacokinetics, and unreliable pharmacological actions in association with toxicities of substrates. Recently, we found that hyperbilirubinemia, which occurs in some disease states, increased intestinal accumulation and toxicity of methotrexate, an MRP substrate, because of the suppression of MRP function by high plasma concentrations of conjugated bilirubin. We also attempted to ameliorate the intestinal toxicity of irinotecan hydrochloride by modulating the hepatic and intestinal functions of MRP2. This review summarizes our findings regarding the role of ABC transporters, especially MRPs, in oral bioavailability and in drug-induced intestinal toxicity. Our approach to treat intestinal toxicity using an MRP2 modulator is also described. PMID:23811769

  12. Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence

    PubMed Central

    Khandelwal, Nitesh Kumar; Kaemmer, Philipp; Förster, Toni M.; Singh, Ashutosh; Coste, Alix T.; Andes, David R.; Hube, Bernhard; Sanglard, Dominique; Chauhan, Neeraj; Kaur, Rupinder; d'Enfert, Christophe; Mondal, Alok Kumar; Prasad, Rajendra

    2016-01-01

    Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans. Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. PMID:27026051

  13. Pleiotropic effects of the vacuolar ABC transporter MLT1 of Candida albicans on cell function and virulence.

    PubMed

    Khandelwal, Nitesh Kumar; Kaemmer, Philipp; Förster, Toni M; Singh, Ashutosh; Coste, Alix T; Andes, David R; Hube, Bernhard; Sanglard, Dominique; Chauhan, Neeraj; Kaur, Rupinder; d'Enfert, Christophe; Mondal, Alok Kumar; Prasad, Rajendra

    2016-06-01

    Among the several mechanisms that contribute to MDR (multidrug resistance), the overexpression of drug-efflux pumps belonging to the ABC (ATP-binding cassette) superfamily is the most frequent cause of resistance to antifungal agents. The multidrug transporter proteins Cdr1p and Cdr2p of the ABCG subfamily are major players in the development of MDR in Candida albicans Because several genes coding for ABC proteins exist in the genome of C. albicans, but only Cdr1p and Cdr2p have established roles in MDR, it is implicit that the other members of the ABC family also have alternative physiological roles. The present study focuses on an ABC transporter of C. albicans, Mlt1p, which is localized in the vacuolar membrane and specifically transports PC (phosphatidylcholine) into the vacuolar lumen. Transcriptional profiling of the mlt1∆/∆ mutant revealed a down-regulation of the genes involved in endocytosis, oxidoreductase activity, virulence and hyphal development. High-throughput MS-based lipidome analysis revealed that the Mlt1p levels affect lipid homoeostasis and thus lead to a plethora of physiological perturbations. These include a delay in endocytosis, inefficient sequestering of reactive oxygen species (ROS), defects in hyphal development and attenuated virulence. The present study is an emerging example where new and unconventional roles of an ABC transporter are being identified. PMID:27026051

  14. 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

  15. Characterization of berberine transport into Coptis japonica cells and the involvement of ABC protein.

    PubMed

    Sakai, Kyoko; Shitan, Nobukazu; Sato, Fumihiko; Ueda, Kazumitsu; Yazaki, Kazufumi

    2002-09-01

    Cultured Coptis japonica cells are able to take up berberine, a benzylisoquinoline alkaloid, from the medium and transport it exclusively into the vacuoles. Uptake activity depends on the growth phase of the cultured cells whereas the culture medium had no effect on uptake. Treatment with several inhibitors suggested that berberine uptake depended on the ATP level. Some inhibitors of P-glycoprotein, an ABC transporter involved in multiple drug resistance in cancer cells, strongly inhibited berberine uptake, whereas a specific inhibitor for glutathione biosynthesis and vacuolar ATPase, bafilomycin A1, had little effect. Vanadate-induced ATP trap experiments to detect ABC proteins expressed in C. japonica cells showed that three membrane proteins of between 120 and 150 kDa were photolabelled with 8-azido-[alpha-32P] ATP. Two revealed the same photoaffinity-labelling pattern as P-glycoprotein, and the interaction of these proteins with berberine was also demonstrated. These results suggest that ABC proteins of the MDR-type are involved in the uptake of berberine from the medium. PMID:12177126

  16. Distribution and Physiology of ABC-Type Transporters Contributing to Multidrug Resistance in Bacteria

    PubMed Central

    Lubelski, Jacek; Konings, Wil N.; Driessen, Arnold J. M.

    2007-01-01

    Summary: Membrane proteins responsible for the active efflux of structurally and functionally unrelated drugs were first characterized in higher eukaryotes. To date, a vast number of transporters contributing to multidrug resistance (MDR transporters) have been reported for a large variety of organisms. Predictions about the functions of genes in the growing number of sequenced genomes indicate that MDR transporters are ubiquitous in nature. The majority of described MDR transporters in bacteria use ion motive force, while only a few systems have been shown to rely on ATP hydrolysis. However, recent reports on MDR proteins from gram-positive organisms, as well as genome analysis, indicate that the role of ABC-type MDR transporters in bacterial drug resistance might be underestimated. Detailed structural and mechanistic analyses of these proteins can help to understand their molecular mode of action and may eventually lead to the development of new strategies to counteract their actions, thereby increasing the effectiveness of drug-based therapies. This review focuses on recent advances in the analysis of ABC-type MDR transporters in bacteria. PMID:17804667

  17. Interdomain regulation of the ATPase activity of the ABC transporter haemolysin B from Escherichia coli.

    PubMed

    Reimann, Sven; Poschmann, Gereon; Kanonenberg, Kerstin; Stühler, Kai; Smits, Sander H J; Schmitt, Lutz

    2016-08-15

    Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB. PMID:27279651

  18. The modulation of ABC transporter-mediated multidrug resistance in cancer: a review of the past decade.

    PubMed

    Kathawala, Rishil J; Gupta, Pranav; Ashby, Charles R; Chen, Zhe-Sheng

    2015-01-01

    ATP-binding cassette (ABC) transporters represent one of the largest and oldest families of membrane proteins in all extant phyla from prokaryotes to humans, which couple the energy derived from ATP hydrolysis essentially to translocate, among various substrates, toxic compounds across the membrane. The fundamental functions of these multiple transporter proteins include: (1) conserved mechanisms related to nutrition and pathogenesis in bacteria, (2) spore formation in fungi, and (3) signal transduction, protein secretion and antigen presentation in eukaryotes. Moreover, one of the major causes of multidrug resistance (MDR) and chemotherapeutic failure in cancer therapy is believed to be the ABC transporter-mediated active efflux of a multitude of structurally and mechanistically distinct cytotoxic compounds across membranes. It has been postulated that ABC transporter inhibitors known as chemosensitizers may be used in combination with standard chemotherapeutic agents to enhance their therapeutic efficacy. The current paper reviews the advance in the past decade in this important domain of cancer chemoresistance and summarizes the development of new compounds and the re-evaluation of compounds originally designed for other targets as transport inhibitors of ATP-dependent drug efflux pumps. PMID:25554624

  19. A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.

    PubMed

    Ryu, Jaewook; Lee, Ukjin; Park, Jiye; Yoo, Do-Hyun; Ahn, Jung Hoon

    2015-03-01

    Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species. PMID:25548043

  20. Use of Baculovirus BacMam Vectors for Expression of ABC Drug Transporters in Mammalian Cells

    PubMed Central

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul

    2012-01-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  1. Use of baculovirus BacMam vectors for expression of ABC drug transporters in mammalian cells.

    PubMed

    Shukla, Suneet; Schwartz, Candice; Kapoor, Khyati; Kouanda, Abdul; Ambudkar, Suresh V

    2012-02-01

    ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters. PMID:22041108

  2. Screening of Streptococcus pneumoniae ABC transporter mutants demonstrates that LivJHMGF, a branched-chain amino acid ABC transporter, is necessary for disease pathogenesis.

    PubMed

    Basavanna, Shilpa; Khandavilli, Suneeta; Yuste, Jose; Cohen, Jonathan M; Hosie, Arthur H F; Webb, Alexander J; Thomas, Gavin H; Brown, Jeremy S

    2009-08-01

    Bacterial ABC transporters are an important class of transmembrane transporters that have a wide variety of substrates and are important for the virulence of several bacterial pathogens, including Streptococcus pneumoniae. However, many S. pneumoniae ABC transporters have yet to be investigated for their role in virulence. Using insertional duplication mutagenesis mutants, we investigated the effects on virulence and in vitro growth of disruption of 9 S. pneumoniae ABC transporters. Several were partially attenuated in virulence compared to the wild-type parental strain in mouse models of infection. For one ABC transporter, required for full virulence and termed LivJHMGF due to its similarity to branched-chain amino acid (BCAA) transporters, a deletion mutant (DeltalivHMGF) was constructed to investigate its phenotype in more detail. When tested by competitive infection, the DeltalivHMGF strain had reduced virulence in models of both pneumonia and septicemia but was fully virulent when tested using noncompetitive experiments. The DeltalivHMGF strain had no detectable growth defect in defined or complete laboratory media. Recombinant LivJ, the substrate binding component of the LivJHMGF, was shown by both radioactive binding experiments and tryptophan fluorescence spectroscopy to specifically bind to leucine, isoleucine, and valine, confirming that the LivJHMGF substrates are BCAAs. These data demonstrate a previously unsuspected role for BCAA transport during infection for S. pneumoniae and provide more evidence that functioning ABC transporters are required for the full virulence of bacterial pathogens. PMID:19470745

  3. The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes

    PubMed Central

    Sheps, Jonathan A; Ralph, Steven; Zhao, Zhongying; Baillie, David L; Ling, Victor

    2004-01-01

    Background Many drugs of natural origin are hydrophobic and can pass through cell membranes. Hydrophobic molecules must be susceptible to active efflux systems if they are to be maintained at lower concentrations in cells than in their environment. Multi-drug resistance (MDR), often mediated by intrinsic membrane proteins that couple energy to drug efflux, provides this function. All eukaryotic genomes encode several gene families capable of encoding MDR functions, among which the ABC transporters are the largest. The number of candidate MDR genes means that study of the drug-resistance properties of an organism cannot be effectively carried out without taking a genomic perspective. Results We have annotated sequences for all 60 ABC transporters from the Caenorhabditis elegans genome, and performed a phylogenetic analysis of these along with the 49 human, 30 yeast, and 57 fly ABC transporters currently available in GenBank. Classification according to a unified nomenclature is presented. Comparison between genomes reveals much gene duplication and loss, and surprisingly little orthology among analogous genes. Proteins capable of conferring MDR are found in several distinct subfamilies and are likely to have arisen independently multiple times. Conclusions ABC transporter evolution fits a pattern expected from a process termed 'dynamic-coherence'. This is an unusual result for such a highly conserved gene family as this one, present in all domains of cellular life. Mechanistically, this may result from the broad substrate specificity of some ABC proteins, which both reduces selection against gene loss, and leads to the facile sorting of functions among paralogs following gene duplication. PMID:15003118

  4. Gangliosides do not affect ABC transporter function in human neuroblastoma cells.

    PubMed

    Dijkhuis, Anne-Jan; Klappe, Karin; Kamps, Willem; Sietsma, Hannie; Kok, Jan Willem

    2006-06-01

    Previous studies have indicated a role for glucosylceramide synthase (GCS) in multidrug resistance (MDR), either related to turnover of ceramide (Cer) or generation of gangliosides, which modulate apoptosis and/or the activity of ABC transporters. This study challenges the hypothesis that gangliosides modulate the activity of ABC transporters and was performed in two human neuroblastoma cell lines, expressing either functional P-glycoprotein (Pgp) or multidrug resistance-related protein 1 (MRP1). Two inhibitors of GCS, D,L-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (t-PPPP) and N-butyldeoxynojirimycin (NB-dNJ), very efficiently depleted ganglioside content in two human neuroblastoma cell lines. This was established by three different assays: equilibrium radiolabeling, cholera toxin binding, and mass analysis. Fluorescence-activated cell sorting (FACS) analysis showed that ganglioside depletion only slightly and in the opposite direction affected Pgp- and MRP1-mediated efflux activity. Moreover, both effects were marginal compared with those of well-established inhibitors of either MRP1 (i.e., MK571) or Pgp (i.e., GF120918). t-PPPP slightly enhanced cellular sensitivity to vincristine, as determined by 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide analysis, in both neuroblastoma cell lines, whereas NB-dNJ was without effect. MRP1 expression and its localization in detergent-resistant membranes were not affected by ganglioside depletion. Together, these results show that gangliosides are not relevant to ABC transporter-mediated MDR in neuroblastoma cells. PMID:16547352

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

    PubMed Central

    Choi, Cheol-Hee

    2005-01-01

    One of the major problems related with anticancer chemotherapy is resistance against anticancer drugs. The ATP-binding cassette (ABC) transporters are a family of transporter proteins that are responsible for drug resistance and a low bioavailability of drugs by pumping a variety of drugs out cells at the expense of ATP hydrolysis. One strategy for reversal of the resistance of tumor cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. In this review, the physiological functions and structures of ABC transporters, and the development of chemosensitizers are described focusing on well-known proteins including P-glycoprotein, multidrug resistance associated protein, and breast cancer resistance protein. PMID:16202168

  6. Genetic identification of three ABC transporters as essential elements for nitrate respiration in Haloferax volcanii.

    PubMed Central

    Wanner, C; Soppa, J

    1999-01-01

    More than 40 nitrate respiration-deficient mutants of Haloferax volcanii belonging to three different phenotypic classes were isolated. All 15 mutants of the null phenotype were complemented with a genomic library of the wild type. Wild-type copies of mutated genes were recovered from complemented mutants using two different approaches. The DNA sequences of 13 isolated fragments were determined. Five fragments were found to overlap; therefore nine different genomic regions containing genes essential for nitrate respiration could be identified. Three genomic regions containing genes coding for subunits of ABC transporters were further characterized. In two cases, genes coding for an ATP-binding subunit and a permease subunit were clustered and overlapped by four nucleotides. The third gene for a permease subunit had no additional ABC transporter gene in proximity. One ABC transporter was found to be glucose specific. The mutant reveals that the ABC transporter solely mediates anaerobic glucose transport. Based on sequence similarity, the second ABC transporter is proposed to be molybdate specific, explaining its essential role in nitrate respiration. The third ABC transporter is proposed to be anion specific. Genome sequencing has shown that ABC transporters are widespread in Archaea. Nevertheless, this study represents only the second example of a functional characterization. PMID:10430572

  7. Imidazolium-containing, hydrophobic-ionic-hydrophilic ABC triblock copolymers: synthesis, ordered phase-separation, and supported membrane fabrication

    SciTech Connect

    Wiesenauer, EF; Nguyen, PT; Newell, BS; Bailey, TS; Nobleb, RD; Gin, DL

    2013-01-01

    Novel ABC triblock copolymers containing hydrophobic, imidazolium ionic liquid (IL)-based ionic, and non-charged hydrophilic blocks were synthesized by direct sequential, ring-opening metathesis polymerization (ROMP) of three chemically immiscible norborene monomers. The resulting ABC triblock copolymers were found by small-angle X-ray scattering to phase-separate into different nanostructures in their pure melt states, depending on their block sequence and compositions. Supported composite membranes of these triblock copolymers were successfully fabricated with defect-free, <= 20 microns thick top coatings. Preliminary CO2/light gas transport studies demonstrated the potential of this new type of IL-based block copolymer material for gas separation applications.

  8. Research Progress on the Role of ABC Transporters in the Drug Resistance Mechanism of Intractable Epilepsy

    PubMed Central

    Xiong, Jie; Mao, Ding-an; Liu, Li-qun

    2015-01-01

    The pathogenesis of intractable epilepsy is not fully clear. In recent years, both animal and clinical trials have shown that the expression of ATP-binding cassette (ABC) transporters is increased in patients with intractable epilepsy; additionally, epileptic seizures can lead to an increase in the number of sites that express ABC transporters. These findings suggest that ABC transporters play an important role in the drug resistance mechanism of epilepsy. ABC transporters can perform the funcions of a drug efflux pump, which can reduce the effective drug concentration at epilepsy lesions by reducing the permeability of the blood brain barrier to antiepileptic drugs, thus causing resistance to antiepileptic drugs. Given the important role of ABC transporters in refractory epilepsy drug resistance, antiepileptic drugs that are not substrates of ABC transporters were used to obtain ABC transporter inhibitors with strong specificity, high safety, and few side effects, making them suitable for long-term use; therefore, these drugs can be used for future clinical treatment of intractable epilepsy. PMID:26491660

  9. Characterization of Two ABC Transporters from Biocontrol and Phytopathogenic Fusarium oxysporus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ABC transporter genes from four strains of Fusarium oxysporum [two biocontrol and two phytopathogenic (f. sp. lycopersici Race 1) isolates] indicated that this gene is well conserved. However, sequences of promoter regions of FoABC1 differed between 8 phytopathogenic and 11 biocontrol strains of F....

  10. The multidrug transporter Pdr5 on the 25th anniversary of its discovery: an important model for the study of asymmetric ABC transporters

    PubMed Central

    Golin, John; Ambudkar, Suresh V.

    2016-01-01

    Asymmetric ABC (ATP-binding cassette) transporters make up a significant proportion of this important superfamily of integral membrane proteins. These proteins contain one canonical (catalytic) ATP-binding site and a second atypical site with little enzymatic capability. The baker’s yeast (Saccharomyces cerevisiae) Pdr5 multidrug transporter is the founding member of the Pdr subfamily of asymmetric ABC transporters, which exist only in fungi and slime moulds. Because these organisms are of considerable medical and agricultural significance, Pdr5 has been studied extensively, as has its medically important homologue Cdr1 from Candida albicans. Genetic and biochemical analyses of Pdr5 have contributed important observations that are likely to be applicable to mammalian asymmetric ABC multidrug transporter proteins, including the basis of transporter promiscuity, the function of the non-catalytic deviant ATP-binding site, the most complete description of an in vivo transmission interface, and the recent discovery that Pdr5 is a molecular diode (one-way gate). In the present review, we discuss the observations made with Pdr5 and compare them with findings from clinically important asymmetric ABC transporters, such as CFTR (cystic fibrosis transmembrane conductance regulator), Cdr1 and Tap1/Tap2. PMID:25886173

  11. The multidrug transporter Pdr5 on the 25th anniversary of its discovery: an important model for the study of asymmetric ABC transporters.

    PubMed

    Golin, John; Ambudkar, Suresh V

    2015-05-01

    Asymmetric ABC (ATP-binding cassette) transporters make up a significant proportion of this important superfamily of integral membrane proteins. These proteins contain one canonical (catalytic) ATP-binding site and a second atypical site with little enzymatic capability. The baker's yeast (Saccharomyces cerevisiae) Pdr5 multidrug transporter is the founding member of the Pdr subfamily of asymmetric ABC transporters, which exist only in fungi and slime moulds. Because these organisms are of considerable medical and agricultural significance, Pdr5 has been studied extensively, as has its medically important homologue Cdr1 from Candida albicans. Genetic and biochemical analyses of Pdr5 have contributed important observations that are likely to be applicable to mammalian asymmetric ABC multidrug transporter proteins, including the basis of transporter promiscuity, the function of the non-catalytic deviant ATP-binding site, the most complete description of an in vivo transmission interface, and the recent discovery that Pdr5 is a molecular diode (one-way gate). In the present review, we discuss the observations made with Pdr5 and compare them with findings from clinically important asymmetric ABC transporters, such as CFTR (cystic fibrosis transmembrane conductance regulator), Cdr1 and Tap1/Tap2. PMID:25886173

  12. 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

  13. Molecular dynamics simulations of the bacterial ABC transporter SAV1866 in the closed form.

    PubMed

    St-Pierre, Jean-François; Bunker, Alex; Róg, Tomasz; Karttunen, Mikko; Mousseau, Normand

    2012-03-01

    The ATP binding cassette (ABC) transporter family of proteins contains members involved in ATP-mediated import or export of ligands at the cell membrane. For the case of exporters, the translocation mechanism involves a large-scale conformational change that involves a clothespin-like motion from an inward-facing open state, able to bind ligands and adenosine triphosphate (ATP), to an outward-facing closed state. Our work focuses on SAV1866, a bacterial member of the ABC transporter family for which the structure is known for the closed state. To evaluate the ability of this protein to undergo conformational changes at physiological temperature, we first performed conventional molecular dynamics (MD) on the cocrystallized adenosine diphosphate (ADP)-bound structure and on a nucleotide-free structure. With this assessment of SAV1866's stability, conformational changes were induced by steered molecular dynamics (SMD), in which the nucleotide binding domains (NBD) were pushed apart, simulating the ATP hydrolysis energy expenditure. We found that the transmembrane domain is not easily perturbed by large-scale motions of the NBDs. PMID:22339391

  14. An asymmetric post-hydrolysis state of the ABC transporter ATPase dimer.

    PubMed

    George, Anthony M; Jones, Peter M

    2013-01-01

    ABC transporters are a superfamily of enzyme pumps that hydrolyse ATP in exchange for translocation of substrates across cellular membranes. Architecturally, ABC transporters are a dimer of transmembrane domains coupled to a dimer of nucleotide binding domains (NBDs): the NBD dimer contains two ATP-binding sites at the intersubunit interface. A current controversy is whether the protomers of the NBD dimer separate during ATP hydrolysis cycling, or remain in constant contact. In order to investigate the ABC ATPase catalytic mechanism, MD simulations using the recent structure of the ADP+Pi-bound MJ0796 isolated NBD dimer were performed. In three independent simulations of the ADP+Pi/apo state, comprising a total of >0.5 µs, significant opening of the apo (empty) active site was observed; occurring by way of intrasubunit rotations between the core and helical subdomains within both NBD monomers. In contrast, in three equivalent simulations of the ATP/apo state, the NBD dimer remained close to the crystal structure, and no opening of either active site occurred. The results thus showed allosteric coupling between the active sites, mediated by intrasubunit conformational changes. Opening of the apo site is exquisitely tuned to the nature of the ligand, and thus to the stage of the reaction cycle, in the opposite site. In addition to this, in also showing how one active site can open, sufficient to bind nucleotide, while the opposite site remains occluded and bound to the hydrolysis products ADP+Pi, the results are consistent with a Constant Contact Model. Conversely, they show how there may be no requirement for the NBD protomers to separate to complete the catalytic cycle. PMID:23573213

  15. A subset of annular lipids is linked to the flippase activity of an ABC transporter

    NASA Astrophysics Data System (ADS)

    Bechara, Chérine; Nöll, Anne; Morgner, Nina; Degiacomi, Matteo T.; Tampé, Robert; Robinson, Carol V.

    2015-03-01

    Lipids are critical components of membranes that could affect the properties of membrane proteins, yet the precise compositions of lipids surrounding membrane-embedded protein complexes is often difficult to discern. Here we report that, for the heterodimeric ABC transporter TmrAB, the extent of delipidation can be controlled by timed exposure to detergent. We subsequently characterize the cohort of endogenous lipids that are extracted in contact with the membrane protein complex, and show that with prolonged delipidation the number of neutral lipids is reduced in favour of their negatively charged counterparts. We show that lipid A is retained by the transporter and that the extent of its binding decreases during the catalytic cycle, implying that lipid A release is linked to adenosine tri-phosphate hydrolysis. Together, these results enable us to propose that a subset of annular lipids is invariant in composition, with negatively charged lipids binding tightly to TmrAB, and imply a role for this exporter in glycolipid translocation.

  16. A subset of annular lipids is linked to the flippase activity of an ABC transporter.

    PubMed

    Bechara, Chérine; Nöll, Anne; Morgner, Nina; Degiacomi, Matteo T; Tampé, Robert; Robinson, Carol V

    2015-03-01

    Lipids are critical components of membranes that could affect the properties of membrane proteins, yet the precise compositions of lipids surrounding membrane-embedded protein complexes is often difficult to discern. Here we report that, for the heterodimeric ABC transporter TmrAB, the extent of delipidation can be controlled by timed exposure to detergent. We subsequently characterize the cohort of endogenous lipids that are extracted in contact with the membrane protein complex, and show that with prolonged delipidation the number of neutral lipids is reduced in favour of their negatively charged counterparts. We show that lipid A is retained by the transporter and that the extent of its binding decreases during the catalytic cycle, implying that lipid A release is linked to adenosine tri-phosphate hydrolysis. Together, these results enable us to propose that a subset of annular lipids is invariant in composition, with negatively charged lipids binding tightly to TmrAB, and imply a role for this exporter in glycolipid translocation. PMID:25698336

  17. The cloning of a human ABC gene (ABC3) mapping to chromosome 16p13.3

    SciTech Connect

    Connors, T.D.; Van Raay, T.J.; Petry, L.R.

    1997-01-15

    The ATP binding cassette (ABC) transporters, or traffic ATPases, constitute a large family of proteins responsible for the transport of a wide variety of substrates across cell membranes in both prokaryotic and eukaryotic cells. We describe a human ABC protein with regions of strong homology to the recently described murine ABC1 and ABC2 transporters. The gene for this novel protein, human ABC3, maps near the polycystic kidney disease type 1 (PKD1) gene on chromosome 16p13.3. The ABC3 gene is expressed at highest levels in lung compared to other tissues. 19 refs., 3 figs.

  18. OXYGEN TRANSPORT CERAMIC MEMBRANES

    SciTech Connect

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2002-04-01

    This report covers the following tasks: Task 1--Design, fabricate and evaluate ceramic to metal seals based on graded ceramic powder/metal braze joints; Task 2--Evaluate the effect of defect configuration on ceramic membrane conductivity and long term chemical and structural stability; Task 3--Determine materials mechanical properties under conditions of high temperatures and reactive atmospheres; Task 4--Evaluate phase stability and thermal expansion of candidate perovskite membranes and develop techniques to support these materials on porous metal structures; Task 5--Assess the microstructure of membrane materials to evaluate the effects of vacancy-impurity association, defect clusters, and vacancy-dopant association on the membrane performance and stability; and Task 6--Measure kinetics of oxygen uptake and transport in ceramic membrane materials under commercially relevant conditions using isotope labeling techniques.

  19. Membrane Transport Phenomena (MTP)

    NASA Technical Reports Server (NTRS)

    Mason, Larry W.

    1997-01-01

    The third semi-annual period of the MTP project has been involved with performing experiments using the Membrane Transport Apparatus (MTA), development of analysis techniques for the experiment results, analytical modeling of the osmotic transport phenomena, and completion of a DC-9 microgravity flight to test candidate fluid cell geometries. Preparations were also made for the MTP Science Concept Review (SCR), held on 13 June 1997 at Lockheed Martin Astronautics in Denver. These activities are detailed in the report.

  20. OXYGEN TRANSPORT CERAMIC MEMBRANES

    SciTech Connect

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2000-10-01

    This is the third quarterly report on oxygen Transport Ceramic Membranes. In the following, the report describes the progress made by our university partners in Tasks 1 through 6, experimental apparatus that was designed and built for various tasks of this project, thermodynamic calculations, where applicable and work planned for the future. (Task 1) Design, fabricate and evaluate ceramic to metal seals based on graded ceramic powder/metal braze joints. (Task 2) Evaluate the effect of defect configuration on ceramic membrane conductivity and long term chemical and structural stability. (Task 3) Determine materials mechanical properties under conditions of high temperatures and reactive atmospheres. (Task 4) Evaluate phase stability and thermal expansion of candidate perovskite membranes and develop techniques to support these materials on porous metal structures. (Task 5) Assess the microstructure of membrane materials to evaluate the effects of vacancy-impurity association, defect clusters, and vacancy-dopant association on the membrane performance and stability. (Task 6) Measure kinetics of oxygen uptake and transport in ceramic membrane materials under commercially relevant conditions using isotope labeling techniques.

  1. Electron transfer between the QmoABC membrane complex and adenosine 5'-phosphosulfate reductase.

    PubMed

    Duarte, Américo G; Santos, André A; Pereira, Inês A C

    2016-04-01

    The dissimilatory adenosine 5'-phosphosulfate reductase (AprAB) is a key enzyme in the sulfate reduction pathway that catalyzes the reversible two electron reduction of adenosine 5'-phosphosulfate (APS) to sulfite and adenosine monophosphate (AMP). The physiological electron donor for AprAB is proposed to be the QmoABC membrane complex, coupling the quinone-pool to sulfate reduction. However, direct electron transfer between these two proteins has never been observed. In this work we demonstrate for the first time direct electron transfer between the Desulfovibrio desulfuricans ATCC 27774 QmoABC complex and AprAB. Cyclic voltammetry conducted with the modified Qmo electrode and AprAB in the electrolyte solution presented the Qmo electrochemical signature with two additional well-defined one electron redox processes, attributed to the AprAB FAD redox behavior. Moreover, experiments performed under catalytic conditions using the QmoABC modified electrode, with AprAB and APS in solution, show a catalytic current peak develop in the cathodic wave, attributed to substrate reduction, and which is not observed in the absence of QmoABC. Substrate dependence conducted with different electrode preparations (with and without immobilized Qmo) demonstrated that the QmoABC complex is essential for efficient electron delivery to AprAB, in order to sustain catalysis. These results confirm the role of Qmo in electron transfer to AprAB. PMID:26768116

  2. 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. PMID:15826647

  3. A New ABC Half-Transporter in Leishmania major Is Involved in Resistance to Antimony

    PubMed Central

    Manzano, J. I.; García-Hernández, R.; Castanys, S.

    2013-01-01

    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. PMID:23716044

  4. Hydrogen transport membranes

    DOEpatents

    Mundschau, Michael V.

    2005-05-31

    Composite hydrogen transport membranes, which are used for extraction of hydrogen from gas mixtures are provided. Methods are described for supporting metals and metal alloys which have high hydrogen permeability, but which are either too thin to be self supporting, too weak to resist differential pressures across the membrane, or which become embrittled by hydrogen. Support materials are chosen to be lattice matched to the metals and metal alloys. Preferred metals with high permeability for hydrogen include vanadium, niobium, tantalum, zirconium, palladium, and alloys thereof. Hydrogen-permeable membranes include those in which the pores of a porous support matrix are blocked by hydrogen-permeable metals and metal alloys, those in which the pores of a porous metal matrix are blocked with materials which make the membrane impervious to gases other than hydrogen, and cermets fabricated by sintering powders of metals with powders of lattice-matched ceramic.

  5. PDR-type ABC transporter mediates cellular uptake of the phytohormone abscisic acid

    PubMed Central

    Kang, Joohyun; Hwang, Jae-Ung; Kim, Yu-Young; Assmann, Sarah M.; Martinoia, Enrico; Lee, Youngsook

    2010-01-01

    Abscisic acid (ABA) is a ubiquitous phytohormone involved in many developmental processes and stress responses of plants. ABA moves within the plant, and intracellular receptors for ABA have been recently identified; however, no ABA transporter has been described to date. Here, we report the identification of the ATP-binding cassette (ABC) transporter Arabidopsis thaliana Pleiotropic drug resistance transporter PDR12 (AtPDR12)/ABCG40 as a plasma membrane ABA uptake transporter. Uptake of ABA into yeast and BY2 cells expressing AtABCG40 was increased, whereas ABA uptake into protoplasts of atabcg40 plants was decreased compared with control cells. In response to exogenous ABA, the up-regulation of ABA responsive genes was strongly delayed in atabcg40 plants, indicating that ABCG40 is necessary for timely responses to ABA. Stomata of loss-of-function atabcg40 mutants closed more slowly in response to ABA, resulting in reduced drought tolerance. Our results integrate ABA-dependent signaling and transport processes and open another avenue for the engineering of drought-tolerant plants. PMID:20133880

  6. Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices.

    PubMed

    Webb, D C; Rosenberg, H; Cox, G B

    1992-12-01

    The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system. PMID:1447208

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

    PubMed

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

    2012-10-19

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

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

    PubMed Central

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

    2012-01-01

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

  9. 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. PMID:26953208

  10. 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. PMID:26520102

  11. ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled.

    PubMed

    Singh, Himansha; Velamakanni, Saroj; Deery, Michael J; Howard, Julie; Wei, Shen L; van Veen, Hendrik W

    2016-01-01

    ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter. PMID:27499013

  12. ATP-dependent substrate transport by the ABC transporter MsbA is proton-coupled

    PubMed Central

    Singh, Himansha; Velamakanni, Saroj; Deery, Michael J.; Howard, Julie; Wei, Shen L.; van Veen, Hendrik W.

    2016-01-01

    ATP-binding cassette transporters mediate the transbilayer movement of a vast number of substrates in or out of cells in organisms ranging from bacteria to humans. Current alternating access models for ABC exporters including the multidrug and Lipid A transporter MsbA from Escherichia coli suggest a role for nucleotide as the fundamental source of free energy. These models involve cycling between conformations with inward- and outward-facing substrate-binding sites in response to engagement and hydrolysis of ATP at the nucleotide-binding domains. Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. These findings introduce ion coupling as a new parameter in the mechanism of this homodimeric ABC transporter. PMID:27499013

  13. Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

    PubMed Central

    Watanabe, Akira; Hiraga, Kazumi; Suda, Masako; Yukawa, Hideaki

    2015-01-01

    The Corynebacterium alkanolyticum xylEFGD gene cluster comprises the xylD gene that encodes an intracellular β-xylosidase next to the xylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover for p-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover for p-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutive tac promoter in the Corynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended on cgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguous xylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability of C. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functional xylEFG-encoded xyloside ABC transporter. The finding that xylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates. PMID:25862223

  14. Molecular Cloning and Analysis of a Putative Siderophore ABC Transporter from Staphylococcus aureus

    PubMed Central

    Morrissey, Julie A.; Cockayne, Alan; Hill, Philip J.; Williams, Paul

    2000-01-01

    From a mass-excised Staphylococcus aureus λZapII expression library, we cloned an operon encoding a novel ABC transporter with significant homology to bacterial siderophore transporter systems. The operon encodes four genes designated sstA, -B, -C, and -D encoding two putative cytoplasmic membrane proteins (sstA and sstB), an ATPase (sstC), and a membrane-bound 38-kDa lipoprotein (sstD). The sst operon is preceded by two putative Fur boxes, which indicated that expression of the sst operon was likely to be iron dependent. SstD was overexpressed in Escherichia coli, purified by Triton X-114 phase partitioning, and used to generate monospecific antisera in rats. Immunoblotting studies located SstD in the membrane fraction of S. aureus and showed that expression of the lipoprotein was reduced under iron-rich growth conditions. Triton X-114 partitioning studies on isolated membranes provided additional biochemical evidence that SstD in S. aureus is a lipoprotein. Immunoreactive polypeptides of approximately 38 kDa were detected in a wide range of staphylococcal species, but no antigenic homolog was detected in Bacillus subtilis. Expression of SstD in vivo was confirmed by immunoblotting studies with S. aureus recovered from a rat intraperitoneal chamber implant model. To further define the contribution of SstD in promoting growth of S. aureus in vitro and in vivo, we used antisense RNA technology to modulate expression of SstD. Expression of antisense sstD RNA in S. aureus resulted in a decrease in SstD expression under both iron-rich and iron-restricted growth conditions. However, this reduction in SstD levels did not affect the growth of S. aureus in vitro in an iron-limited growth medium or when grown in an intraperitoneal rat chamber implant model in vivo. PMID:11035736

  15. ABC transporters: one, two or four extracytoplasmic substrate-binding sites?

    PubMed Central

    van der Heide, Tiemen; Poolman, Bert

    2002-01-01

    Two families of ATP-binding cassette (ABC) transporters in which one or two extracytoplasmic substrate-binding domains are fused to either the N- or C-terminus of the translocator protein have been detected. This suggests that two, or even four, substrate-binding sites may function in the ABC transporter complex. This domain organization in ABC transporters, widely represented among microorganisms, raises new possibilities for how the substrate-binding protein(s) (SBPs) might interact with the translocator. One appealing hypothesis is that multiple substrate-binding sites in proximity to the entry site of the translocation pore enhance the transport capacity. We also discuss the implications of multiple substrate-binding sites in close proximity to the translocator in terms of broadened substrate specificity and possible cooperative interactions between SBPs and the translocator. PMID:12370206

  16. OXYGEN TRANSPORT CERAMIC MEMBRANES

    SciTech Connect

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2003-01-01

    In the present quarter, the possibility of using a more complex interfacial engineering approach to the development of reliable and stable oxygen transport perovskite ceramic membranes/metal seals is discussed. Experiments are presented and ceramic/metal interactions are characterized. Crack growth and fracture toughness of the membrane in the reducing conditions are also discussed. Future work regarding this approach is proposed are evaluated for strength and fracture in oxygen gradient conditions. Oxygen gradients are created in tubular membranes by insulating the inner surface from the reducing environment by platinum foils. Fracture in these test conditions is observed to have a gradient in trans and inter-granular fracture as opposed to pure trans-granular fracture observed in homogeneous conditions. Fracture gradients are reasoned to be due to oxygen gradient set up in the membrane, variation in stoichiometry across the thickness and due to varying decomposition of the parent perovskite. The studies are useful in predicting fracture criterion in actual reactor conditions and in understanding the initial evolution of fracture processes.

  17. Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light

    PubMed Central

    Sidler, M; Hassa, P; Hasan, S; Ringli, C; Dudler, R

    1998-01-01

    In the dark, plant seedlings follow the skotomorphogenetic developmental program, which results in hypocotyl cell elongation. When the seedlings are exposed to light, a switch to photomorphogenetic development occurs, and hypocotyl cell elongation is inhibited. We have manipulated the expression of the AtPGP1 (for Arabidopsis thaliana P glycoprotein1) gene in transgenic Arabidopsis plants by using sense and antisense constructs. We show that within a certain light fluence rate window, overexpression of the AtPGP1 gene under the control of the cauliflower mosaic virus 35S promoter causes plants to develop longer hypocotyls, whereas expression of the gene in antisense orientation results in hypocotyls shorter than those occurring in the wild type. In the dark, hypocotyls of transgenic and wild-type plants are indistinguishable. Because the AtPGP1 gene encodes a member of the superfamily of ATP binding cassette-containing (ABC) transporters, these results imply that a transport process is involved in a hypocotyl cell elongation pathway active in the light. The AtPGP1 transporter is localized in the plasmalemma, as indicated by immunohistochemical techniques and biochemical membrane separation methods. Analysis of the AtPGP1 expression pattern by using reporter gene constructs and in situ hybridization shows that in wild-type seedlings, AtPGP1 is expressed in both the root and shoot apices. PMID:9761790

  18. OXYGEN TRANSPORT CERAMIC MEMBRANES

    SciTech Connect

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2003-01-01

    In the present quarter, experiments are presented on ceramic/metal interactions of Zirconia/Ni-B-Si system and with a thin Ti coating deposited on zirconia surface. Processing of perovskites of LSC, LSF and LSCF composition for evaluation of mechanical properties as a function of environment are begun. The studies are to be in parallel with LSFCO composition to characterize the segregation of cations and slow crack growth in environmental conditions. La{sub 1-x}Sr{sub x}FeO{sub 3-d} has also been characterized for paramagnetic ordering at room temperature and the evolution of magnetic moments as a function of temperature are investigated. Investigation on the thermodynamic properties of the membrane materials are continued to develop a complete model for the membrane transport.

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

    PubMed Central

    Yang, Janet G.; Rees, Douglas C.

    2015-01-01

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

  20. Plasma Membrane Transporters in Modern Liver Pharmacology

    PubMed Central

    Marin, Jose J. G.

    2012-01-01

    The liver plays a crucial role in the detoxification of drugs used in the treatment of many diseases. The liver itself is the target for drugs aimed to modify its function or to treat infections and tumours affecting this organ. Both detoxification and pharmacological processes occurring in the liver require the uptake of the drug by hepatic cells and, in some cases, the elimination into bile. These steps have been classified as detoxification phase 0 and phase III, respectively. Since most drugs cannot cross the plasma membrane by simple diffusion, the involvement of transporters is mandatory. Several members of the superfamilies of solute carriers (SLC) and ATP-binding cassette (ABC) proteins, with a minor participation of other families of transporters, account for the uptake and efflux, respectively, of endobiotic and xenobiotic compounds across the basolateral and apical membranes of hepatocytes and cholangiocytes. These transporters are also involved in the sensitivity and refractoriness to the pharmacological treatment of liver tumours. An additional interesting aspect of the role of plasma membrane transporters in liver pharmacology regards the promiscuity of many of these carriers, which accounts for a variety of drug-drug, endogenous substances-drug and food components-drug interactions with clinical relevance. PMID:24278693

  1. OXYGEN TRANSPORT CERAMIC MEMBRANES

    SciTech Connect

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2001-12-01

    Conversion of natural gas to liquid fuels and chemicals is a major goal for the Nation as it enters the 21st Century. Technically robust and economically viable processes are needed to capture the value of the vast reserves of natural gas on Alaska's North Slope, and wean the Nation from dependence on foreign petroleum sources. Technologies that are emerging to fulfill this need are all based syngas as an intermediate. Syngas (a mixture of hydrogen and carbon monoxide) is a fundamental building block from which chemicals and fuels can be derived. Lower cost syngas translates directly into more cost-competitive fuels and chemicals. The currently practiced commercial technology for making syngas is either steam methane reforming (SMR) or a two-step process involving cryogenic oxygen separation followed by natural gas partial oxidation (POX). These high-energy, capital-intensive processes do not always produce syngas at a cost that makes its derivatives competitive with current petroleum-based fuels and chemicals. This project has the following 6 main tasks: Task 1--Design, fabricate and evaluate ceramic to metal seals based on graded ceramic powder/metal braze joints. Task 2--Evaluate the effect of defect configuration on ceramic membrane conductivity and long term chemical and structural stability. Task 3--Determine materials mechanical properties under conditions of high temperatures and reactive atmospheres. Task 4--Evaluate phase stability and thermal expansion of candidate perovskite membranes and develop techniques to support these materials on porous metal structures. Task 5--Assess the microstructure of membrane materials to evaluate the effects of vacancy-impurity association, defect clusters, and vacancy-dopant association on the membrane performance and stability. Task 6--Measure kinetics of oxygen uptake and transport in ceramic membrane materials under commercially relevant conditions using isotope labeling techniques.

  2. An ABC transporter involved in the control of streptomycin production in Streptomyces griseus.

    PubMed

    Takano, Hideaki; Toriumi, Naoe; Hirata, Mariko; Amano, Taisuke; Ohya, Takaaki; Shimada, Reona; Kusada, Hiroyuki; Amano, Sho-Ichi; Matsuda, Ko-Ichi; Beppu, Teruhiko; Ueda, Kenji

    2016-07-01

    We screened for a gene that inhibits streptomycin production in Streptomyces griseus when it is introduced on a high-copy-number plasmid pIJ702, and obtained a plasmid pKM545. The introduction of pKM545 abolished streptomycin production on all media tested including YMP-sugar and Nutrient broth. S1 protection analysis demonstrated that the introduction of this plasmid downregulated the transcriptional activity of the promoter preceding strR, the pathway-specific transcriptional regulator for streptomycin biosynthesis. The 2.8-kb BamHI fragment cloned onto pKM545 contained two coding sequences SGR_5442 and 5443. These coding sequences and the two downstream ones (SGR_5444 and 5445) constituted a possible operon structure designated to be rspABCD (regulation of streptomycin production). RspB and RspC exhibited a marked similarity with an ATP-binding domain and a membrane-associating domain of an ABC-2 type transporter, respectively, suggesting that the Rsp proteins comprise a membrane exporter. The gene cluster consisting of the rsp operon and the upstream divergent small coding sequence (SGR_5441) was widely distributed to Streptomyces genome. An rspB mutant of S. griseus produced 3-fold streptomycin of the parental strain in YMP liquid medium. The evidence implies that the Rsp translocator is involved in the export of a substance that specifies the expression level of streptomycin biosynthesis genes in S. griseus. PMID:27268270

  3. An ABC transporter from Bacillus thuringiensis is essential for beta-exotoxin I production.

    PubMed

    Espinasse, Sylvain; Gohar, Michel; Lereclus, Didier; Sanchis, Vincent

    2002-11-01

    beta-Exotoxin I is a nonspecific insecticidal metabolite secreted by some Bacillus thuringiensis strains. Several studies of B. thuringiensis strains that have lost the capacity to produce beta-exotoxin I have suggested that there is a strong correlation between high levels of beta-exotoxin I production and the ability to synthesize crystal proteins. In this study, we showed that a mutant strain, B. thuringiensis 407-1(Cry(-))(Pig(+)), with no crystal gene, produced considerable amounts of beta-exotoxin I together with a soluble brown melanin pigment. Therefore, beta-exotoxin I production can take place after a strain has lost the plasmids bearing the cry genes, which suggests that these curable plasmids probably contain determinants involved in the regulation of beta-exotoxin I production. Using a mini-Tn10 transposon, we constructed a library of strain 407-1(Cry(-))(Pig(+)) mutants. We screened for nonpigmented mutants with impaired beta-exotoxin I production and identified a genetic locus harboring two genes (berA and berB) essential for beta-exotoxin I production. The deduced amino acid sequence of the berA gene displayed significant similarity to the ATP-binding domains of the DRI (drug resistance and immunity) family of ATP-binding cassette (ABC) proteins involved in drug resistance and immunity to bacteriocins and lantibiotics. The berB gene encodes a protein with six putative transmembrane helices, which probably constitutes the integral membrane component of the transporter. The demonstration that berAB is required for beta-exotoxin I production and/or resistance in B. thuringiensis adds an adenine nucleotide analog to the wide range of substrates of the superfamily of ABC proteins. We suggest that berAB confers beta-exotoxin I immunity in B. thuringiensis, through active efflux of the molecule. PMID:12374817

  4. Alterations in function and expression of ABC transporters at blood-brain barrier under diabetes and the clinical significances

    PubMed Central

    Liu, Li; Liu, Xiao-Dong

    2014-01-01

    Diabetes is a systematic metabolic disease, which often develops a number of well-recognized vascular complications including brain complications which may partly result from the dysfunction of blood-brain barrier (BBB). BBB is generally considered as a mechanism for protecting the brain from unwanted actions resulting from substances in the blood and maintaining brain homeostasis via monitoring the entry or efflux of compounds. ATP-binding cassette (ABC) family of transporters including P-glycoprotein (P-GP) and breast cancer-related protein (BCRP), widely expressed in the luminal membrane of the microvessel endothelium and in the apical membrane of the choroids plexus epithelium, play important roles in the function of BBB. However, these transporters are easily altered by some diseases. The present article was focused on the alteration in expression and function of both P-GP and BCRP at BBB by diabetes and the clinical significances. PMID:25540622

  5. Microarray-based detection and expression analysis of ABC and SLC transporters in drug-resistant ovarian cancer cell lines.

    PubMed

    Januchowski, Radosław; Zawierucha, Piotr; Andrzejewska, Małgorzata; Ruciński, Marcin; Zabel, Maciej

    2013-04-01

    Multiple drug resistance of cancer cells is multifactorial. A microarray technique may provide information about new candidate genes playing a role in drug resistance. Drug membrane transporters from ABC and SLC families play a main role in this phenomenon. This study demonstrates alterations in ABC and SLC gene expression levels in methotrexate, cisplatin, doxorubicin, vincristine, topotecan and paclitaxel-resistant variant of W1 ovarian cancer cell line. Resistant W1 cell lines were derived by stepwise selection of cells in increasing concentration of drugs. Affymetrix GeneChip(®) Human Genome U219 Array Strip was used for hybridizations. Statistical significance was determined by independent sample t-test. The genes having altered expression levels in drug-resistant sublines were selected and filtered by scater plot. Genes up/downregulated more than threefolds were selected and listed. Among ABC genes, seven were upregulated and three were downregulated. Three genes: ABCB1, ABCB4 and ABCG2 were upregulated very significantly (over tenfold). One ABCA8 was significantly downregulated. Among 38 SLC genes, 18 were upregulated, 16 were downregulated and four were up- or downregulated dependent on the cell line. Expression of 10 SLC genes was changed very significantly (over tenfold). Four genes were significantly increased: SLC6A1, SLC9A2, SLC12A1, SLC16A6 and six genes were significantly decreased: SLC2A14, SLC7A3, SLC7A8, SLC7A11, SLC16A14, SLC38A9. Based on the expression profiles, our results provide a preliminary insight into the relationship between drug resistance and expression of membrane transporters involved in drug resistance. Correlation of specific drug transporter with drug resistance requires further analysis. PMID:23462296

  6. The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages

    PubMed Central

    Macedo, Auricelio A.; Silva, Ana P. C.; Mol, Juliana P. S.; Costa, Luciana F.; Garcia, Luize N. N.; Araújo, Marcio S.; Martins Filho, Olindo A.; Paixão, Tatiane A.; Santos, Renato L.

    2015-01-01

    Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment. PMID:26366863

  7. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; N. Nagabhushana; T. Nithyanantham; X.-D Zhou; Y-W. Sin; H.U. Anderson; Alan Jacobson; C.A. Mims

    2005-02-01

    under air separation mode (small gradient) were completed on the membrane of LSCrF-2828 at 900 C. Low pO{sub 2} atmospheres based on with CO-CO{sub 2} mixtures have also been admitted to the delivery side of the LSCrF-2828 membrane to produce the gradients which exist under syngas generation conditions. The CO-CO{sub 2} mixtures have normal isotopic {sup 18}O abundances. The evolution of {sup 18}O on the delivery side in these experiments after an {sup 18}O pulse on the air side reveals a wealth of information about the oxygen transport processes.

  8. The role of ABCG-type ABC transporters in phytohormone transport

    PubMed Central

    Borghi, Lorenzo; Kang, Joohyun; Ko, Donghwi; Lee, Youngsook; Martinoia, Enrico

    2015-01-01

    Plant hormones (phytohormones) integrate endogenous and exogenous signals thus synchronizing plant growth with environmental and developmental changes. Similar to animals, phytohormones have distinct source and target tissues, hence controlled transport and focused targeting are required for their functions. Many evidences accumulated in the last years about the regulation of long-distance and directional transport of phytohormones. ATP-binding cassette (ABC) transporters turned out to play major roles in routing phytohormones not only in the plant body but also towards the outer environment. The ABCG-type proteins ABCG25 and ABCG40 are high affinity abscisic acid (ABA) transporters. ABCG14 is highly co-expressed with cytokinin biosynthesis and is the major root-to-shoot cytokinin transporter. Pleiotropic drug resistance1 (PDR1) from Petunia hybrida transports strigolactones (SLs) from the root tip to the plant shoot but also outside to the rhizosphere, where SLs are the main attractants to mycorrhizal fungi. Last but not least, ABCG36 and ABCG37 possibly play a dual role in coumarine and IBA transport. PMID:26517905

  9. Membrane Transport Phenomena (MTP)

    NASA Technical Reports Server (NTRS)

    Mason, Larry W.

    1997-01-01

    The activities during the fourth semi-annual period of the MTP project have involved the completion of the Science Concept Review (SCR) presentation and peer review, continuation of analyses for the mass transfer coefficients measured from MTA experiment data, and development of the second generation (MTP-II) instrument. The SCR panel members were generated several recommendations for the MTP project recommendations are : Table 1 Summary of Primary SCR Panel Recommendations (1) Continue and refine development of mass transfer coefficient analyses (2) Refine and upgrade analytical modeling associated with the MTP experiment. (3) Increase resolution of measurements in proximity of the membrane interface. (4) Shift emphasis to measurement of coupled transport effects (i.e., development of MTP phase II experiment concept).

  10. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; N. Nagabhushana; X.-D Zhou; Q. Cai; J. Yang; W.B. Yelon; W.J. James; H.U. Anderson; Alan Jacobson; C.A. Mims

    2004-05-01

    The present quarterly report describes some of the investigations on the structural properties of dense OTM bars provided by Praxair and studies on newer composition of Ti doped LSF. In this report, in situ neutron diffraction was used to characterize the chemical and structural properties of La{sub 0.2}Sr{sub 0.8}Fe{sub 0.55}Ti{sub 0.45}O{sub 3-{delta}} (here after as L2SF55T) specimen, which was subject to measurements of neutron diffraction from room temperature to 900 C. It was found that space group of R3c yielded a better refinement than a cubic structure of Pm3m. Oxygen occupancy was nearly 3 in the region from room temperature to 700 C, above which the occupancy decreased due to oxygen loss. Dense OTM bars provided by Praxair were loaded to fracture at varying stress rates. Studies were done at room temperature in air and at 1000 C in a specified environment to evaluate slow crack growth behavior. The X-Ray data and fracture mechanisms points to non-equilibrium decomposition of the LSFCO OTM membrane. The non-equilibrium conditions could probably be due to the nature of the applied stress field (stressing rates) and leads to transition in crystal structures and increased kinetics of decomposition. The formations of a Brownmillerite or Sr2Fe2O5 type structures, which are orthorhombic are attributed to the ordering of oxygen vacancies. The cubic to orthorhombic transitions leads to 2.6% increase in strains and thus residual stresses generated could influence the fracture behavior of the OTM membrane. Continued investigations on the thermodynamic properties (stability and phase-separation behavior) and total conductivity of prototype membrane materials were carried out. The data are needed together with the kinetic information to develop a complete model for the membrane transport. Previously characterization, stoichiometry and conductivity measurements for samples of La{sub 0.2}Sr{sub 0.8}Fe{sub 0.55}Ti{sub 0.45}O{sub 3-{delta}} were reported. In this report

  11. A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deoxynivalenol (DON) is a mycotoxin produced by Fusarium fungi which acts as a disease virulence factor, aiding fungal pathogenesis of cereals spikelets and spread of the economically important Fusarium head blight (FHB) disease. Previously, a fragment of a wheat ABC transporter gene was shown to be...

  12. Tissue-Specific Transcript Profiling for ABC Transporters in the Sequestering Larvae of the Phytophagous Leaf Beetle Chrysomela populi

    PubMed Central

    Gretscher, René R.; Groth, Marco; Boland, Wilhelm; Burse, Antje

    2014-01-01

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

  13. ABC transporter and metallothionein expression affected by NI and Epichloe endophyte infection in tall fescue.

    PubMed

    Mirzahossini, Zahra; Shabani, Leila; Sabzalian, Mohammad R; Sharifi-Tehrani, Majid

    2015-10-01

    Epichloe endophytes are symbiotic fungi which unlike mycorrhiza grow within aerial parts of host plants. The fungi may increase host tolerance to both biotic and abiotic stresses. In this study, the effect of endophyte infection on growth and tolerance, carbohydrate contents and ABC (ABC transporter) and MET (metallothionein) expression in the leaves of tall fescue (Festuca arundinacea) plants cultivated in Ni polluted soil were evaluated. The endophyte infected (E+) and non-infected (E-) fescue plants were cultivated in soil under different Ni concentrations (30, 90 and 180mgkg(-1)). Growth parameters including root, shoot, total biomass, tiller number and total chlorophyll content of plants and H2O2 content of shoots were measured at the end of experiment. Ni translocation to the shoots, carbohydrate contents in roots and expression of ABC and MET of the leaves were also measured after 10 weeks of growth. Results demonstrated the beneficial effect of endophyte association on growth and Ni tolerance of tall fescue under Ni stress through an avoidance mechanism (reduction of Ni accumulation and translocation to the shoots). Endophyte infected plants showed less ABC and MET expression compared to the endophyte free plants. In endophyte free plants, H2O2 production had a significant positive correlation with genes expression, indicating that an increase in H2O2 might be involved in the up-regulation of ABC and MET under Ni stress. PMID:26024809

  14. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; N. Nagabhushana

    2003-08-07

    In the present quarter, experiments are presented on ceramic/metal interactions of Zirconia/ Ni-B-Si system and with a thin Ti coating deposited on zirconia surface. Existing facilities were modified for evaluation of environmental assisted slow crack growth and creep in flexural mode. Processing of perovskites of LSC, LSF and LSCF composition were continued for evaluation of mechanical properties as a function of environment. These studies in parallel to those on the LSFCO composition is expect to yield important information on questions such as the role of cation segregation and the stability of the perovskite structure on crack initiation vs. crack growth. Studies have been continued on the La{sub 1-x}Sr{sub x}FeO{sub 3-d} composition using neutron diffraction and TGA studies. A transition from p-type to n-type of conductor was observed at relative low pO{sub 2}, at which the majority carriers changed from the holes to electrons because of the valence state decreases in Fe due to the further loss of oxygen. Investigation on the thermodynamic properties of the membrane materials are continued to develop a complete model for the membrane transport. Data obtained at 850 C show that the stoichiometry in La{sub 0.2}Sr{sub 0.8}Fe{sub 0.8}Cr{sub 0.2}O{sub 3-x} vary from {approx}2.85 to 2.6 over the pressure range studied. From the stoichiometry a lower limit of 2.6 corresponding to the reduction of all Fe{sup 4+} to Fe{sup 3+} and no reduction of Cr{sup 3+} is expected.

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

    PubMed Central

    Escudero, Leticia; Mariscal, Vicente

    2015-01-01

    ABSTRACT 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. IMPORTANCE Membrane transporters are essential for many aspects of cellular life, from uptake and export of substances in unicellular organisms to intercellular

  16. Regulation of ABC efflux transporters at blood-brain barrier in health and neurological disorders.

    PubMed

    Qosa, Hisham; Miller, David S; Pasinelli, Piera; Trotti, Davide

    2015-12-01

    The strength of the blood-brain barrier (BBB) in providing protection to the central nervous system from exposure to circulating chemicals is maintained by tight junctions between endothelial cells and by a broad range of transporter proteins that regulate exchange between CNS and blood. The most important transporters that restrict the permeability of large number of toxins as well as therapeutic agents are the ABC transporters. Among them, P-gp, BCRP, MRP1 and MRP2 are the utmost studied. These efflux transporters are neuroprotective, limiting the brain entry of neurotoxins; however, they could also restrict the entry of many therapeutics and contribute to CNS pharmacoresistance. Characterization of several regulatory pathways that govern expression and activity of ABC efflux transporters in the endothelium of brain capillaries have led to an emerging consensus that these processes are complex and contain several cellular and molecular elements. Alterations in ABC efflux transporters expression and/or activity occur in several neurological diseases. Here, we review the signaling pathways that regulate expression and transport activity of P-gp, BCRP, MRP1 and MRP2 as well as how their expression/activity changes in neurological diseases. This article is part of a Special Issue entitled SI: Neuroprotection. PMID:26187753

  17. Evidence for an ABC-Type Riboflavin Transporter System in Pathogenic Spirochetes

    PubMed Central

    Deka, Ranjit K.; Brautigam, Chad A.; Biddy, Brent A.; Liu, Wei Z.; Norgard, Michael V.

    2013-01-01

    ABSTRACT Bacterial transporter proteins are involved in the translocation of many essential nutrients and metabolites. However, many of these key bacterial transport systems remain to be identified, including those involved in the transport of riboflavin (vitamin B2). Pathogenic spirochetes lack riboflavin biosynthetic pathways, implying reliance on obtaining riboflavin from their hosts. Using structural and functional characterizations of possible ligand-binding components, we have identified an ABC-type riboflavin transport system within pathogenic spirochetes. The putative lipoprotein ligand-binding components of these systems from three different spirochetes were cloned, hyperexpressed in Escherichia coli, and purified to homogeneity. Solutions of all three of the purified recombinant proteins were bright yellow. UV-visible spectra demonstrated that these proteins were likely flavoproteins; electrospray ionization mass spectrometry and thin-layer chromatography confirmed that they contained riboflavin. A 1.3-Å crystal structure of the protein (TP0298) encoded by Treponema pallidum, the syphilis spirochete, demonstrated that the protein’s fold is similar to the ligand-binding components of ABC-type transporters. The structure also revealed other salient details of the riboflavin binding site. Comparative bioinformatics analyses of spirochetal genomes, coupled with experimental validation, facilitated the discovery of this new ABC-type riboflavin transport system(s). We denote the ligand-binding component as riboflavin uptake transporter A (RfuA). Taken together, it appears that pathogenic spirochetes have evolved an ABC-type transport system (RfuABCD) for survival in their host environments, particularly that of the human host. PMID:23404400

  18. Oxygen Transport Membranes

    SciTech Connect

    S. Bandopadhyay

    2008-08-30

    The focus of this research was to develop new membrane materials by synthesizing different compounds and determining their defect structures, crystallographic structures and electrical properties. In addition to measuring electrical conductivity, oxygen vacancy concentration was also evaluated using thermogravimetry, Neutron diffraction and Moessbauer Spectroscopy. The reducing conditions (CO{sub 2}/CO/H{sub 2} gas mixtures with steam) as encountered in a reactor environment can be expected to have significant influence on the mechanical properties of the oxides membranes. Various La based materials with and without Ti were selected as candidate membrane materials for OTM. The maximum electrical conductivity of LSF in air as a function of temperature was achieved at < 600 C and depends on the concentration of Sr (acceptor dopant). Oxygen occupancy in LSF was estimated using Neutron diffractometry and Moessbauer Spectroscopy by measuring magnetic moment changes depending on the Fe{sup 3+} and Fe{sup 4+} ratio. After extensive studies of candidate materials, lanthanum ferrites (LSF and LSFT) were selected as the favored materials for the oxygen transport membrane (OTM). LSF is a very good material for an OTM because of its high electronic and oxygen ionic conductivity if long term stability and mechanical strength are improved. LSFT not only exhibits p-type behavior in the high oxygen activity regime, but also has n-type conduction in reducing atmospheres. Higher concentrations of oxygen vacancies in the low oxygen activity regime may improve the performance of LSFT as an OTM. The hole concentration is related to the difference in the acceptor and donor concentration by the relation p = [Sr'{sub La}]-[Ti{sm_bullet}{sub Fe}]. The chemical formulation predicts that the hole concentration is, p = 0.8-0.45 or 0.35. Experimental measurements indicated that p is about {approx} 0.35. The activation energy of conduction is 0.2 eV which implies that LSCF conducts via the

  19. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; N. Nagabhushana; X.-D Zhou; Q. Cai; J. Yang; W.B. Yelon; W.J. James; H.U. Anderson; Alan Jacobson; C.A. Mims

    2004-10-01

    The present quarterly report describes some of the investigations on the structural properties of dense OTM bars provided by Praxair and studies on newer composition of Ti doped LSF. In this report, Moessbauer spectroscopy was used to study the local environmentals of LSFT with various level of oxygen deficiency. Ionic valence state, magnetic interaction and influence of Ti on superexchange are discussed Stable crack growth studies on Dense OTM bars provided by Praxair were done at elevated temperature, pressure and elevated conditions. Post-fracture X-ray data of the OTM fractured at 1000 C in environment were refined by FullProf code and results indicate a distortion of the parent cubic perovskite to orthorhombic structure with reduced symmetry. TGA-DTA studies on the post-fracture samples also indicated residual effect arising from the thermal and stress history of the samples. An electrochemical cell has been designed and built for measurements of the Seebeck coefficient as a function of temperature and pressure. The initial measurements on La{sub 0.2}Sr{sub 0.8}Fe{sub 0.55}Ti{sub 0.45}O{sub 3-{delta}} are reported. Neutron diffraction measurements of the same composition are in agreement with both the stoichiometry and the kinetic behavior observed in coulometric titration measurements. A series of isotope transients under air separation mode (small gradient) were completed on the membrane of LSCrF-2828 at 900 C. Low pO{sub 2} atmospheres based on with CO-CO{sub 2} mixtures have also been admitted to the delivery side of the LSCrF-2828 membrane to produce the gradients which exist under syngas generation conditions. The COCO{sub 2} mixtures have normal isotopic {sup 18}O abundances. The evolution of {sup 18}O on the delivery side in these experiments after an {sup 18}O pulse on the air side reveals a wealth of information about the oxygen transport processes.

  20. Radiopharmaceuticals for assessing ABC transporters at the blood-brain barrier.

    PubMed

    Raaphorst, R M; Windhorst, A D; Elsinga, P H; Colabufo, N A; Lammertsma, A A; Luurtsema, G

    2015-04-01

    ABC transporters protect the brain by transporting neurotoxic compounds from the brain back into the blood. P-glycoprotein (P-gp) is the most investigated ABC (efflux) transporter, as it is implicated in neurodegenerative diseases such as Alzheimer's disease. Altered function of P-gp can be studied in vivo, using Positron Emission Tomography (PET). To date, several radiopharmaceuticals have been developed to image P-gp function in vivo. So far, attempts to image expression levels of P-gp using radiolabeled P-gp inhibitors have not been successful. Improved knowledge of compound behavior toward P-gp from in vitro studies should increase predictability of in vivo outcome. PMID:25669763

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  2. 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

  3. Alkylrhodamines enhance the toxicity of clotrimazole and benzalkonium chloride by interfering with yeast pleiotropic ABC-transporters.

    PubMed

    Knorre, Dmitry A; Besedina, Elizaveta; Karavaeva, Iuliia E; Smirnova, Ekaterina A; Markova, Olga V; Severin, Fedor F

    2016-06-01

    ABC-transporters with broad substrate specificity are responsible for pathogenic yeast resistance to antifungal compounds. Here we asked whether highly hydrophobic chemicals with delocalized positive charge can be used to overcome the resistance. Such molecules efficiently penetrate the plasma membrane and accumulate inside the cells. We reasoned that these properties can convert an active efflux of the compounds into a futile cycle thus interfering with the extrusion of the antibiotics. To test this, we studied the effects of several alkylated rhodamines on the drug resistance of yeast Saccharomyces cerevisiae We found that octylrhodamine synergetically increases toxicity of Pdr5p substrate-clotrimazole, while the others were less effective. Next, we compared the contributions of three major pleiotropic ABC-transporters (Pdr5p, Yor1p, Snq2p) on the accumulation of the alkylated rhodamines. While all of the tested compounds were extruded by Pdr5p, Yor1p and Snq2p showed narrower substrate specificity. Interestingly, among the tested alkylated rhodamines, inactivation of Pdr5p had the strongest effect on the accumulation of octylrhodamine inside the cells, which is consistent with the fact that clotrimazole is a substrate of Pdr5p. As alkylated rhodamines were shown to be non-toxic on mice, our study makes them potential components of pharmacological antifungal compositions. PMID:27044313

  4. Nanoengineered membranes for controlled transport

    DOEpatents

    Doktycz, Mitchel J [Oak Ridge, TN; Simpson, Michael L [Knoxville, TN; McKnight, Timothy E [Greenback, TN; Melechko, Anatoli V [Oak Ridge, TN; Lowndes, Douglas H [Knoxville, TN; Guillorn, Michael A [Knoxville, TN; Merkulov, Vladimir I [Oak Ridge, TN

    2010-01-05

    A nanoengineered membrane for controlling material transport (e.g., molecular transport) is disclosed. The membrane includes a substrate, a cover definining a material transport channel between the substrate and the cover, and a plurality of fibers positioned in the channel and connected to an extending away from a surface of the substrate. The fibers are aligned perpendicular to the surface of the substrate, and have a width of 100 nanometers or less. The diffusion limits for material transport are controlled by the separation of the fibers. In one embodiment, chemical derivitization of carbon fibers may be undertaken to further affect the diffusion limits or affect selective permeability or facilitated transport. For example, a coating can be applied to at least a portion of the fibers. In another embodiment, individually addressable carbon nanofibers can be integrated with the membrane to provide an electrical driving force for material transport.

  5. Membrane Transport Phenomena (MTP)

    NASA Technical Reports Server (NTRS)

    Mason, Larry W.

    1996-01-01

    The development of the seal between the membrane and the Fluid Optical Cells (FOC) has been a high priority activity. This seal occurs at an interface in the instrument where three key functions must be realized: (1) physical membrane support, (2) fluid sealing, and (3) unobscured optical transmission.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25504146

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

    PubMed

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

    2014-01-01

    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. PMID:25504146

  8. 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.

  9. ABC transporters and xenobiotic defense systems in early life stages of rainbow trout (Oncorhynchus mykiss).

    PubMed

    Kropf, Christian; Segner, Helmut; Fent, Karl

    2016-01-01

    Embryos of oviparous fish, in contrast to (ovo) viviparous species, develop in the aquatic environment, and therefore need solute transport systems at their body surfaces for maintaining internal homeostasis and defending against potentially harmful substances. We hypothesized that solute transporters undergo changes in tissue distribution from the embryo to the larval stage. We therefore studied the mRNA profiles of eight ABC transporters (abcb1a, abcb1b, abcc1, abcc2, abcc3, abcc4, abcc5, abcg2) and three solute carriers (oatp1d, putative oatp2 putative, mate1) in different body regions (head, yolk sac epithelium, abdominal viscera, skin/muscles) of developing rainbow trout. Additionally, we investigated mRNA levels of phase I (cyp1a, cyp3a) and phase II (gstp, putative ugt1, putative ugt2) biotransformation enzymes. The study covered the developmental period from the eleuthero-embryo stage to the first-feeding larval stage (1-20days post-hatch, dph). At 1dph, transcripts of abcc2, abcc4, abcg2, cyp3a, gstp, putative mate1, and putative oatp2 occurred primarily in the yolk sac epithelium, whereas at later stages expression of these genes was predominantly observed in the abdominal viscera. The functional activity of ABC transporters in fish early life stages was assessed by rhodamine B accumulation assays. Finally, we investigated the potential impact of xenobiotics (clotrimazole, clofibric acid) on the ABC and biotransformation systems of trout early life stages. While clofibric acid had no effect, clotrimazole lead to an increased rhodamine B accumulation. The results provide evidence that the transition from the eleuthero-embryo to the larval stage is accompanied by a major alteration in tissue expression of ABC transporters. PMID:26945521

  10. 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

  11. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; N. Nagabhushana; X.-D Zhou; Q. Cai; J. Yang; W.B. Yelon; W.J. James; H.U. Anderson; Alan Jacobson; C.A. Mims

    2004-05-01

    the LSCrF-2828 membrane to produce the gradients which exist under syngas generation conditions. The CO-CO{sub 2} mixtures have normal isotopic {sup 18}O abundances. The evolution of {sup 18}O on the delivery side in these experiments after an {sup 18}O pulse on the air side reveals a wealth of information about the oxygen transport processes.

  12. The putative ABC transporter encoded by the orf19.4531 plays a role in the sensitivity of Candida albicans cells to azole antifungal drugs.

    PubMed

    Jiang, Linghuo; Xu, Dayong; Chen, Zhen; Cao, Yongbing; Gao, Pinghui; Jiang, Yuanying

    2016-05-01

    ATP-binding cassette (ABC) transporters constitute a large superfamily of integral membrane proteins in prokaryotic and eukaryotic cells. In the human fungal pathogen Candida albicans, there are 28 genes encoding ABC transporters and many of them have not been characterized so far. The orf19.4531 (also known as IPF7530) encodes a putative ABC transporter. In this study, we have demonstrated that disruption of orf19.4531 causes C. albicans cells to become tolerant to azoles, but not to polyene antifungals and terbinafine. Therefore, the protein encoded by orf19.4531 is involved in azole sensitivity and we name it as ROA1, the regulator of azole sensitivity 1 gene. Consistently, we show that the expression of ROA1 is responsive to treatment of either fluconazole or ketoconazole inC. albicans In addition, through a GFP tagging approach, Roa1 is localized in a small punctuate compartment adjacent to the vacuolar membrane. However, ROA1 is not essential for the in vitro filamentation of C. albicans cells. PMID:26975389

  13. Direct Observation of a Gate Tunable Band Gap in Electrical Transport in ABC-Trilayer Graphene.

    PubMed

    Khodkov, Tymofiy; Khrapach, Ivan; Craciun, Monica Felicia; Russo, Saverio

    2015-07-01

    Few layer graphene systems such as Bernal stacked bilayer and rhombohedral (ABC-) stacked trilayer offer the unique possibility to open an electric field tunable energy gap. To date, this energy gap has been experimentally confirmed in optical spectroscopy. Here we report the first direct observation of the electric field tunable energy gap in electronic transport experiments on doubly gated suspended ABC-trilayer graphene. From a systematic study of the nonlinearities in current versus voltage characteristics and the temperature dependence of the conductivity, we demonstrate that thermally activated transport over the energy-gap dominates the electrical response of these transistors. The estimated values for energy gap from the temperature dependence and from the current voltage characteristics follow the theoretically expected electric field dependence with critical exponent 3/2. These experiments indicate that high quality few-layer graphene are suitable candidates for exploring novel tunable terahertz light sources and detectors. PMID:26079989

  14. An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR.

    PubMed

    Randak, Christoph; Welsh, Michael J

    2003-12-26

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption. PMID:14697202

  15. Spin Labeling Studies of Transmembrane Signaling and Transport: Applications to Phototaxis, ABC Transporters and Symporters.

    PubMed

    Klare, Johann P; Steinhoff, Heinz-Jürgen

    2015-01-01

    Membrane proteins still represent a major challenge for structural biologists. This chapter will focus on the application of continuous wave and pulsed EPR spectroscopy on spin-labeled membrane proteins. Site-directed spin labeling EPR spectroscopy has evolved as a powerful tool to study the structure and dynamics of proteins, especially membrane proteins, as this method works largely independently of the size and complexity of the biological system under investigation. This chapter describes applications of this technique to three different systems: the archaeal photoreceptor/-transducer complex SRII/HtrII as an example for transmembrane signaling and two transport systems, the histidine ATP-binding cassette transporter HisQMP, and the sodium-proline symporter PutP. PMID:26477256

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

    PubMed

    Saurin, W; Hofnung, M; Dassa, E

    1999-01-01

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

  17. Antitubercular Agent Delamanid and Metabolites as Substrates and Inhibitors of ABC and Solute Carrier Transporters.

    PubMed

    Sasabe, Hiroyuki; Shimokawa, Yoshihiko; Shibata, Masakazu; Hashizume, Kenta; Hamasako, Yusuke; Ohzone, Yoshihiro; Kashiyama, Eiji; Umehara, Ken

    2016-06-01

    Delamanid (Deltyba, OPC-67683) is the first approved drug in a novel class of nitro-dihydro-imidazooxazoles developed for the treatment of multidrug-resistant tuberculosis. Patients with tuberculosis require treatment with multiple drugs, several of which have known drug-drug interactions. Transporters regulate drug absorption, distribution, and excretion; therefore, the inhibition of transport by one agent may alter the pharmacokinetics of another, leading to unexpected adverse events. Therefore, it is important to understand how delamanid affects transport activity. In the present study, the potencies of delamanid and its main metabolites as the substrates and inhibitors of various transporters were evaluated in vitro Delamanid was not transported by the efflux ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp; MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), solute carrier (SLC) transporters, organic anion-transporting polypeptides, or organic cation transporter 1. Similarly, metabolite 1 (M1) was not a substrate for any of these transporters except P-gp. Delamanid showed no inhibitory effect on ABC transporters MDR1, BCRP, and bile salt export pump (BSEP; ABCB11), SLC transporters, or organic anion transporters. M1 and M2 inhibited P-gp- and BCRP-mediated transport but did so only at the 50% inhibitory concentrations (M1, 4.65 and 5.71 μmol/liter, respectively; M2, 7.80 and 6.02 μmol/liter, respectively), well above the corresponding maximum concentration in plasma values observed following the administration of multiple doses in clinical trials. M3 and M4 did not affect the activities of any of the transporters tested. These in vitro data suggest that delamanid is unlikely to have clinically relevant interactions with drugs for which absorption and disposition are mediated by this group of transporters. PMID:27021329

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

    PubMed

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

    2016-09-01

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

  19. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; T. Nithyanantham; X.-D Zhou; Y-W. Sin; H.U. Anderson; Alan Jacobson; C.A. Mims

    2005-11-01

    The present quarterly report describes some of the investigations on the structural properties of dense OTM bars provided by Praxair and studies on newer composition of Ti doped LSF. In the current research, the electrical conductivity and Seebeck coefficient were measured as a function of temperature in air. Based on these measurements, the charge carrier concentration, net acceptor dopant concentration, activation energy of conduction and mobility were estimated. The studies on the fracture toughness of the LSFT and dual phase membranes at room temperature have been completed and reported previously. The membranes that are exposed to high temperatures at an inert and a reactive atmosphere undergo many structural and chemical changes which affects the mechanical properties. To study the effect of temperature on the membranes when exposed to an inert environment, the membranes (LAFT and Dual phase) were heat treated at 1000 C in air and N{sub 2} atmosphere and hardness and fracture toughness of the membranes were studied after the treatment. The indentation method was used to find the fracture toughness and the effect of the heat treatment on the mechanical properties of the membranes. Further results on the investigation of the origin of the slow kinetics on reduction of ferrites have been obtained. The slow kinetics appears to be related to a non-equilibrium reduction pathway that initially results in the formation of iron particles. At long times, equilibrium can be reestablished with recovery of the perovskite phase. 2-D modeling of oxygen movement has been undertaken in order to fit isotope data. The model will serve to study ''frozen'' profiles in patterned or composite membranes.

  20. 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

  1. Role of the Zinc Uptake ABC Transporter of Moraxella catarrhalis in Persistence in the Respiratory Tract

    PubMed Central

    Brauer, Aimee L.; Kirkham, Charmaine; Johnson, Antoinette; Koszelak-Rosenblum, Mary; Malkowski, Michael G.

    2013-01-01

    Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. We have identified and characterized a zinc uptake ABC transporter that is present in all strains of M. catarrhalis tested. A mutant in which the znu gene cluster is knocked out shows markedly impaired growth compared to the wild type in medium that contains trace zinc; growth is restored to wild-type levels by supplementing medium with zinc but not with other divalent cations. Thermal-shift assays showed that the purified recombinant substrate binding protein ZnuA binds zinc but does not bind other divalent cations. Invasion assays with human respiratory epithelial cells demonstrated that the zinc ABC transporter of M. catarrhalis is critical for invasion of respiratory epithelial cells, an observation that is especially relevant because an intracellular reservoir of M. catarrhalis is present in the human respiratory tract and this reservoir is important for persistence. The znu knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the zinc uptake ABC transporter mediates uptake of zinc in environments with very low zinc concentrations and is critical for full virulence of M. catarrhalis in the respiratory tract in facilitating intracellular invasion of epithelial cells and persistence in the respiratory tract. PMID:23817618

  2. Maltose transport system of Escherichia coli: an ABC-type transporter.

    PubMed

    Nikaido, H

    1994-06-01

    The maltose transport system of E. coli is composed of a periplasmic maltose-binding protein (MBP), the presumed transmembrane channel made up of MalF and MalG proteins, and two copies of the ATPase subunit, MalK. The membrane-associated transporter complex was purified in a functional form both from the wild-type strain and from mutants that do not require MBP for transport, and was reconstituted into proteoliposomes. A major function of MBP is to send a transmembrane signal, in the presence of ligands, to the ATPase subunits on the inner side of the membrane. In addition, MBP performs a special function in the translocation of the larger ligands, maltodextrins, perhaps by aligning them for entry into the channel. PMID:8206159

  3. Membrane transport of antineoplastic agents

    SciTech Connect

    Goldman, I.D. )

    1986-01-01

    This book contains 13 chapters. Some of the chapter titles are: Methods for Quantifying the Transport of Drugs Across Brain Barrier Systems; Liposomes as Drug Carriers in Cancer Chemotherapy; Genetic and Bioochemical Characterization of Multidrug Resistance; Membrane Transport of Anthracyclines; and The Cellular Pharmacology of Methotrexate.

  4. Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies

    PubMed Central

    Kunert, Britta; Gardiennet, Carole; Lacabanne, Denis; Calles-Garcia, Daniel; Falson, Pierre; Jault, Jean-Michel; Meier, Beat H.; Penin, François; Böckmann, Anja

    2014-01-01

    We present solid-state NMR sample preparation and first 2D spectra of the Bacillus subtilis ATP-binding cassette (ABC) transporter BmrA, a membrane protein involved in multidrug resistance. The homodimeric 130-kDa protein is a challenge for structural characterization due to its membrane-bound nature, size, inherent flexibility and insolubility. We show that reconstitution of this protein in lipids from Bacillus subtilis at a lipid-protein ratio of 0.5 w/w allows for optimal protein insertion in lipid membranes with respect to two central NMR requirements, high signal-to-noise in the spectra and sample stability over a time period of months. The obtained spectra point to a well-folded protein and a highly homogenous preparation, as witnessed by the narrow resonance lines and the signal dispersion typical for the expected secondary structure distribution of BmrA. This opens the way for studies of the different conformational states of the transporter in the export cycle, as well as on interactions with substrates, via chemical-shift fingerprints and sequential resonance assignments. PMID:25988146

  5. Engineering of Ion Sensing by the Cystathionine β-Synthase Module of the ABC Transporter OpuA*

    PubMed Central

    Mahmood, Nik A. B. N.; Biemans-Oldehinkel, Esther; Poolman, Bert

    2009-01-01

    We have previously shown that the C-terminal cystathionine β-synthase (CBS) domains of the nucleotide-binding domains of the ABC transporter OpuA, in conjunction with an anionic membrane surface function, act as sensor of internal ionic strength (Iin). Here, we show that a surface-exposed cationic region in the CBS module domain is critical for ion sensing. The consecutive substitution of up to five cationic residues led to a gradual decrease of the ionic strength dependence of transport. In fact, a 5-fold mutant was essentially independent of salt in the range from 0 to 250 mm KCl (or NaCl), supplemented to medium of 30 mm potassium phosphate. Importantly, the threshold temperature for transport was lowered by 5–7 °C and the temperature coefficient Q10 was lowered from 8 to ∼1.5 in the 5-fold mutant, indicating that large conformational changes are accompanying the CBS-mediated regulation of transport. Furthermore, by replacing the anionic C-terminal tail residues that extend the CBS module with histidines, the transport of OpuA became pH-dependent, presumably by additional charge interactions of the histidine residues with the membrane. The pH dependence was not observed at high ionic strength. Altogether the analyses of the CBS mutants support the notion that the osmotic regulation of OpuA involves a simple biophysical switching mechanism, in which nonspecific electrostatic interactions of a protein module with the membrane are sufficient to lock the transporter in the inactive state. PMID:19329426

  6. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; T. Nithyanantham; X.-D Zhou; Y-W. Sin; H.U. Anderson; Alan Jacobson; C.A. Mims

    2006-05-01

    In this quarter a systematic analysis on the decomposition behavior of the OTM membranes at air and nitrogen were initiated to understand the structural and stoichiometric changes associated with elevated temperatures. Evaluation of the flexural strengths using 4-point bend test was also started for the dual phase membranes. Initial results on the synthesis of dual phase composite materials have been obtained. The measurements have focused on the compatibility of mixed conductors with the pure ionic conductors yttria stabilized zirconia (YSZ) and gadolinium doped ceria (GDC). The initial results obtained for three different mixed conductors suggest that (GDC) is the better choice. A new membrane permeation system has been designed and tested and sintering studies of biphasic systems are in progress.

  7. 3D cryo-electron reconstruction of BmrA, a bacterial multidrug ABC transporter in an inward-facing conformation and in a lipidic environment.

    PubMed

    Fribourg, Pierre Frederic; Chami, Mohamed; Sorzano, Carlos Oscar S; Gubellini, Francesca; Marabini, Roberto; Marco, Sergio; Jault, Jean-Michel; Lévy, Daniel

    2014-05-15

    ABC (ATP-binding cassette) membrane exporters are efflux transporters of a wide diversity of molecule across the membrane at the expense of ATP. A key issue regarding their catalytic cycle is whether or not their nucleotide-binding domains (NBDs) are physically disengaged in the resting state. To settle this controversy, we obtained structural data on BmrA, a bacterial multidrug homodimeric ABC transporter, in a membrane-embedded state. BmrA in the apostate was reconstituted in lipid bilayers forming a mixture of ring-shaped structures of 24 or 39 homodimers. Three-dimensional models of the ring-shaped structures of 24 or 39 homodimers were calculated at 2.3 nm and 2.5 nm resolution from cryo-electron microscopy, respectively. In these structures, BmrA adopts an inward-facing open conformation similar to that found in mouse P-glycoprotein structure with the NBDs separated by 3 nm. Both lipidic leaflets delimiting the transmembrane domains of BmrA were clearly resolved. In planar membrane sheets, the NBDs were even more separated. BmrA in an ATP-bound conformation was determined from two-dimensional crystals grown in the presence of ATP and vanadate. A projection map calculated at 1.6 nm resolution shows an open outward-facing conformation. Overall, the data are consistent with a mechanism of drug transport involving large conformational changes of BmrA and show that a bacterial ABC exporter can adopt at least two open inward conformations in lipid membrane. PMID:24630999

  8. 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...

  9. Composite oxygen transport membrane

    SciTech Connect

    Christie, Gervase Maxwell; Lane, Jonathan A.

    2014-08-05

    A method of producing a composite oxygen ion membrane and a composite oxygen ion membrane in which a porous fuel oxidation layer and a dense separation layer and optionally, a porous surface exchange layer are formed on a porous support from mixtures of (Ln.sub.1-xA.sub.x).sub.wCr.sub.1-yB.sub.yO.sub.3-.delta. and a doped zirconia. In the porous fuel oxidation layer and the optional porous surface exchange layer, A is Calcium and in the dense separation layer A is not Calcium and, preferably is Strontium. Preferred materials are (La.sub.0.8Ca.sub.0.2).sub.0.95Cr.sub.0.5Mn.sub.0.5O.sub.3-.delta. for the porous fuel oxidation and optional porous surface exchange layers and (La.sub.0.8Sr.sub.0.2).sub.0.95Cr.sub.0.5Fe.sub.0.5O.sub.3-.delta. for the dense separation layer. The use of such materials allows the membrane to sintered in air and without the use of pore formers to reduce membrane manufacturing costs. The use of materials, as described herein, for forming the porous layers have application for forming any type of porous structure, such as a catalyst support.

  10. Arabidopsis mutant of AtABCG26, an ABC transporter gene, is defective in pollen maturation.

    PubMed

    Kuromori, Takashi; Ito, Takuya; Sugimoto, Eriko; Shinozaki, Kazuo

    2011-11-01

    In plants, pollen is the male gametophyte that is generated from microspores, which are haploid cells produced after meiosis of diploid pollen mother cells in floral anthers. In normal maturation, microspores interact with the tapetum, which consists of one layer of metabolically active cells enclosing the locule in anthers. The tapetum plays several important roles in the maturation of microspores. ATP-binding cassette (ABC) transporters are a highly conserved protein super-family that uses the energy released in ATP hydrolysis to transport substrates. The ABC transporter gene family is more diverse in plants than in animals. Previously, we reported that an Arabidopsis half-size type ABC transporter gene, COF1/AtWBC11/AtABCG11, is involved in lipid transport for the construction of cuticle layers and pollen coats in normal organ formation, as compared to CER5/AtWBC12/AtABCG12. However, physiological functions of most other ABCG members are unknown. Here, we identified another family gene, AtABCG26, which is required for pollen development in Arabidopsis. An AtABCG26 mutant developed very few pollen grains, resulting in a male-sterile phenotype. By investigating microspore and pollen development in this mutant, we observed that there was a slight abnormality in tetrad morphology prior to the formation of haploid microspores. At a later stage, we could not detect exine deposition on the microspore surface. During pollen maturation, many grains in the mutant anthers got aborted, and surviving grains were found to be defective in mitosis. Transmission of the mutant allele through male gametophytes appeared to be normal in genetic transmission analysis, supporting the view that the pollen function was disturbed by sporophytic defects in the AtABCG26 mutant. AtABCG26 can be expected to be involved in the transport of substrates such as sporopollenin monomers from tapetum to microspores, which both are plant-specific structures critical to pollen development. PMID

  11. Enhanced exposure of phosphatidylserine in human gastric carcinoma cells overexpressing the half-size ABC transporter BCRP (ABCG2).

    PubMed Central

    Woehlecke, Holger; Pohl, Antje; Alder-Baerens, Nele; Lage, Hermann; Herrmann, Andreas

    2003-01-01

    Members of the ABC (ATP-binding cassette) transporter super-family are emerging to be involved in lipid transport. In the present study, we studied the organization of phospholipids in the plasma membrane of EPG85-257 human gastric carcinoma cells overexpressing BCRP (breast cancer resistance protein, ABCG-2), a half-size transporter belonging to the ABCG subfamily. A significantly increased plasma membrane association of the PS (phosphatidylserine)-binding probe FITC-Annexin V in comparison with control cells was observed. Treatment of BCRP -overexpressing cells with the inhibitor Tryprostatin A decreased FITC-Annexin V binding almost to the control level. This suggests an enhanced exposure of PS in BCRP -overexpressing cells, which is dependent on functional BCRP. A role of BCRP in the transverse distribution of lipids in the plasma membrane is supported by the increased outward transport of the lipid analogue C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-PS in BCRP -overexpressing EPG85-257 cells and MCF-7 human breast cancer cells. As shown for BCRP -overexpressing EPG85-257 cells, enhanced outward redistribution of the lipid analogue is inhibited by Tryprostatin A as well as by reduction of BCRP expression on transfection with an anti- BCRP -ribozyme. We also observed an enhanced outward transport of C6- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine in BCRP -overexpressing EPG85-257 cells, suggesting that the influence of BCRP on transverse lipid organization is not highly specific. PMID:12946267

  12. OXYGEN TRANSPORT CERAMIC MEMBRANES

    SciTech Connect

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2000-07-01

    This is the fourth quarterly report on a new study to develop a ceramic membrane/metal joint. The first experiments using the La-Sr-Fe-O ceramic are reported. Some of the analysis performed on the samples obtained are commented upon. A set of experiments to characterize the mechanical strength and thermal fatigue properties of the joints has been designed and begun. Finite element models of joints used to model residual stresses are described.

  13. The Crystal Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens

    PubMed Central

    Wang, Lulu; Jiang, Rui; Jin, Xiaoling; Liu, Jing; Fan, Shengdi; Quan, Chun-Shan; Ha, Nam-Chul

    2016-01-01

    Bacillus possesses the peptide toxin Sporulation-Delaying Protein (SDP), which can kill cells within a biofilm to support continued growth, thereby delaying the onset of biofilm sporulation. The four-component transporter YknWXYZ acts as a major SDP efflux pump to protect cells against the endogenous SDP toxin, for which YknYZ is a non-canonical ATP-binding cassette (ABC)-type transporter. YknYZ consists of the following two components: (1) an individual protein (YknY) and (2) a respective permease (YknZ). To date, the crystal structure, molecular function, and mechanism of action of the integral membrane protein YknZ remain to be elucidated. In this study, to characterize the structural and biochemical roles of YknZ in the functional assembly of YknWXYZ, we predicted and overexpressed the YknZ extracellular domain. We determined the crystal structure of B. amyloliquefaciens YknZ at a resolution of 2.0 Å. The structure revealed that the YknZ extracellular region exhibits significant structural similarity with the MacB periplasmic domain, which is a non-canonical ABC-type transporter in the tripartite macrolide-specific efflux pump in Gram-negative bacteria. We also found that the YknZ extracellular domain can directly bind to an extracellular component of YknX. This structural and biochemical study provides insights into the assembly of YknWXYZ, which may be relevant to understanding cannibalistic peptide toxin resistance in Bacillus and controlling bacterial growth. PMID:27243566

  14. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; T. Nithyanantham; X.-D Zhou; Y-W. Sin; H.U. Anderson; Alan Jacobson; C.A. Mims

    2005-08-01

    The present quarterly report describes some of the investigations on the structural properties of dense OTM bars provided by Praxair and studies on newer composition of Ti doped LSF. In the previous research, the reference point of oxygen occupancy was determined and verified. In the current research, the oxygen occupancy was investigated at 1200 C as a function of oxygen activity and compared with that at 1000 C. The cause of bumps at about 200 C was also investigated by using different heating and cooling rates during TGA. The fracture toughness of LSFT and dual phase membranes at room temperature is an important mechanical property. Vicker's indentation method was used to evaluate this toughness. Through this technique, a K{sub Ic} (Mode-I Fracture Toughness) value is attained by means of semi-empirical correlations between the indentation load and the length of the cracks emanating from the corresponding Vickers indentation impression. In the present investigation, crack propagation behavior was extensively analyzed in order to understand the strengthening mechanisms involved in the non-transforming La based ceramic composites. Cracks were generated using Vicker's indenter and used to identify and evaluate the toughening mechanisms involved. Preliminary results of an electron microscopy study of the origin of the slow kinetics on reduction of ferrites have been obtained. The slow kinetics appear to be related to a non-equilibrium reduction pathway that initially results in the formation of iron particles. At long times, equilibrium can be reestablished with recovery of the perovskite phase. Modeling of the isotopic transients on operating membranes (LSCrF-2828 at 900 C) and a ''frozen'' isotope profile have been analyzed in conjunction with a 1-D model to reveal the gradient in oxygen diffusivity through the membrane under conditions of high chemical gradients.

  15. Membranes, mechanics, and intracellular transport

    NASA Astrophysics Data System (ADS)

    Parthasarathy, Raghuveer

    2012-10-01

    Cellular membranes are remarkable materials -- self-assembled, flexible, two-dimensional fluids. Understanding how proteins manipulate membrane curvature is crucial to understanding the transport of cargo in cells, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical-trap based assay involving dynamic deformation of biomimetic membranes, we have examined the behavior of Sar1, a key component of the COPII family of transport proteins. We find that Sar1 from yeast (S. cerevisiae) lowers membrane rigidity by up to 100% as a function of its concentration, thereby lowering the energetic cost of membrane deformation. Human Sar1 proteins can also lower the mechanical rigidity of the membranes to which they bind. However, unlike the yeast proteins, the rigidity is not a monotonically decreasing function of concentration but rather shows increased rigidity and decreased mobility at high concentrations that implies interactions between proteins. In addition to describing this study of membrane mechanics, I'll also discuss some topics relevant to a range of biophysical investigations, such as the insights provided by imaging methods and open questions in the dynamics of multicellular systems.

  16. The Riboswitch Regulates a Thiamine Pyrophosphate ABC Transporter of the Oral Spirochete Treponema denticola ▿ †

    PubMed Central

    Bian, Jiang; Shen, Hongwu; Tu, Youbin; Yu, Aiming; Li, Chunhao

    2011-01-01

    Thiamine pyrophosphate (TPP), a biologically active form of thiamine (vitamin B1), is an essential cofactor in all living systems. Microorganisms either synthesize TPP via de novo biosynthesis pathways or uptake exogenous thiamine from the environment via specific transporters. The oral spirochete Treponema denticola is an important pathogen that is associated with human periodontal diseases. It lacks a de novo TPP biosynthesis pathway and needs exogenous TPP for growth, suggesting that it may obtain exogenous TPP via a thiamine transporter. In this study, we identified a gene cluster that encodes a TPP ABC transporter which consists of a TPP-binding protein (TDE0143), a transmembrane permease (TDE0144), and a cytosolic ATPase (TDE0145). Transcriptional and translational analyses showed that the genes encoding these three proteins are cotranscribed and form an operon (tbpABCTd) that is initiated by a σ70-like promoter. The expression level of this operon is negatively regulated by exogenous TPP and is mediated by a TPP-sensing riboswitch (Tdthi-box). Genetic and biochemical studies revealed that the TDE0143 deletion mutant (T. denticola ΔtbpA) had a decreased ability to transport exogenous TPP, and the mutant failed to grow when exogenous TPP was insufficient. These results taken together indicate that the tbpABCTd operon encodes an ABC transporter that is required for the uptake of exogenous TPP and that the expression of this operon is regulated by a TPP-binding riboswitch via a feedback inhibition mechanism. PMID:21622748

  17. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; T. Nithyanantham; X.-D Zhou; Y-W. Sin; H.U. Anderson; Alan Jacobson; C.A. Mims

    2005-05-01

    The present quarterly report describes some of the investigations on the structural properties of dense OTM bars provided by Praxair and studies on newer composition of Ti doped Ti-substituted perovskites, La{sub 0.7}Sr{sub 0.3}Mn{sub 1-x}Ti{sub x}O{sub 3}, with 0 {le} x {le} 0.20, were investigated by neutron diffraction, magnetization, electric resistivity, and magnetoresistance (MR) measurements. All samples show a rhombohedral structure (space group R3C) from 10 K to room temperature. At room temperature, the cell parameters a, c and the unit cell volume increase with increasing Ti content. However, at 10 K, the cell parameter a has a maximum value for x = 0.10, and decreases for x > 0.10, while the unit cell volume remains nearly constant for x > 0.10. The average (Mn,Ti)-O bond length increases up to x = 0.15, and the (Mn,Ti)-O-(Mn,Ti) bond angle decreases with increasing Ti content to its minimum value at x = 0.15 at room temperature. Below the Curie temperature TC, the resistance exhibits metallic behavior for the x {le} 0.05 samples. A metal (semiconductor) to insulator transition is observed for the x {ge} 0.10 samples. A peak in resistivity appears below TC for all samples, and shifts to a lower temperature as x increases. The substitution of Mn by Ti decreases the 2p-3d hybridization between O and Mn ions, reduces the bandwidth W, and increases the electron-phonon coupling. Therefore, the TC shifts to a lower temperature and the resistivity increases with increasing Ti content. A field-induced shift of the resistivity maximum occurs at x {le} 0.10 compounds. The maximum MR effect is about 70% for La{sub 0.7}Sr{sub 0.3}Mn{sub 0.8}Ti{sub 0.2}O{sub 3}. The separation of TC and the resistivity maximum temperature T{sub {rho},max} enhances the MR effect in these compounds due to the weak coupling between the magnetic ordering and the resistivity as compared with La{sub 0.7}Sr{sub 0.3}MnO{sub 3}. The bulk densities of the membranes were determined using the

  18. Mutations in the white gene of Drosophila melanogaster affecting ABC transporters that determine eye colouration.

    PubMed

    Mackenzie, S M; Brooker, M R; Gill, T R; Cox, G B; Howells, A J; Ewart, G D

    1999-07-15

    The white, brown and scarlet genes of Drosophila melanogaster encode proteins which transport guanine or tryptophan (precursors of the red and brown eye colour pigments) and belong to the ABC transporter superfamily. Current models envisage that the white and brown gene products interact to form a guanine specific transporter, while white and scarlet gene products interact to form a tryptophan transporter. In this study, we report the nucleotide sequence of the coding regions of five white alleles isolated from flies with partially pigmented eyes. In all cases, single amino acid changes were identified, highlighting residues with roles in structure and/or function of the transporters. Mutations in w(cf) (G589E) and w(sat) (F590G) occur at the extracellular end of predicted transmembrane helix 5 and correlate with a major decrease in red pigments in the eyes, while brown pigments are near wild-type levels. Therefore, those residues have a more significant role in the guanine transporter than the tryptophan transporter. Mutations identified in w(crr) (H298N) and w(101) (G243S) affect amino acids which are highly conserved among the ABC transporter superfamily within the nucleotide binding domain. Both cause substantial and similar decreases of red and brown pigments indicating that both tryptophan and guanine transport are impaired. The mutation identified in w(Et87) alters an amino acid within an intracellular loop between transmembrane helices 2 and 3 of the predicted structure. Red and brown pigments are reduced to very low levels by this mutation indicating this loop region is important for the function of both guanine and tryptophan transporters. PMID:10407069

  19. Role of ABC and Solute Carrier Transporters in the Placental Transport of Lamivudine.

    PubMed

    Ceckova, Martina; Reznicek, Josef; Ptackova, Zuzana; Cerveny, Lukas; Müller, Fabian; Kacerovsky, Marian; Fromm, Martin F; Glazier, Jocelyn D; Staud, Frantisek

    2016-09-01

    Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of drug efflux transporters P-glycoprotein (P-gp) (MDR1 [ABCB1]), BCRP (ABCG2), MRP2 (ABCC2), and MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of lamivudine. We employed in vitro accumulation and transport experiments on MDCK cells overexpressing drug efflux transporters, in situ-perfused rat term placenta, and vesicular uptake in microvillous plasma membrane (MVM) vesicles isolated from human term placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-MDR1, MDCK-MRP2, and MDCK-BCRP monolayers. MATE1-mediated efflux of lamivudine appeared to be a low-affinity process (apparent Km of 4.21 mM and Vmax of 5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP, or MRP2. However, lamivudine transfer across dually perfused rat placenta and the uptake of lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible involvement of MATE1 in the fetal-to-maternal efflux of the drug. To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2, or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction. We suggest that MATE1 might be, at least partly, responsible for this transport. PMID:27401571

  20. ABC transporters coordinately expressed during lignification of Arabidopsis stems include a set of ABCBs associated with auxin transport

    PubMed Central

    Kaneda, M.; Schuetz, M.; Lin, B.S.P.; Chanis, C.; Hamberger, B.; Western, T.L.; Ehlting, J.; Samuels, A.L.

    2011-01-01

    The primary inflorescence stem of Arabidopsis thaliana is rich in lignified cell walls, in both vascular bundles and interfascicular fibres. Previous gene expression studies demonstrated a correlation between expression of phenylpropanoid biosynthetic genes and a subset of genes encoding ATP-binding cassette (ABC) transporters, especially in the ABCB/multi-drug resistance/P-glycoprotein (ABCB/MDR/PGP) and ABCG/pleiotropic drug resistance (ABCG/PDR) subfamilies. The objective of this study was to characterize these ABC transporters in terms of their gene expression and their function in development of lignified cells. Based on in silico analyses, four ABC transporters were selected for detailed investigation: ABCB11/MDR8, ABCB14/MDR12, ABCB15/MDR13, and ABCG33/PDR5. Promoter::glucuronidase reporter assays for each gene indicated that promoters of ABCB11, ABCB14, ABCB15, and ABCG33 transporters are active in the vascular tissues of primary stem, and in some cases in interfascicular tissues as well. Homozygous T-DNA insertion mutant lines showed no apparent irregular xylem phenotype or alterations in interfascicular fibre lignification or morphology in comparison with wild type. However, in abcb14-1 mutants, stem vascular morphology was slightly disorganized, with decreased phloem area in the vascular bundle and decreased xylem vessel lumen diameter. In addition, abcb14-1 mutants showed both decreased polar auxin transport through whole stems and altered auxin distribution in the procambium. It is proposed that both ABCB14 and ABCB15 promote auxin transport since inflorescence stems in both mutants showed a reduction in polar auxin transport, which was not observed for any of the ABCG subfamily mutants tested. In the case of ABCB14, the reduction in auxin transport is correlated with a mild disruption of vascular development in the inflorescence stem. PMID:21239383

  1. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; T. Nithyanantham

    2006-12-31

    Ti doping on La{sub 1-x}Sr{sub x}FeO{sub 3-{delta}} (LSF) tends to increase the oxygen equilibration kinetics of LSF in lower oxygen activity environment because of the high valence state of Ti. However, the addition of Ti decreases the total conductivity because the acceptor ([Sr{prime}{sub La}]) is compensated by the donor ([Ti{sub Fe}{sup {sm_bullet}}]) which decreases the carrier concentration. The properties of La{sub 0.2}Sr{sub 0.8}Fe{sub 1-x}Ti{sub x}O{sub 3-{delta}} (LSFT, x = 0.45) have been experimentally and theoretically investigated to elucidate (1) the dependence of oxygen occupancy and electrochemical properties on temperature and oxygen activity by thermogravimetric analysis (TGA) and (2) the electrical conductivity and carrier concentration by Seebeck coefficient and electrical measurements. In the present study, dual phase (La{sub 0.2}Sr{sub 0.8}Fe{sub 0.6}Ti{sub 0.4}O{sub 3-{delta}}/Ce{sub 0.9}Gd{sub 0.1}O{sub 2-{delta}}) membranes have been evaluated for structural properties such as hardness, fracture toughness and flexural strength. The effect of high temperature and slightly reducing atmosphere on the structural properties of the membranes was studied. The flexural strength of the membrane decreases upon exposure to slightly reducing conditions at 1000 C. The as-received and post-fractured membranes were characterized using XRD, SEM and TG-DTA to understand the fracture mechanisms. Changes in structural properties of the composite were sought to be correlated with the physiochemical features of the two-phases. We have reviewed the electrical conductivity data and stoichiometry data for La{sub 0.2}Sr{sub 0.8}Cr{sub 0.2}Fe{sub 0.8}O{sub 3-{delta}} some of which was reported previously. Electrical conductivity data for La{sub 0.2}Sr{sub 0.8}Cr{sub 0.2}Fe{sub 0.8}O{sub 3-{delta}} (LSCrF) were obtained in the temperature range, 752 {approx} 1055 C and in the pO{sub 2} range, 10{sup -18} {approx} 0.5 atm. The slope of the plot of log {sigma} vs

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  4. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; T. Nithyanantham

    2006-06-30

    A non-agglomerated and nanocrystalline-sized powder was successfully produced using ethylene glycol nitrate methods. The LSFT powder prepared using this method exhibits well dispersed and nano-sized particles about 100-200 nm. The density of LSFT sintered at 1300 C was about 90% of the theoretical density at which is 100 C less than that of the previous LSFT which was sintered at 1400 C. The sample sintered at 1400 C exhibited the evidence of a liquid phase at the grain boundaries and 2nd phase formation which probably caused low mechanical stability. The electrical conductivity and Seebeck coefficient were measured as a function of temperature. The LSFT-CGO specimens were cut from the as sintered bars and used for the evaluation of Mechanical Properties after polishing. The effect of strain rate on the flexural strength of the LSFT-CGO test specimens was studied. Three strain rates 6, 60 and 600 {micro}m/ min were chosen for this study. It is observed from the results that with increasing cross head speed the membrane takes higher loads to fail. A reduction in the strength of the membrane was observed at 1000 C in N{sub 2}. Two different routes were investigated to synthesis GDC using either formate or carbonate precursors. The precursor and CGO particle morphologies were examined by scanning electron microscopy. The thermal decomposition behaviors of Ce(Gd)(HCOO){sub 3} and Ce(Gd)(CO{sub 3})(OH) were determined by thermogravimetric analysis (TGA) at a rate of 3 C/min in air. The X-ray powder diffraction patterns of the precursor and CGO were collected and nitrogen adsorption isotherms were measured. Conductivity measurements were made by AC impedance spectroscopy on sintered disks in air using platinum electrodes.

  5. Membrane transporter proteins: a challenge for CNS drug development

    PubMed Central

    Girardin, François

    2006-01-01

    Drug transporters are membrane proteins present in various tissues such as the lymphocytes, intestine, liver, kidney, testis, placenta, and central nervous system. These transporters play a significant role in drug absorption and distribution to organic systems, particularly if the organs are protected by blood-organ barriers, such as the blood-brain barrier or the maternal-fetal barrier. In contrast to neurotransmitters and receptor-coupled transporters or other modes of interneuronal transmission, drug transporters are not directly involved in specific neuronal functions, but provide global protection to the central nervous system. The lack of capillary fenestration, the low pinocytic activity, and the tight junctions between brain capillary and choroid plexus endothelial cells represent further gatekeepers limiting the entrance of endogenous and exogenous compounds into the central nervous system. Drug transport is a result of the concerted action of efflux and influx pumps (transporters) located both in the basolateral and apical membranes of brain capillary and choroid plexus endothelial cells. By regulating efflux and influx of endogenous or exogenous substances, the blood-brain barrier and, to a lesser extent, the blood-cerebrospinal barrier in the ventricles, represents the main interface between the central nervous system and the blood, ie, the rest of the body. As drug distribution to organs is dependent on the affinity of a substrate for a specific transport system, membrane transporter proteins are increasingly recognized as a key determinant of drug disposition. Many drug transporters are members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter superfamily or the solute-linked carrier (SLC) class. The multidrug resistance protein MDR1 (ABCB1), also called P-glycoprotein, the multidrug resistance-associated proteins MRP1 (ABCC1) and MRP2 (ABCC2), and the breast cancer-resistance protein BCRP (ABCG2) are ATP-dependent efflux

  6. Asymmetric localizations of the ABC transporter PaPDR1 trace paths of directional strigolactone transport.

    PubMed

    Sasse, Joëlle; Simon, Sibu; Gübeli, Christian; Liu, Guo-Wei; Cheng, Xi; Friml, Jiří; Bouwmeester, Harro; Martinoia, Enrico; Borghi, Lorenzo

    2015-03-01

    Strigolactones, first discovered as germination stimulants for parasitic weeds [1], are carotenoid-derived phytohormones that play major roles in inhibiting lateral bud outgrowth and promoting plant-mycorrhizal symbiosis [2-4]. Furthermore, strigolactones are involved in the regulation of lateral and adventitious root development, root cell division [5, 6], secondary growth [7], and leaf senescence [8]. Recently, we discovered the strigolactone transporter Petunia axillaris PLEIOTROPIC DRUG RESISTANCE 1 (PaPDR1), which is required for efficient mycorrhizal colonization and inhibition of lateral bud outgrowth [9]. However, how strigolactones are transported through the plant remained unknown. Here we show that PaPDR1 exhibits a cell-type-specific asymmetric localization in different root tissues. In root tips, PaPDR1 is co-expressed with the strigolactone biosynthetic gene DAD1 (CCD8), and it is localized at the apical membrane of root hypodermal cells, presumably mediating the shootward transport of strigolactone. Above the root tip, in the hypodermal passage cells that form gates for the entry of mycorrhizal fungi, PaPDR1 is present in the outer-lateral membrane, compatible with its postulated function as strigolactone exporter from root to soil. Transport studies are in line with our localization studies since (1) a papdr1 mutant displays impaired transport of strigolactones out of the root tip to the shoot as well as into the rhizosphere and (2) DAD1 expression and PIN1/PIN2 levels change in plants deregulated for PDR1 expression, suggestive of variations in endogenous strigolactone contents. In conclusion, our results indicate that the polar localizations of PaPDR1 mediate directional shootward strigolactone transport as well as localized exudation into the soil. PMID:25683808

  7. Carbon Dioxide Transport through Membranes*

    PubMed Central

    Missner, Andreas; Kügler, Philipp; Saparov, Sapar M.; Sommer, Klaus; Mathai, John C.; Zeidel, Mark L.; Pohl, Peter

    2008-01-01

    Several membrane channels, like aquaporin-1 (AQP1) and the RhAG protein of the rhesus complex, were hypothesized to be of physiological relevance for CO2 transport. However, the underlying assumption that the lipid matrix imposes a significant barrier to CO2 diffusion was never confirmed experimentally. Here we have monitored transmembrane CO2 flux (JCO2) by imposing a CO2 concentration gradient across planar lipid bilayers and detecting the resulting small pH shift in the immediate membrane vicinity. An analytical model, which accounts for the presence of both carbonic anhydrase and buffer molecules, was fitted to the experimental pH profiles using inverse problems techniques. At pH 7.4, the model revealed that JCO2 was entirely rate-limited by near-membrane unstirred layers (USL), which act as diffusional barriers in series with the membrane. Membrane tightening by sphingomyelin and cholesterol did not alter JCO2 confirming that membrane resistance was comparatively small. In contrast, a pH-induced shift of the CO2 hydration-dehydration equilibrium resulted in a relative membrane contribution of about 15% to the total resistance (pH 9.6). Under these conditions, a membrane CO2 permeability (3.2 ± 1.6 cm/s) was estimated. It indicates that cellular CO2 uptake (pH 7.4) is always USL-limited, because the USL size always exceeds 1 μm. Consequently, facilitation of CO2 transport by AQP1, RhAG, or any other protein is highly unlikely. The conclusion was confirmed by the observation that CO2 permeability of epithelial cell monolayers was always the same whether AQP1 was overexpressed in both the apical and basolateral membranes or not. PMID:18617525

  8. 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.

  9. Evidence that Bacterial ABC-Type Transporter Imports Free EDTA for Metabolism

    SciTech Connect

    Zhang, Hua; Herman, Jacob P.; Bolton, Harvey; Zhang, Zhicheng; Clark, Sue B.; Xun, Luying

    2007-11-01

    Ethylenediaminetetraacetic acid (EDTA), a common chelating agent, is becoming a major organic pollutant in the form of metal-EDTA complexes in surface waters, partly due to its recalcitrance to biodegradation. Even an EDTA-degrading bacterium BNC1 does not degrade stable metal-EDTA complexes. An ABC-type transporter was identified for possible uptake of EDTA because the transporter genes and EDTA monooxygenase gene were expressed in a single operon in BNC1. The ABC-type transporter had a periplasmic binding protein (EppA) that should confer the substrate specificity for the transporter; therefore, EppA was produced in Escherichia coli,purified, and characterized. EppA was shown to bind free EDTA with a dissociation constant as low as 25 nM by using isothermal titration calorimetry. When unstable metal-EDTA complexes, e.g. MgEDTA2-, were added to the EppA solution, binding was also observed. However, experimental data and theoretical analysis only supported EppA binding of free EDTA. When stable metal-EDTA complexes, e.g. CuEDTA2-, are titrated into the EppA solution, no binding was observed. Since EDTA monooxygenase in the cytoplasm uses some of the stable metal-EDTA complexes as substrates, we suggest that the lack of EppA binding and EDTA uptake are responsible for the failure of BNC1 cells to degrade the stable complexes.

  10. Oxygen Transport Ceramic Membranes

    SciTech Connect

    S. Bandopadhyay; N. Nagabhushana; X.-D Zhou; W.B. Yelon; H.U. Anderson; Alan Jacobson; C.A. Mims

    2004-02-01

    The present quarterly report describes some of the investigations on the structural properties of dense OTM bars provided by Praxair and initial studies on newer composition of Ti doped LSF. Dense OTM bars provided by Praxair were loaded to fracture at varying stress rates. Studies were done at room temperature in air and at 1000 C in a specified environment to evaluate slow crack growth behavior. In addition, studies were also begun to obtain reliable estimates of fracture toughness and stable crack growth in specific environments. Newer composition of Ti doped LSF membranes were characterized by neutron diffraction analysis. Quench studies indicated an apparent correlation between the unit cell volume and oxygen occupancy. The studies however, indicated an anomaly of increasing Fe/Ti ratio with change in heat treatment. Ti doped LSF was also characterized for stoichiometry as a function of temp and pO{sub 2}. The non stoichiometry parameter {delta} was observed to increase almost linearly on lowering pO{sub 2} until a ideal stoichiometric composition of {delta} = 0.175 was approached.

  11. The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

    PubMed Central

    Hürlimann, Lea M.; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V.; Tieleman, D. Peter

    2016-01-01

    Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis. In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell. PMID:27381387

  12. The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis.

    PubMed

    Hürlimann, Lea M; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V; Tieleman, D Peter; Seeger, Markus A

    2016-09-01

    Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell. PMID:27381387

  13. Effect of dietary polyunsaturated fatty acids on the expression of peroxisomal ABC transporters.

    PubMed

    Leclercq, Sabrina; Skrzypski, Jérémy; Courvoisier, Anne; Gondcaille, Catherine; Bonnetain, Franck; André, Agnès; Chardigny, Jean-Michel; Bellenger, Sandrine; Bellenger, Jérôme; Narce, Michel; Savary, Stéphane

    2008-10-01

    Peroxisomal ABC transporters encoded by the ABCD genes are thought to participate in the import of specific fatty acids in the peroxisomal matrix. ABCD1 deficiency is associated with X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder which is characterized by the accumulation of saturated very-long-chain fatty acids (VLCFA). ABCD2 (the closest homolog of ABCD1) and ABCD3 have been shown to have partial functional redundancy with ABCD1; only when overexpressed, they can compensate for VLCFA accumulation. Other lipids, for instance polyunsaturated fatty acids (PUFA), should be possible candidate substrates for the ABCD2 and ABCD3 gene products, ALDRP and PMP70 respectively. Moreover, PUFA, which are known regulators of gene expression, could therefore represent potent inducers of the ABCD genes. To test this hypothesis, littermates of n-3-deficient rats were subjected to an n-3-deficient diet or equilibrated diets containing ALA (alpha-linolenic acid, 18:3n-3) as unique source of n-3 fatty acids or ALA plus DHA (docosahexaenoic acid, 22:6n-3) at two different doses. We analyzed the expression of peroxisomal ABC transporters and of the peroxisomal acyl-CoA oxidase gene 1 (Acox1) in adrenals, brain and liver. Whatever the diet, we did not observe any difference in gene expression in adrenals and brain. However, the hepatic expression level of Abcd2 and Abcd3 genes was found to be significantly higher in the n-3-deficient rats than in the rats fed the ALA diet or the DHA supplemented diets. This was accompanied by important changes in hepatic fatty acid composition. In summary, the hepatic expression of Abcd2 and Abcd3 but not of Abcd1 and Abcd4 appears to be highly sensitive towards dietary PUFA. This difference could be linked to the substrate specificity of the peroxisomal ABC transporters and a specific involvement of Abcd2 and Abcd3 in PUFA metabolism. PMID:18585430

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

    PubMed

    Tydén, Eva; Skarin, Moa; Höglund, Johan

    2014-12-01

    The most widespread helminth parasites of grazing cattle in northern Europe are the gastrointestinal nematodes Ostertagia ostertagi and Cooperia oncophora. Heavy reliance on the use of macrocyclic lactone (ML) in cattle has led to world-wide emergence of resistance to this drug class in C. oncophora. There is evidence that members of the ATP-binding cassette (ABC) transporter family, such as P-glycoproteins (P-gp) and multidrug-resistant proteins (MRP), play a role in resistance to ML. In this study gene expression of Con-pgp9, Con-pgp11, Con-pgp12, Con-pgp16 and Con-mrp1 was examined in two isolates of C. oncophora sharing the same genetic background but exposed to ML differently. For isolate one (Laboratory-selected), adult worms were recovered before and after treatment with ML in vivo. For isolate two (Field-selected), adult worms were collected from tracer animals that had never received anthelmintics themselves. One group grazed together with untreated animals and one group grazed with animals that received suppressive prophylactic treatment with ML at monthly intervals for up to two consecutive grazing seasons. Real-time PCR data demonstrated differences in gene expression after ML selection, with the highest constitutive expression levels for Con-pgp16 and Con-mrp1. Remarkably, the same pattern of increasing expression levels of the ABC transport genes was observed in both Laboratory- and Field-selected isolates, despite the Field-selected isolate not being directly exposed to ML. The higher expression levels of ABC transporters observed in the Field-selected isolate was thus not a response to direct exposure to ML, but rather appeared to reflect a genetic characteristic inherited from worms in the previous generation which had survived exposure to ML in the co-grazing treated animals. PMID:25619799

  15. 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

  16. Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog.

    PubMed

    Hardham, J M; Stamm, L V; Porcella, S F; Frye, J G; Barnes, N Y; Howell, J K; Mueller, S L; Radolf, J D; Weinstock, G M; Norris, S J

    1997-09-15

    We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp. PMID:9332349

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

    DOE PAGESBeta

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

    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

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

    SciTech Connect

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

    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, which coordinates the ATP-binding site, decreases NBD dimerization affinity and turns the unidirectional primary active pump into a passive bidirectional nucleotide-gated facilitator. Thus, ATP hydrolysis is not required for translocation per se, but is essential for both active and unidirectional transport. As a result, our data provide detailed mechanistic insight into how heterodimeric ABC exporters operate.

  19. ABC-Transporter Expression Does Not Correlate with Response to Irinotecan in Patients with Metastatic Colorectal Cancer

    PubMed Central

    Trumpi, K.; Emmink, B.L.; Prins, A.M.; van Oijen, M.G.H.; van Diest, P.J.; Punt, C.J.A.; Koopman, M.; Kranenburg, O.; Rinkes, I.H.M. Borel

    2015-01-01

    Background: Active efflux of irinotecan by ATP-binding cassette (ABC)-transporters, in particular ABCB1 and ABCG2, is a well-established drug resistance mechanism in vitro and in pre-clinical mouse models, but its relevance in colorectal cancer (CRC) patients is unknown. Therefore, we assessed the association between ABC-transporter expression and tumour response to irinotecan in patients with metastatic CRC. Methods: Tissue microarrays of a large cohort of metastatic CRC patients treated with irinotecan in a prospective study (CAIRO study; n=566) were analysed for expression of ABCB1 and ABCG2 by immunohistochemistry. Kaplan-Meier and Cox proportional hazard regression analyses were performed to assess the association of ABC transporter expression with irinotecan response. Gene expression profiles of 17 paired tumours were used to assess the concordance of ABCB1/ABCG2 expression in primary CRC and corresponding metastases. Results: The response to irinotecan was not significantly different between primary tumours with positive versus negative expression of ABCB1 (5.8 vs 5.7 months, p=0.696) or ABCG2 (5.7 vs 6.1 months, p=0.811). Multivariate analysis showed neither ABCB1 nor ABCG2 were independent predictors for progression free survival. There was a mediocre to poor concordance between ABC-transporter expression in paired tumours. Conclusion: In metastatic CRC, ABC-transporter expression in the primary tumour does not predict irinotecan response. PMID:26516354

  20. Lysimachia foenum-graecum Herba Extract, a Novel Biopesticide, Inhibits ABC Transporter Genes and Mycelial Growth of Magnaporthe oryzae.

    PubMed

    Lee, Youngjin

    2016-02-01

    To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration (IC50) of 39.28 μg/ml, which is lower than the IC50 of blasticidin S (63.06 μg/ml), a conventional fungicide for rice blast disease. When treatments were combined, the IC50 of blasticidin S was dramatically reduced to 10.67 μg/ml. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn't show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide. PMID:26889110

  1. Lysimachia foenum-graecum Herba Extract, a Novel Biopesticide, Inhibits ABC Transporter Genes and Mycelial Growth of Magnaporthe oryzae

    PubMed Central

    Lee, Youngjin

    2016-01-01

    To identify a novel biopesticide controlling rice blast disease caused by Magnaporthe oryzae, 700 plant extracts were evaluated for their inhibitory effects on mycelial growth of M. oryzae. The L. foenum-graecum Herba extract showed the lowest inhibition concentration (IC50) of 39.28 μg/ml, which is lower than the IC50 of blasticidin S (63.06 μg/ml), a conventional fungicide for rice blast disease. When treatments were combined, the IC50 of blasticidin S was dramatically reduced to 10.67 μg/ml. Since ABC transporter genes are involved in fungicide resistance of many organisms, we performed RT-PCR to investigate the transcriptional changes of 40 ABC transporter family genes of M. oryzae treated with the plant extract, blasticidin S, and tetrandrine, a recognized ABC transporter inhibitor. Four ABC transporter genes were prominently activated by blasticidin S treatment, but were suppressed by combinational treatment of blasticidin S with the plant extract, or with tetrandrine that didn’t show cellular toxicity by itself in this study. Mycelial death was detected via confocal microscopy at 24 h after plant extract treatment. Finally, subsequent rice field study revealed that the plant extract had high control efficacy of 63.3% and should be considered a biopesticide for rice blast disease. These results showed that extract of L. foenum graecum Herba suppresses M. oryzae ABC transporter genes inducing mycelial death and therefore may be a potent novel biopesticide. PMID:26889110

  2. 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

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

    PubMed

    Chen, Mingli; Yin, Huancai; Bai, Pengli; Miao, Peng; Deng, Xudong; Xu, Yingxue; Hu, Jun; Yin, Jian

    2016-07-15

    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 530nm). 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 of 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 CdCl2 at equivalent concentrations of cadmium, indicating that the effects of QDs should be a combination of free Cd(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. PMID:27131644

  4. 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

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

    PubMed

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

    2012-07-01

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

  6. The ABC Transporter ABCG1 Is Required for Suberin Formation in Potato Tuber Periderm[W

    PubMed Central

    Landgraf, Ramona; Smolka, Ulrike; Altmann, Simone; Eschen-Lippold, Lennart; Senning, Melanie; Sonnewald, Sophia; Weigel, Benjamin; Frolova, Nadezhda; Strehmel, Nadine; Hause, Gerd; Scheel, Dierk; Böttcher, Christoph; Rosahl, Sabine

    2014-01-01

    The lipid biopolymer suberin plays a major role as a barrier both at plant-environment interfaces and in internal tissues, restricting water and nutrient transport. In potato (Solanum tuberosum), tuber integrity is dependent on suberized periderm. Using microarray analyses, we identified ABCG1, encoding an ABC transporter, as a gene responsive to the pathogen-associated molecular pattern Pep-13. Further analyses revealed that ABCG1 is expressed in roots and tuber periderm, as well as in wounded leaves. Transgenic ABCG1-RNAi potato plants with downregulated expression of ABCG1 display major alterations in both root and tuber morphology, whereas the aerial part of the ABCG1-RNAi plants appear normal. The tuber periderm and root exodermis show reduced suberin staining and disorganized cell layers. Metabolite analyses revealed reduction of esterified suberin components and hyperaccumulation of putative suberin precursors in the tuber periderm of RNA interference plants, suggesting that ABCG1 is required for the export of suberin components. PMID:25122151

  7. Inhibition of ABC transport proteins by oil sands process affected water.

    PubMed

    Alharbi, Hattan A; Saunders, David M V; Al-Mousa, Ahmed; Alcorn, Jane; Pereira, Alberto S; Martin, Jonathan W; Giesy, John P; Wiseman, Steve B

    2016-01-01

    The ATP-binding cassette (ABC) superfamily of transporter proteins is important for detoxification of xenobiotics. For example, ABC transporters from the multidrug-resistance protein (MRP) subfamily are important for excretion of polycyclic aromatic hydrocarbons (PAHs) and their metabolites. Effects of chemicals in the water soluble organic fraction of relatively fresh oil sands process affected water (OSPW) from Base Mine Lake (BML-OSPW) and aged OSPW from Pond 9 (P9-OSPW) on the activity of MRP transporters were investigated in vivo by use of Japanese medaka at the fry stage of development. Activities of MRPs were monitored by use of the lipophilic dye calcein, which is transported from cells by ABC proteins, including MRPs. To begin to identify chemicals that might inhibit activity of MRPs, BML-OSPW and P9-OSPW were fractionated into acidic, basic, and neutral fractions by use of mixed-mode sorbents. Chemical compositions of fractions were determined by use of ultrahigh resolution orbitrap mass spectrometry in ESI(+) and ESI(-) mode. Greater amounts of calcein were retained in fry exposed to BML-OSPW at concentration equivalents greater than 1× (i.e., full strength). The neutral and basic fractions of BML-OSPW, but not the acidic fraction, caused greater retention of calcein. Exposure to P9-OSPW did not affect the amount of calcein in fry. Neutral and basic fractions of BML-OSPW contained relatively greater amounts of several oxygen-, sulfur, and nitrogen-containing chemical species that might inhibit MRPs, such as O(+), SO(+), and NO(+) chemical species, although secondary fractionation will be required to conclusively identify the most potent inhibitors. Naphthenic acids (O2(-)), which were dominant in the acidic fraction, did not appear to be the cause of the inhibition. This is the first study to demonstrate that chemicals in the water soluble organic fraction of OSPW inhibit activity of this important class of proteins. However, aging of OSPW attenuates

  8. Membrane transporters in drug development

    PubMed Central

    2011-01-01

    Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labeling. PMID:20190787

  9. ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium--Effects of Prochloraz.

    PubMed

    Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H; Tallkvist, Jonas

    2016-01-01

    Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers. PMID:27028005

  10. ABC- and SLC-Transporters in Murine and Bovine Mammary Epithelium - Effects of Prochloraz

    PubMed Central

    Yagdiran, Yagmur; Oskarsson, Agneta; Knight, Christopher H.; Tallkvist, Jonas

    2016-01-01

    Some chemicals are ligands to efflux transporters which may result in high concentrations in milk. Limited knowledge is available on the influence of maternal exposure to chemicals on the expression and function of transporters in the lactating mammary gland. We determined gene expression of ABC and SLC transporters in murine mammary tissue of different gestation and lactation stages, in murine mammary cells (HC11) featuring resting and secreting phenotypes and in bovine mammary tissue and cells (BME-UV). Effects on transporter expression and function of the imidazole fungicide prochloraz, previously reported to influence BCRP in mammary cells, was investigated on transporter expression and function in the two cell lines. Transporters studied were BCRP, MDR1, MRP1, OATP1A5/OATP1A2, OCTN1 and OCT1. Gene expressions of BCRP and OCT1 in murine mammary glands were increased during gestation and lactation, whereas MDR1, MRP1, OATP1A5 and OCTN1 were decreased, compared to expressions in virgins. All transporters measured in mammary glands of mice were detected in bovine mammary tissue and in HC11 cells, while only MDR1 and MRP1 were detected in BME-UV cells. Prochloraz treatment induced MDR1 gene and protein expression in both differentiated HC11 and BME-UV cells and increased protein function in HC11 cells, resulting in decreased accumulation of the MDR1 substrate digoxin. In conclusion, our results demonstrate that murine (HC11) and bovine (BME-UV) mammary epithelial cells can be applied to characterize expression and function of transporters as well as effects of contaminants on the mammary transporters. An altered expression, induced by a drug or toxic chemical, on any of the transporters expressed in the mammary epithelial cells during lactation may modulate the well-balanced composition of nutrients and/or secretion of contaminants in milk with potential adverse effects on breast-fed infants and dairy consumers. PMID:27028005

  11. 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

  12. Characterisation of the membrane transport of pilocarpine in cell suspension cultures of Pilocarpus microphyllus.

    PubMed

    Andreazza, Nathalia Luiza; Abreu, Ilka Nacif; Sawaya, Alexandra Christine Helena Frankland; Mazzafera, Paulo

    2015-03-01

    Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results. PMID:25474486

  13. The CydDC ABC transporter of Escherichia coli: new roles for a reductant efflux pump.

    PubMed

    Shepherd, Mark

    2015-10-01

    The CydDC complex of Escherichia coli is a heterodimeric ATP-binding cassette (ABC) transporter that exports cysteine and glutathione to the periplasm. These reductants are thought to modulate periplasmic redox poise, impacting upon the disulfide folding of periplasmic and secreted proteins involved in bacterial virulence. Diminished CydDC activity abolishes the assembly of functional bd-type respiratory oxidases and perturbs haem ligation during the assembly of c-type cytochromes. The focus herein is upon a newly-discovered interaction of the CydDC complex with a haem cofactor; haem has recently been shown to modulate CydDC activity and structural modelling reveals a potential haem-binding site on the periplasmic surface of the complex. These findings have important implications for future investigations into the potential roles for the CydDC-bound haem in redox sensing and tolerance to nitric oxide (NO). PMID:26517902

  14. Role of the ABC transporter Mdr49 in Hedgehog signaling and germ cell migration.

    PubMed

    Deshpande, Girish; Manry, Diane; Jourjine, Nicholas; Mogila, Vladic; Mozes, Henny; Bialistoky, Tzofia; Gerlitz, Offer; Schedl, Paul

    2016-06-15

    Coalescence of the embryonic gonad in Drosophila melanogaster requires directed migration of primordial germ cells (PGCs) towards somatic gonadal precursor cells (SGPs). It was recently proposed that the ATP-binding cassette (ABC) transporter Mdr49 functions in the embryonic mesoderm to facilitate the transmission of the PGC attractant from the SGPs; however, the precise molecular identity of the Mdr49-dependent guidance signal remained elusive. Employing the loss- and gain-of-function strategies, we show that Mdr49 is a component of the Hedgehog (hh) pathway and it potentiates the signaling activity. This function is direct because in Mdr49 mutant embryos the Hh ligand is inappropriately sequestered in the hh-expressing cells. Our data also suggest that the role of Mdr49 is to provide cholesterol for the correct processing of the Hh precursor protein. Supporting this conclusion, PGC migration defects in Mdr49 embryos are substantially ameliorated by a cholesterol-rich diet. PMID:27122170

  15. 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...

  16. An ABC Transporter Mutation Alters Root Exudation of Phytochemicals that Provoke an Overhaul of Natural Soil Microbiota.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    It has been shown that Arabidopsis root exudates can support the fungal community in native soils but not in non-native soils and recent evidence demonstrates the involvement of ABC transporters in the root secretion of phytochemicals. In this paper we examined differences in the root exudate profil...

  17. 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

    2016-01-01

    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. PMID:27456980

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

    PubMed Central

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

    2016-01-01

    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. PMID:27456980

  19. Involvement of the Carboxyl-Terminal Region of the Yeast Peroxisomal Half ABC Transporter Pxa2p in Its Interaction with Pxa1p and in Transporter Function

    PubMed Central

    Chuang, Cheng-Yi; Chen, Ling-Yun; Fu, Ru-Huei; Chen, Shih-Ming; Ho, Ming-Hua; Huang, Jie-Mau; Hsu, Chia-Chi; Wang, Chien-Cheng; Chen, Meng-Shian; Tsai, Rong-Tzong

    2014-01-01

    Background The peroxisome is a single membrane-bound organelle in eukaryotic cells involved in lipid metabolism, including β-oxidation of fatty acids. The human genetic disorder X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene (encoding ALDP, a peroxisomal half ATP-binding cassette [ABC] transporter). This disease is characterized by defective peroxisomal β-oxidation and a large accumulation of very long-chain fatty acids in brain white matter, adrenal cortex, and testis. ALDP forms a homodimer proposed to be the functional transporter, whereas the peroxisomal transporter in yeast is a heterodimer comprising two half ABC transporters, Pxa1p and Pxa2p, both orthologs of human ALDP. While the carboxyl-terminal domain of ALDP is engaged in dimerization, it remains unknown whether the same region is involved in the interaction between Pxa1p and Pxa2p. Methods/Principal Findings Using a yeast two-hybrid assay, we found that the carboxyl-terminal region (CT) of Pxa2p, but not of Pxa1p, is required for their interaction. Further analysis indicated that the central part of the CT (designated CT2) of Pxa2p was indispensable for its interaction with the carboxyl terminally truncated Pxa1_NBD. An interaction between the CT of Pxa2p and Pxa1_NBD was not detected, but could be identified in the presence of Pxa2_NBD-CT1. A single mutation of two conserved residues (aligned with X-ALD-associated mutations at the same positions in ALDP) in the CT2 of the Pxa2_NBD-CT protein impaired its interaction with Pxa1_NBD or Pxa1_NBD-CT, resulting in a mutant protein that exhibited a proteinase K digestion profile different from that of the wild-type protein. Functional analysis of these mutant proteins on oleate plates indicated that they were defective in transporter function. Conclusions/Significance The CT of Pxa2p is involved in its interaction with Pxa1p and in transporter function. This concept may be applied to human ALDP studies, helping to establish

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

    PubMed Central

    Jones, Peter M.; George, Anthony M.

    2015-01-01

    ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs), which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR) transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration ‘sandwich’ dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD ‘Switch’ mechanism in which the NBD monomers separate in between ATP hydrolysis cycles. PMID:26120849

  1. Optimized Purification of a Heterodimeric ABC Transporter in a Highly Stable Form Amenable to 2-D Crystallization

    PubMed Central

    Galián, Carmen; Manon, Florence; Dezi, Manuela; Torres, Cristina; Ebel, Christine; Lévy, Daniel; Jault, Jean-Michel

    2011-01-01

    Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (kcat∼5 s−1). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment. PMID:21602923

  2. Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.

    PubMed

    Galián, Carmen; Manon, Florence; Dezi, Manuela; Torres, Cristina; Ebel, Christine; Lévy, Daniel; Jault, Jean-Michel

    2011-01-01

    Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment. PMID:21602923

  3. 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

  4. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Noel, Gregory; Stieger, Bruno; Fardel, Olivier

    2015-08-01

    Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies. PMID:25862123

  5. Catalyst containing oxygen transport membrane

    DOEpatents

    Christie, Gervase Maxwell; Wilson, Jamie Robyn; van Hassel, Bart Antonie

    2012-12-04

    A composite oxygen transport membrane having a dense layer, a porous support layer and an intermediate porous layer located between the dense layer and the porous support layer. Both the dense layer and the intermediate porous layer are formed from an ionic conductive material to conduct oxygen ions and an electrically conductive material to conduct electrons. The porous support layer has a high permeability, high porosity, and a high average pore diameter and the intermediate porous layer has a lower permeability and lower pore diameter than the porous support layer. Catalyst particles selected to promote oxidation of a combustible substance are located in the intermediate porous layer and in the porous support adjacent to the intermediate porous layer. The catalyst particles can be formed by wicking a solution of catalyst precursors through the porous support toward the intermediate porous layer.

  6. ABC transporter functions as a pacemaker for sequestration of plant glucosides in leaf beetles

    PubMed Central

    Strauss, Anja S; Peters, Sven; Boland, Wilhelm; Burse, Antje

    2013-01-01

    Plant-herbivore interactions dominate the planet’s terrestrial ecology. When it comes to host–plant specialization, insects are among the most versatile evolutionary innovators, able to disarm multiple chemical plant defenses. Sequestration is a widespread strategy to detoxify noxious metabolites, frequently for the insect’s own benefit against predation. In this study, we describe the broad-spectrum ATP-binding cassette transporter CpMRP of the poplar leaf beetle, Chrysomela populi as the first candidate involved in the sequestration of phytochemicals in insects. CpMRP acts in the defensive glands of the larvae as a pacemaker for the irreversible shuttling of pre-selected metabolites from the hemolymph into defensive secretions. Silencing CpMRP in vivo creates a defenseless phenotype, indicating its role in the secretion process is crucial. In the defensive glands of related leaf beetle species, we identified sequences similar to CpMRP and assume therefore that exocrine gland-based defensive strategies, evolved by these insects to repel their enemies, rely on ABC transporters as a key element. DOI: http://dx.doi.org/10.7554/eLife.01096.001 PMID:24302568

  7. ABC transporter functions as a pacemaker for sequestration of plant glucosides in leaf beetles.

    PubMed

    Strauss, Anja S; Peters, Sven; Boland, Wilhelm; Burse, Antje

    2013-01-01

    Plant-herbivore interactions dominate the planet's terrestrial ecology. When it comes to host-plant specialization, insects are among the most versatile evolutionary innovators, able to disarm multiple chemical plant defenses. Sequestration is a widespread strategy to detoxify noxious metabolites, frequently for the insect's own benefit against predation. In this study, we describe the broad-spectrum ATP-binding cassette transporter CpMRP of the poplar leaf beetle, Chrysomela populi as the first candidate involved in the sequestration of phytochemicals in insects. CpMRP acts in the defensive glands of the larvae as a pacemaker for the irreversible shuttling of pre-selected metabolites from the hemolymph into defensive secretions. Silencing CpMRP in vivo creates a defenseless phenotype, indicating its role in the secretion process is crucial. In the defensive glands of related leaf beetle species, we identified sequences similar to CpMRP and assume therefore that exocrine gland-based defensive strategies, evolved by these insects to repel their enemies, rely on ABC transporters as a key element. DOI: http://dx.doi.org/10.7554/eLife.01096.001. PMID:24302568

  8. The dynamics of the MBP-MalFGK(2) interaction: a prototype for binding protein dependent ABC-transporter systems.

    PubMed

    Shilton, Brian H

    2008-09-01

    This review is focused on the interaction between maltose binding protein (MBP) and the maltose transporter complex, MalFGK(2), which is a member of the ATP Binding Cassette (ABC) superfamily. The interaction between MBP and MalFGK(2) has a critical role in maltose transport, but a coherent description of the interaction is complicated because both MBP and MalFGK(2) can adopt multiple conformations. Drawing on genetic, structural, and biochemical data, the different conformations of MBP and MalFGK(2) are described and incorporated into a model for their interaction. The most important feature of this model is that ligand-bound MBP initiates the process of ATP-dependent maltose transport by stabilizing a high-energy conformation of MalFGK(2). In this model of the MBP-MalFGK(2) interaction, stabilization of a high-energy conformation of MalFGK(2) allows ATP to drive conformational changes in the system - in particular the opening of bound MBP - that leads to formation of a transition state for ATP hydrolysis. Such a role for ligand-bound MBP explains how MBP-independent MalFGK(2) mutants work, and represents a general mechanism for binding-protein dependent ABC import systems. In ABC export systems, which do not use a binding protein, the substrate itself is expected to play a role similar to ligand-bound MBP in the maltose transport system. The mechanistic model for the maltose transporter suggests that ABC-type import systems evolved to make use of a peripheral binding protein so that the transport process is essentially irreversible. PMID:17950243

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

    PubMed

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

    2016-06-01

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

  10. 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

  11. The ABCC4 membrane transporter modulates platelet aggregation.

    PubMed

    Cheepala, Satish B; Pitre, Aaron; Fukuda, Yu; Takenaka, Kazumasa; Zhang, Yuanyuan; Wang, Yao; Frase, Sharon; Pestina, Tamara; Gartner, T Kent; Jackson, Carl; Schuetz, John D

    2015-11-12

    Controlling the activation of platelets is a key strategy to mitigate cardiovascular disease. Previous studies have suggested that the ATP-binding cassette (ABC) transporter, ABCC4, functions in platelet-dense granules. Using plasma membrane biotinylation and super-resolution microscopy, we demonstrate that ABCC4 is primarily expressed on the plasma membrane of both mouse and human platelets. Platelets lacking ABCC4 have unchanged dense-granule function, number, and volume, but harbor a selective impairment in collagen-induced aggregation. Accordingly, Abcc4 knockout (KO) platelet attachment to a collagen substratum was also faulty and associated with elevated intracellular cyclic AMP (cAMP) and reduced plasma membrane localization of the major collagen receptor, GPVI. In the ferric-chloride vasculature injury model, Abcc4 KO mice exhibited markedly impaired thrombus formation. The attenuation of platelet aggregation by the phosphodiesterase inhibitor EHNA (a non-ABCC4 substrate), when combined with Abcc4 deficiency, illustrated a crucial functional interaction between phosphodiesterases and ABCC4. This was extended in vivo where EHNA dramatically prolonged the bleeding time, but only in Abcc4 KO mice. Further, we demonstrated in human platelets that ABCC4 inhibition, when coupled with phosphodiesterase inhibition, strongly impaired platelet aggregation. These findings have important clinical implications because they directly highlight an important relationship between ABCC4 transporter function and phosphodiesterases in accounting for the cAMP-directed activity of antithrombotic agents. PMID:26405223

  12. β-Cyclodextrins Decrease Cholesterol Release and ABC-Associated Transporter Expression in Smooth Muscle Cells and Aortic Endothelial Cells

    PubMed Central

    Coisne, Caroline; Hallier-Vanuxeem, Dorothée; Boucau, Marie-Christine; Hachani, Johan; Tilloy, Sébastien; Bricout, Hervé; Monflier, Eric; Wils, Daniel; Serpelloni, Michel; Parissaux, Xavier; Fenart, Laurence; Gosselet, Fabien

    2016-01-01

    Atherosclerosis is an inflammatory disease that leads to an aberrant accumulation of cholesterol in vessel walls forming atherosclerotic plaques. During this process, the mechanism regulating complex cellular cholesterol pools defined as the reverse cholesterol transport (RCT) is altered as well as expression and functionality of transporters involved in this process, namely ABCA1, ABCG1, and SR-BI. Macrophages, arterial endothelial and smooth muscle cells (SMCs) have been involved in the atherosclerotic plaque formation. As macrophages are widely described as the major cell type forming the foam cells by accumulating intracellular cholesterol, RCT alterations have been poorly studied at the arterial endothelial cell and SMC levels. Amongst the therapeutics tested to actively counteract cellular cholesterol accumulation, the methylated β-cyclodextrin, KLEPTOSE® CRYSMEβ, has recently shown promising effects on decreasing the atherosclerotic plaque size in atherosclerotic mouse models. Therefore we investigated in vitro the RCT process occurring in SMCs and in arterial endothelial cells (ABAE) as well as the ability of some modified β-CDs with different methylation degree to modify RCT in these cells. To this aim, cells were incubated in the presence of different methylated β-CDs, including KLEPTOSE® CRYSMEβ. Both cell types were shown to express basal levels of ABCA1 and SR-BI whereas ABCG1 was solely found in ABAE. Upon CD treatments, the percentage of membrane-extracted cholesterol correlated to the methylation degree of the CDs independently of the lipid composition of the cell membranes. Decreasing the cellular cholesterol content with CDs led to reduce the expression levels of ABCA1 and ABCG1. In addition, the cholesterol efflux to ApoA-I and HDL particles was significantly decreased suggesting that cells forming the blood vessel wall are able to counteract the CD-induced loss of cholesterol. Taken together, our observations suggest that methylated

  13. Ion transport membrane module and vessel system

    DOEpatents

    Stein, VanEric Edward; Carolan, Michael Francis; Chen, Christopher M.; Armstrong, Phillip Andrew; Wahle, Harold W.; Ohrn, Theodore R.; Kneidel, Kurt E.; Rackers, Keith Gerard; Blake, James Erik; Nataraj, Shankar; van Doorn, Rene Hendrik Elias; Wilson, Merrill Anderson

    2008-02-26

    An ion transport membrane system comprising (a) a pressure vessel having an interior, an exterior, an inlet, and an outlet; (b) a plurality of planar ion transport membrane modules disposed in the interior of the pressure vessel and arranged in series, each membrane module comprising mixed metal oxide ceramic material and having an interior region and an exterior region, wherein any inlet and any outlet of the pressure vessel are in flow communication with exterior regions of the membrane modules; and (c) one or more gas manifolds in flow communication with interior regions of the membrane modules and with the exterior of the pressure vessel.The ion transport membrane system may be utilized in a gas separation device to recover oxygen from an oxygen-containing gas or as an oxidation reactor to oxidize compounds in a feed gas stream by oxygen permeated through the mixed metal oxide ceramic material of the membrane modules.

  14. Ion transport membrane module and vessel system

    DOEpatents

    Stein, VanEric Edward; Carolan, Michael Francis; Chen, Christopher M.; Armstrong, Phillip Andrew; Wahle, Harold W.; Ohrn, Theodore R.; Kneidel, Kurt E.; Rackers, Keith Gerard; Blake, James Erik; Nataraj, Shankar; van Doorn, Rene Hendrik Elias; Wilson, Merrill Anderson

    2007-02-20

    An ion transport membrane system comprising (a) a pressure vessel having an interior, an exterior, an inlet, and an outlet; (b) a plurality of planar ion transport membrane modules disposed in the interior of the pressure vessel and arranged in series, each membrane module comprising mixed metal oxide ceramic material and having an interior region and an exterior region, wherein any inlet and any outlet of the pressure vessel are in flow communication with exterior regions of the membrane modules; and (c) one or more gas manifolds in flow communication with interior regions of the membrane modules and with the exterior of the pressure vessel. The ion transport membrane system may be utilized in a gas separation device to recover oxygen from an oxygen-containing gas or as an oxidation reactor to oxidize compounds in a feed gas stream by oxygen permeated through the mixed metal oxide ceramic material of the membrane modules.

  15. Ion transport membrane module and vessel system

    DOEpatents

    Stein, VanEric Edward; Carolan, Michael Francis; Chen, Christopher M.; Armstrong, Phillip Andrew; Wahle, Harold W.; Ohrn, Theodore R.; Kneidel, Kurt E.; Rackers, Keith Gerard; Blake, James Erik; Nataraj, Shankar; Van Doorn, Rene Hendrik Elias; Wilson, Merrill Anderson

    2012-02-14

    An ion transport membrane system comprising (a) a pressure vessel having an interior, an exterior, an inlet, and an outlet; (b) a plurality of planar ion transport membrane modules disposed in the interior of the pressure vessel and arranged in series, each membrane module comprising mixed metal oxide ceramic material and having an interior region and an exterior region, wherein any inlet and any outlet of the pressure vessel are in flow communication with exterior regions of the membrane modules; and (c) one or more gas manifolds in flow communication with interior regions of the membrane modules and with the exterior of the pressure vessel. The ion transport membrane system may be utilized in a gas separation device to recover oxygen from an oxygen-containing gas or as an oxidation reactor to oxidize compounds in a feed gas stream by oxygen permeated through the mixed metal oxide ceramic material of the membrane modules.

  16. Placental Transfer of Maraviroc in an Ex Vivo Human Cotyledon Perfusion Model and Influence of ABC Transporter Expression

    PubMed Central

    Vinot, C.; Gavard, L.; Tréluyer, J. M.; Manceau, S.; Courbon, E.; Scherrmann, J. M.; Declèves, X.; Duro, D.; Peytavin, G.; Mandelbrot, L.

    2013-01-01

    Nowadays, antiretroviral therapy is recommended during pregnancy to prevent mother-to-child transmission of HIV. However, for many antiretroviral drugs, including maraviroc, a CCR5 antagonist, very little data exist regarding placental transfer. Besides, various factors may modulate this transfer, including efflux transporters belonging to the ATP-binding cassette (ABC) transporter superfamily. We investigated maraviroc placental transfer and the influence of ABC transporter expression on this transfer using the human cotyledon perfusion model. Term placentas were perfused ex vivo for 90 min with maraviroc (600 ng/ml) either in the maternal-to-fetal (n = 10 placentas) or fetal-to-maternal (n = 6 placentas) direction. Plasma concentrations were determined by ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Fetal transfer rates (FTR) and clearance indexes (CLI) were calculated as ratios of fetal to maternal concentrations at steady state (mean values between 30 and 90 min) and ratios of FTR of maraviroc to that of antipyrine, respectively. ABC transporter gene expression levels were determined by quantitative reverse transcription (RT)-PCR and ABCB1 protein expression by Western blotting. For the maternal-to-fetal direction, the mean FTR and CLI were 8.0% ± 3.0 and 0.26 ± 0.07, respectively, whereas the mean CLI was 0.52 ± 0.23 for the fetal-to-maternal direction. We showed a significant inverse correlation between maraviroc CLI and ABCC2, ABCC10, and ABCC11 placental gene expression levels (P < 0.05). To conclude, we report a low maraviroc placental transfer probably involving ABC efflux transporters and thus in all likelihood associated with a limited fetal exposition. Nevertheless, these results would need to be supported by in vivo data obtained from paired maternal and cord blood samples. PMID:23295922

  17. Fluid transport by active elastic membranes

    NASA Astrophysics Data System (ADS)

    Evans, Arthur A.; Lauga, Eric

    2011-09-01

    A flexible membrane deforming its shape in time can self-propel in a viscous fluid. Alternatively, if the membrane is anchored, its deformation will lead to fluid transport. Past work in this area focused on situations where the deformation kinematics of the membrane were prescribed. Here we consider models where the deformation of the membrane is not prescribed, but instead the membrane is internally forced. Both the time-varying membrane shape and the resulting fluid motion result then from a balance between prescribed internal active stresses, internal passive resistance, and external viscous stresses. We introduce two specific models for such active internal forcing: one where a distribution of active bending moments is prescribed, and one where active inclusions exert normal stresses on the membrane by pumping fluid through it. In each case, we asymptotically calculate the membrane shape and the fluid transport velocities for small forcing amplitudes, and recover our results using scaling analysis.

  18. The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter.

    PubMed Central

    Balan, I; Alarco, A M; Raymond, M

    1997-01-01

    We report the cloning and functional analysis of a third member of the CDR gene family in Candida albicans, named CDR3. This gene codes for an ABC (ATP-binding cassette) transporter of 1,501 amino acids highly homologous to Cdr1p and Cdr2p (56 and 55% amino acid sequence identity, respectively), two transporters involved in fluconazole resistance in C. albicans. The predicted structure of Cdr3p is typical of the PDR/CDR family, with two similar halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six predicted transmembrane segments. Northern analysis showed that CDR3 expression is regulated in a cell-type-specific manner, with low levels of CDR3 mRNA in CAI4 yeast and hyphal cells, high levels in WO-1 opaque cells, and undetectable levels in WO-1 white cells. Disruption of both alleles of CDR3 in CAI4 resulted in no obvious changes in cell morphology, growth rate, or susceptibility to fluconazole. Overexpression of Cdr3p in C. albicans did not result in increased cellular resistance to fluconazole, cycloheximide, and 4-nitroquinoline-N-oxide, which are known substrates for different transporters of the PDR/CDR family. These results indicate that despite a high degree of sequence conservation with C. albicans Cdr1p and Cdr2p, Cdr3p does not appear to be involved in drug resistance, at least to the compounds tested which include the clinically relevant antifungal agent fluconazole. Rather, the high level of Cdr3p expression in WO-1 opaque cells suggests an opaque-phase-associated biological function which remains to be identified. PMID:9393682

  19. The Staphylococcus aureus ABC-Type Manganese Transporter MntABC Is Critical for Reinitiation of Bacterial Replication Following Exposure to Phagocytic Oxidative Burst

    PubMed Central

    Coady, Alison; Xu, Min; Phung, Qui; Cheung, Tommy K.; Bakalarski, Corey; Alexander, Mary Kate; Lehar, Sophie M.; Kim, Janice; Park, Summer; Tan, Man-Wah; Nishiyama, Mireille

    2015-01-01

    Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for Staphylococcus aureus, a major human pathogen. Here, we show that a mntC mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the mntC mutant was pre-exposed to oxidative stress. Notably, the mntC mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the mntC mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the mntC mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for S. aureus viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for S. aureus pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host. PMID:26379037

  20. Absolute immunoquantification of the expression of ABC transporters P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 2 in human liver and duodenum.

    PubMed

    Tucker, Theodora G H A; Milne, Alison M; Fournel-Gigleux, Sylvie; Fenner, Katherine S; Coughtrie, Michael W H

    2012-01-15

    The ATP-binding cassette (ABC) transporters breast cancer resistance protein (BCRP), multidrug resistance-associated protein 2 (MRP2), and P-glycoprotein (Pgp) are important in the distribution and elimination of many drugs and endogenous metabolites. Due to their membrane location and hydrophobicity it is difficult to generate purified protein standards to quantify these transporters in human tissues. The present study generated transporter proteins fused with the S-peptide of ribonuclease for use as standards in immunoquantification in human liver and small intestine. Quantification of the S•tag™, a 15 amino acid peptide, is based on the formation of a functional ribonuclease activity upon its high affinity reconstitution with ribonuclease S-protein. S-tagged transporters were used as full-length protein standards in the immunoquantification of endogenous BCRP, MRP2, and Pgp levels in 14 duodenum and 13 liver human tissue samples. Expression levels in the duodenum were 305±248 (BCRP), 66±70 (MRP2), and 275±205 (Pgp) fmoles per cm(2). Hepatic levels were 2.6±0.9 (BCRP), 19.8±10.5 (MRP2), and 26.1±10.1 (total Pgp) pmoles per g of liver. The mean hepatic scaling factor was 35.8mg crude membrane per g of liver, and the mean duodenal scaling factor was 1.3mg crude membrane per cm(2) mucosal lining. Interindividual variability was greater in duodenal samples than liver samples. It is hoped that this innovative method of quantifying these transporters (and other membrane proteins) will improve in vivo-in vitro extrapolation and in silico prediction of drug absorption and elimination, thus supporting drug development. PMID:22062654

  1. Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5.

    PubMed

    Urzúa, Lucia Soto; Vázquez-Candanedo, Ada P; Sánchez-Espíndola, Adriana; Ramírez, Carlos Ávila; Baca, Beatriz E

    2013-06-01

    Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus. PMID:23624722

  2. Lysophosphatidylinositol: a novel link between ABC transporters and G-protein-coupled receptors.

    PubMed

    Ruban, Emily L; Ferro, Riccardo; Arifin, Syamsul Ahmad; Falasca, Marco

    2014-10-01

    Lysophosphatidylinositol (LPI) is a well-known bioactive lipid that is able to activate signalling cascades relevant to cell proliferation, migration, survival and tumorigenesis. Our previous work suggested that LPI is involved in cancer progression since it can be released in the medium of Ras-transformed fibroblasts and can function as an autocrine modulator of cell growth. Different research groups have established that LPI is the specific and functional ligand for G-protein-coupled receptor 55 (GPR55) and that this GPR55-LPI axis is able to activate signalling cascades that are relevant for different cell functions. Work in our laboratory has recently unravelled an autocrine loop, by which LPI synthesized by cytosolic phospholipase A₂ (cPLA₂) is pumped out of the cell by ATP-binding cassette (ABC) transporter C1 (ABCC1)/multidrug resistance protein 1 (MRP1), initiating a signalling cascade downstream of GPR55. Our current work suggests that blockade of this pathway may represent a novel strategy to inhibit cancer cell proliferation. PMID:25233417

  3. CD4+ T cell immunity to the Burkholderia pseudomallei ABC transporter LolC in melioidosis

    PubMed Central

    Chu, Karen K.; Tippayawat, Patcharaporn; Walker, Nicola J.; Harding, Sarah V.; Atkins, Helen S.; Maillere, Bernard; Bancroft, Gregory J.; Lertmemongkolchai, Ganjana; Altmann, Daniel M.

    2011-01-01

    Burkholderia pseudomallei (Bp) causes melioidosis, a disease with a wide range of possible outcomes, from seroconversion and dormancy to sepsis and death. This spectrum of host-pathogen interactions poses challenging questions about heterogeneity in immunity to Bp. Models show protection to be dependent on CD4+ cells and IFNγ, but little is known about specific target antigens. Having previously implicated the ABC transporter, LolC, in protective immunity, we here use epitope prediction, HLA binding studies, HLA-transgenic models and studies of T cells from seropositive individuals to characterize HLA-restricted LolC responses. Immunized mice showed long-lasting memory to the protein, while predictive algorithms identified epitopes within LolC that subsequently demonstrated strong HLA class II binding. Immunization of HLA-DR transgenics with LolC stimulated T cell responses to four of these epitopes. Furthermore, responsiveness of HLA-transgenics to LolC revealed a hierarchy supportive of HLA polymorphism-determined differential susceptibility. Seropositive human donors of diverse HLA class II types showed T cell responses to LolC epitopes which are conserved among Burkholderia species including B. cenocepacia, associated with life-threatening cepacia complex in cystic fibrosis patients and B. mallei, which causes glanders. These findings suggest a role for LolC epitopes in multiepitope vaccine design for melioidosis and related diseases. PMID:21182082

  4. Pharmacogenetics of human ABC transporter ABCC11: new insights into apocrine gland growth and metabolite secretion

    PubMed Central

    Ishikawa, Toshihisa; Toyoda, Yu; Yoshiura, Koh-ichiro; Niikawa, Norio

    2013-01-01

    Cell secretion is an important physiological process that ensures smooth metabolic activities and tissue repair as well as growth and immunological functions in the body. Apocrine secretion occurs when the secretory process is accomplished with a partial loss of cell cytoplasm. The secretory materials are contained within secretory vesicles and are released during secretion as cytoplasmic fragments into the glandular lumen or interstitial space. The recent finding that the non-synonymous single nucleotide polymorphisms (SNP) 538G > A (rs17822931; Gly180Arg) in the ABCC11 gene determines the type of earwax in humans has shed light on the novel function of this ABC (ATP-binding cassette) transporter in apocrine glands. The wild-type (Gly180) of ABCC11 is associated with wet-type earwax, axillary osmidrosis, and colostrum secretion from the mammary gland as well as the potential risk of mastopathy. Furthermore, the SNP (538G > A) in the ABCC11 gene is suggested to be a clinical biomarker for the prediction of chemotherapeutic efficacy. The aim of this review article is to provide an overview on the discovery and characterization of genetic polymorphisms in the human ABCC11 gene and to explain the impact of ABCC11 538G > A on the apocrine phenotype as well as the anthropological aspect of this SNP in the ABCC11 gene and patients’ response to nucleoside-based chemotherapy. PMID:23316210

  5. A two-component system regulates the expression of an ABC transporter for xylo-oligosaccharides in Geobacillus stearothermophilus.

    PubMed

    Shulami, Smadar; Zaide, Galia; Zolotnitsky, Gennady; Langut, Yael; Feld, Geoff; Sonenshein, Abraham L; Shoham, Yuval

    2007-02-01

    Geobacillus stearothermophilus T-6 utilizes an extensive and highly regulated hemicellulolytic system. The genes comprising the xylanolytic system are clustered in a 39.7-kb chromosomal segment. This segment contains a 6-kb transcriptional unit (xynDCEFG) coding for a potential two-component system (xynDC) and an ATP-binding cassette (ABC) transport system (xynEFG). The xynD promoter region contains a 16-bp inverted repeat resembling the operator site for the xylose repressor, XylR. XylR was found to bind specifically to this sequence, and binding was efficiently prevented in vitro in the presence of xylose. The ABC transport system was shown to comprise an operon of three genes (xynEFG) that is transcribed from its own promoter. The nonphosphorylated fused response regulator, His6-XynC, bound to a 220-bp fragment corresponding to the xynE operator. DNase I footprinting analysis showed four protected zones that cover the -53 and the +34 regions and revealed direct repeat sequences of a GAAA-like motif. In vitro transcriptional assays and quantitative reverse transcription-PCR demonstrated that xynE transcription is activated 140-fold in the presence of 1.5 microM XynC. The His6-tagged sugar-binding lipoprotein (XynE) of the ABC transporter interacted with different xylosaccharides, as demonstrated by isothermal titration calorimetry. The change in the heat capacity of binding (DeltaCp) for XynE with xylotriose suggests a stacking interaction in the binding site that can be provided by a single Trp residue and a sugar moiety. Taken together, our data show that XynEFG constitutes an ABC transport system for xylo-oligosaccharides and that its transcription is negatively regulated by XylR and activated by the response regulator XynC, which is part of a two-component sensing system. PMID:17142383

  6. Crystallization and preliminary X-ray crystallographic studies of an oligomeric species of a refolded C39 peptidase-like domain of the Escherichia coli ABC transporter haemolysin B

    PubMed Central

    Schwarz, Christian K. W.; Tschapek, Britta; Jumpertz, Thorsten; Jenewein, Stefan; Lecher, Justin; Willbold, Dieter; Panjikar, Santosh; Holland, I. Barry; Smits, Sander H. J.; Schmitt, Lutz

    2011-01-01

    The ABC transporter haemolysin B (HlyB) from Escherichia coli is part of a type I secretion system that translocates a 110 kDa toxin in one step across both membranes of this Gram-negative bacterium in an ATP-dependent manner. Sequence analysis indicates that HlyB contains a C39 peptidase-like domain at its N-terminus. C39 domains are thiol-dependent peptidases that cleave their substrates after a GG motif. Interestingly, the catalytically invariant cysteine is replaced by a tyrosine in the C39-like domain of HlyB. Here, the overexpression, purification and crystallization of the isolated C39-like domain are described as a first step towards obtaining structural insights into this domain and eventually answering the question concerning the function of a degenerated C39 domain in the ABC transporter HlyB. PMID:21543878

  7. A burst of ABC genes in the genome of the polyphagous spider mite Tetranychus urticae

    PubMed Central

    2013-01-01

    Background The ABC (ATP-binding cassette) gene superfamily is widespread across all living species. The majority of ABC genes encode ABC transporters, which are membrane-spanning proteins capable of transferring substrates across biological membranes by hydrolyzing ATP. Although ABC transporters have often been associated with resistance to drugs and toxic compounds, within the Arthropoda ABC gene families have only been characterized in detail in several insects and a crustacean. In this study, we report a genome-wide survey and expression analysis of the ABC gene superfamily in the spider mite, Tetranychus urticae, a chelicerate ~ 450 million years diverged from other Arthropod lineages. T. urticae is a major agricultural pest, and is among of the most polyphagous arthropod herbivores known. The species resists a staggering array of toxic plant secondary metabolites, and has developed resistance to all major classes of pesticides in use for its control. Results We identified 103 ABC genes in the T. urticae genome, the highest number discovered in a metazoan species to date. Within the T. urticae ABC gene set, all members of the eight currently described subfamilies (A to H) were detected. A phylogenetic analysis revealed that the high number of ABC genes in T. urticae is due primarily to lineage-specific expansions of ABC genes within the ABCC, ABCG and ABCH subfamilies. In particular, the ABCC subfamily harbors the highest number of T. urticae ABC genes (39). In a comparative genomic analysis, we found clear orthologous relationships between a subset of T. urticae ABC proteins and ABC proteins in both vertebrates and invertebrates known to be involved in fundamental cellular processes. These included members of the ABCB-half transporters, and the ABCD, ABCE and ABCF families. Furthermore, one-to-one orthologues could be distinguished between T. urticae proteins and human ABCC10, ABCG5 and ABCG8, the Drosophila melanogaster sulfonylurea receptor and ecdysone

  8. Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system.

    PubMed

    Długołecka, Anna; Cieśliński, Hubert; Turkiewicz, Marianna; Białkowska, Aneta M; Kur, Józef

    2008-12-01

    Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli. PMID:18700165

  9. 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

  10. 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

  11. Identification of ABC Transporter Interaction of a Novel Cyanoquinoline Radiotracer and Implications for Tumour Imaging by Positron Emission Tomography

    PubMed Central

    Slade, Rozanna L.; Pisaneschi, Federica; Nguyen, Quang-De; Smith, Graham; Carroll, Laurence; Beckley, Alice; Kaliszczak, Maciej A.; Aboagye, Eric O.

    2016-01-01

    Background The epidermal growth factor receptor (EGFR) is overexpressed in many cancers including lung, ovarian, breast, head and neck and brain. Mutation of this receptor has been shown to play a crucial role in the response of non-small cell lung carcinoma (NSCLC) to EGFR-targeted therapies. It is envisaged that imaging of EGFR using positron emission tomography (PET) could aid in selection of patients for treatment with novel inhibitors. We recognised multi-drug resistant phenotype as a threat to development of successful imaging agents. In this report, we describe discovery of a novel cyanoquinoline radiotracer that lacks ABC transporter activity. Methods Cellular retention of the prototype cyanoquinoline [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-({[1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl]methyl}amino)-but-2-enamide ([18F]FED6) and [18F](2E)-N-{4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxyquinolin-6-yl}-4-[({1-[(2R,5S)-3-fluoro-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H-1,2,3-triazol-4-yl}methyl)amino]but-2-enamide ([18F]FED20) were evaluated to establish potential for imaging specificity. The substrate specificity of a number of cyanoquinolines towards ABC transporters was investigated in cell lines proficient or deficient in ABCB1 or ABCG2. Results FED6 demonstrated substrate specificity for both ABCG2 and ABCB1, a property that was not observed for all cyanoquinolines tested, suggesting scope for designing novel probes. ABC transporter activity was confirmed by attenuating the activity of transporters with drug inhibitors or siRNA. We synthesized a more hydrophilic compound [18F]FED20 to overcome ABC transporter activity. FED20 lacked substrate specificity for both ABCB1 and ABCG2, and maintained a strong affinity for EGFR. Furthermore, FED20 showed higher inhibitory affinity for active mutant EGFR versus wild-type or resistant mutant EGFR; this property resulted in higher [18F]FED20 cellular retention in active

  12. Modulation of Biotransformation Systems and ABC Transporters by Benznidazole in Rats

    PubMed Central

    Perdomo, Virginia G.; Rigalli, Juan P.; Villanueva, Silvina S. M.; Ruiz, María L.; Luquita, Marcelo G.; Echenique, Claudia G.

    2013-01-01

    The effect of antichagasic benznidazole (BZL; 100 mg/kg body weight/day, 3 consecutive days, intraperitoneally) on biotransformation systems and ABC transporters was evaluated in rats. Expression of cytochrome P-450 (CYP3A), UDP-glucuronosyltransferase (UGT1A), glutathione S-transferases (alpha glutathione S-transferase [GST-α], GST-μ, and GST-π), multidrug-resistance-associated protein 2 (Mrp2), and P glycoprotein (P-gp) in liver, small intestine, and kidney was estimated by Western blotting. Increases in hepatic CYP3A (30%) and GST-μ (40%) and in intestinal GST-α (72% in jejunum and 136% in ileum) were detected. Significant increases in Mrp2 (300%) and P-gp (500%) proteins in liver from BZL-treated rats were observed without changes in kidney. P-gp and Mrp2 were also increased by BZL in jejunum (170% and 120%, respectively). In ileum, only P-gp was increased by BZL (50%). The activities of GST, P-gp, and Mrp2 correlated well with the upregulation of proteins in liver and jejunum. Plasma decay of a test dose of BZL (5 mg/kg body weight) administered intraduodenally was faster (295%) and the area under the concentration-time curve (AUC) was lower (41%) for BZL-pretreated rats than for controls. The biliary excretion of BZL was higher (60%) in the BZL group, and urinary excretion of BZL did not show differences between groups. The amount of absorbed BZL in intestinal sacs was lower (25%) in pretreated rats than in controls. In conclusion, induction of biotransformation enzymes and/or transporters by BZL could increase the clearance and/or decrease the intestinal absorption of coadministered drugs that are substrates of these systems, including BZL itself. PMID:23877690

  13. An ABCG/WBC-type ABC transporter is essential for transport of sporopollenin precursors for exine formation in developing pollen.

    PubMed

    Choi, Hyunju; Jin, Jun-Young; Choi, Setbyoul; Hwang, Jae-Ung; Kim, Yu-Young; Suh, Mi Chung; Lee, Youngsook

    2011-01-01

    The exine of the pollen wall shows an intricate pattern, primarily comprising sporopollenin, a polymer of fatty acids and phenolic compounds. A series of enzymes synthesize sporopollenin precursors in tapetal cells, and the precursors are transported from the tapetum to the pollen surface. However, the mechanisms underlying the transport of sporopollenin precursors remain elusive. Here, we provide evidence that strongly suggests that the Arabidopsis ABC transporter ABCG26/WBC27 is involved in the transport of sporopollenin precursors. Two independent mutations at ABCG26 coding region caused drastic decrease in seed production. This defect was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollen, observed initially as a defect in pollen-wall development. The reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It showed high co-expression with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. Similar to two other mutants with defects in pollen-wall deposition, abcg26 tapetal cells accumulated numerous vesicles and granules. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface. PMID:21223384

  14. Liners for ion transport membrane systems

    DOEpatents

    Carolan, Michael Francis; Miller, Christopher Francis

    2010-08-10

    Ion transport membrane system comprising (a) a pressure vessel comprising an interior, an exterior, an inlet, an inlet conduit, an outlet, and an outlet conduit; (b) a plurality of planar ion transport membrane modules disposed in the interior of the pressure vessel and arranged in series, each membrane module comprising mixed metal oxide ceramic material and having an interior region and an exterior region, wherein the inlet and the outlet of the pressure vessel are in flow communication with exterior regions of the membrane modules; (c) a gas manifold having an interior surface wherein the gas manifold is in flow communication with the interior region of each of the planar ion transport membrane modules and with the exterior of the pressure vessel; and (d) a liner disposed within any of the inlet conduit, the outlet conduit, and the interior surface of the gas manifold.

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

    PubMed

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

    2015-01-01

    The ATP-binding cassette (ABC) proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree). A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 'full-size', 21 'half-size' and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress) stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis. PMID:25615936

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

    PubMed Central

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

    2015-01-01

    The ATP-binding cassette (ABC) proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree). A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 ‘full-size’, 21 ‘half-size’ and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress) stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis. PMID:25615936

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

    PubMed Central

    2013-01-01

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

  18. Host ABC transporter proteins may influence the efficacy of ivermectin and possibly have broader implications for the development of resistance in parasitic nematodes.

    PubMed

    Dooley, L A; Froese, E A; Chung, Y T; Burkman, E J; Moorhead, A R; Ardelli, B F

    2015-10-01

    ABC transporter proteins function to extrude compounds from the cell. These proteins present an obstacle for treatment and for overcoming drug resistance as they are expressed by both host and parasite, and function similarly. The contribution of host ABC proteins to drug efficacy was examined using ivermectin and a Brugia malayi model system. Parallel in vitro and in vivo experiments were conducted using equal concentrations of ivermectin. The motilities and fecundity of B. malayi exposed to ivermectin in vitro were significantly lower than those treated in vivo. The higher motilities were correlated with low concentrations of ivermectin in worms extracted from treated hosts. The expression of ABC proteins was significantly higher in worms treated in vitro compared to those treated in vivo as well as in gerbils treated with ivermectin than in non-treated controls. The results suggest that host ABC transporters may influence the efficacy of ivermectin. PMID:26143231

  19. Solute transporters in plant thylakoid membranes

    PubMed Central

    Schoefs, Benoît

    2010-01-01

    Plants utilize sunlight to drive photosynthetic energy conversion in the chloroplast thylakoid membrane. Here are located four major photosynthetic complexes, about which we have great knowledge in terms of structure and function. However, much less we know about auxiliary proteins, such as transporters, ensuring an optimum function and turnover of these complexes. The most prominent thylakoid transporter is the proton-translocating ATP-synthase. Recently, four additional transporters have been identified in the thylakoid membrane of Arabidopsis thaliana, namely one copper-transporting P-ATPase, one chloride channel, one phosphate transporter, and one ATP/ADP carrier. Here, we review the current knowledge on the function and physiological role of these transporters during photosynthesis and light stress in plants. Subsequently, we make a survey on the outlook of thylakoid activities awaiting identification of responsible proteins. Such knowledge is necessary to understand the thylakoid network of transporters, and to design strategies for bioengineering crop plants in the future. PMID:20585503

  20. Molecular mechanism of {alpha}-tocopheryl-phosphate transport across the cell membrane

    SciTech Connect

    Negis, Yesim; Meydani, Mohsen; Zingg, Jean-Marc; Azzi, Angelo . E-mail: angelo.azzi@tufts.edu

    2007-07-27

    {alpha}-Tocopheryl-phosphate ({alpha}-TP) is synthesized and hydrolyzed in animal cells and tissues where it modulates several functions. {alpha}-TP is more potent than {alpha}-T in inhibiting cell proliferation, down-regulating CD36 transcription, inhibiting atherosclerotic plaque formation. Administration of {alpha}-TP to cells or animals requires its transfer through membranes, via a transporter. We show here that {alpha}-TP is passing the plasma membrane via a system that is inhibited by glibenclamide and probenecid, inhibitors of a number of transporters. Glibenclamide and probenecid prevent dose-dependently {alpha}-TP inhibition of cell proliferation. The two inhibitors act on ATP binding cassette (ABC) and organic anion transporters (OAT). Since ABC transporters function to export solutes and {alpha}-TP is transported into cells, it may be concluded that {alpha}-TP transport may occur via an OAT family member. Due to the protection by glibenclamide and probenecid on the {alpha}-TP induced cell growth inhibition it appears that {alpha}-TP acts after its uptake inside cells.

  1. 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. PMID:27033456

  2. NMR studies of cation transport across membranes

    SciTech Connect

    Shochet, N.R.

    1985-01-01

    /sup 23/Na NMR Studies of cation transport across membranes were conducted both on model and biological membranes. Two ionophores, the carrier monensin and the channel-former gramicidin, were chosen to induce cation transport in large unilamellar phosphatidylcholine vesicles. The distinction between the NMR signals arising from the two sides of the membrane was achieved by the addition of an anionic paramagnetic shift reagent to the outer solution. The kinetics of the cation transport across the membrane was observed simultaneously monitoring the changes in the /sup 23/Na NMR signals of both compartments. Two mathematical models were developed for the estimation of the transport parameters of the monensin- and gramicidin-induced cation transport. The models were able to fit the experimental data very well. A new method for the estimation of the volume trapped inside the vesicles was developed. The method uses the relative areas of the intra- and extravesicular NMR signals arising from a suspension of vesicles bathed in the same medium they contain, as a measure for the relative volumes of these compartments. Sodium transport across biological membranes was studied by /sup 23/ NMR, using suspensions of cultured nerve cells. The sodium influx through voltage-gated channels was studied using the channel modifier batrachotoxin in combination with scorpion toxin.

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

    PubMed

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

    1996-03-01

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

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

    PubMed Central

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

    1996-01-01

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

  5. Localized frameshift mutation generates selective, high-frequency phase variation of a surface lipoprotein encoded by a mycoplasma ABC transporter operon.

    PubMed Central

    Theiss, P; Wise, K S

    1997-01-01

    The wall-less mycoplasmas have revealed unusual microbial strategies for adaptive variation of antigenic membrane proteins exposed during their surface colonization of host cells. In particular, high-frequency mutations affecting the expression of selected surface lipoproteins have been increasingly documented for this group of organisms. A novel manifestation of mutational phase variation is shown here to occur in Mycoplasma fermentans, a chronic human infectious agent and possible AIDS-associated pathogen. A putative ABC type transport operon encoding four gene products is identified. The 3' distal gene encoding P78, a known surface-exposed antigen and the proposed substrate-binding lipoprotein of the transporter, is subject to localized hypermutation in a short homopolymeric tract of adenine residues located in the N-terminal coding region of the mature product. High-frequency, reversible insertion/deletion frameshift mutations lead to selective phase variation in P78 expression, whereas the putative nucleotide-binding protein, P63, encoded by the most 5' gene of the operon, is continually expressed. Mutation-based phase variation in specific surface-exposed microbial transporter components may provide an adaptive advantage for immune evasion, while continued expression of other elements of the same transporter may preserve essential metabolic functions and confer alternative substrate specificity. These features could be critical in mycoplasmas, where limitations in both transcriptional regulators and transport systems may prevail. This study also documents that P63 contains an uncharacteristic hydrophobic sequence between predicted nucleotide binding motifs and displays an amphiphilic character in detergent fractionation. Both features are consistent with an evolutionary adaptation favoring integral association of this putative energy-transducing component with the single mycoplasma membrane. PMID:9190819

  6. Tivantinib (ARQ 197) exhibits antitumor activity by directly interacting with tubulin and overcomes ABC transporter-mediated drug resistance.

    PubMed

    Aoyama, Aki; Katayama, Ryohei; Oh-Hara, Tomoko; Sato, Shigeo; Okuno, Yasushi; Fujita, Naoya

    2014-12-01

    Tivantinib (ARQ197) was first reported as a highly selective inhibitor of c-MET and is currently being investigated in a phase III clinical trial. However, as recently reported by us and another group, tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules, we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Consistent with our previous report, tivantinib reduced tubulin polymerization in cells and in mouse xenograft tumors in vivo. To determine if tivantinib directly binds to tubulin, we performed an in vitro competition assay. Tivantinib competitively inhibited colchicine but not vincristine or vinblastine binding to purified tubulin. These results imply that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin, we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fitted into the colchicine binding pocket of tubulin without steric hindrance. Furthermore, tivantinib showed similar IC50 values against parental and multidrug-resistant cells. In contrast, other microtubule-targeting drugs, such as vincristine, paclitaxel, and colchicine, could not suppress the growth of cells overexpressing ABC transporters. Moreover, the expression level of ABC transporters did not correlate with the apoptosis-inducing ability of tivantinib different from other microtubule inhibitor. These results suggest that tivantinib can overcome ABC transporter-mediated multidrug-resistant tumor cells and is potentially useful against various tumors. PMID:25313010

  7. 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

  8. 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. PMID:25517996

  9. Understanding transport in model water desalination membranes

    NASA Astrophysics Data System (ADS)

    Chan, Edwin

    Polyamide based thin film composites represent the the state-of-the-art nanofiltration and reverse osmosis membranes used in water desalination. The performance of these membranes is enabled by the ultrathin (~100 nm) crosslinked polyamide film in facilitating the selective transport of water over salt ions. While these materials have been refined over the last several decades, understanding the relationships between polyamide structure and membrane performance remains a challenge because of the complex and heterogeneous nature of the polyamide film. In this contribution, we present our approach to addressing this challenge by studying the transport properties of model polyamide membranes synthesized via molecular layer-by-layer (mLbL) assembly. First, we demonstrate that mLbL can successfully construct polyamide membranes with well-defined nanoscale thickness and roughness using a variety of monomer formulations. Next, we present measurement tools for characterizing the network structure and transport of these model polyamide membranes. Specifically, we used X-ray and neutron scattering techniques to characterize their structure as well as a recently-developed indentation based poromechanics approach to extrapolate their water diffusion coefficient. Finally, we illustrate how these measurements can provide insight into the original problem by linking the key polyamide network properties, i.e. water-polyamide interaction parameter and characteristic network mesh size, to the membrane performance.

  10. A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance.

    PubMed

    Walter, Stephanie; Kahla, Amal; Arunachalam, Chanemoughasoundharam; Perochon, Alexandre; Khan, Mojibur R; Scofield, Steven R; Doohan, Fiona M

    2015-05-01

    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

  11. Polyene antibiotic that inhibits membrane transport proteins

    PubMed Central

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-01-01

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains. PMID:22733749

  12. Polyene antibiotic that inhibits membrane transport proteins.

    PubMed

    te Welscher, Yvonne Maria; van Leeuwen, Martin Richard; de Kruijff, Ben; Dijksterhuis, Jan; Breukink, Eefjan

    2012-07-10

    The limited therapeutic arsenal and the increase in reports of fungal resistance to multiple antifungal agents have made fungal infections a major therapeutic challenge. The polyene antibiotics are the only group of antifungal antibiotics that directly target the plasma membrane via a specific interaction with the main fungal sterol, ergosterol, often resulting in membrane permeabilization. In contrast to other polyene antibiotics that form pores in the membrane, the mode of action of natamycin has remained obscure but is not related to membrane permeabilization. Here, we demonstrate that natamycin inhibits growth of yeasts and fungi via the immediate inhibition of amino acid and glucose transport across the plasma membrane. This is attributable to ergosterol-specific and reversible inhibition of membrane transport proteins. It is proposed that ergosterol-dependent inhibition of membrane proteins is a general mode of action of all the polyene antibiotics, of which some have been shown additionally to permeabilize the plasma membrane. Our results imply that sterol-protein interactions are fundamentally important for protein function even for those proteins that are not known to reside in sterol-rich domains. PMID:22733749

  13. The Membrane QmoABC Complex Interacts Directly with the Dissimilatory Adenosine 5′-Phosphosulfate Reductase in Sulfate Reducing Bacteria

    PubMed Central

    Ramos, Ana Raquel; Keller, Kimberly L.; Wall, Judy D.; Pereira, Inês A. Cardoso

    2012-01-01

    The adenosine 5′-phosphosulfate reductase (AprAB) is the enzyme responsible for the reduction of adenosine 5′-phosphosulfate (APS) to sulfite in the biological process of dissimilatory sulfate reduction, which is carried out by a ubiquitous group of sulfate reducing prokaryotes. The electron donor for AprAB has not been clearly identified, but was proposed to be the QmoABC membrane complex, since an aprBA–qmoABC gene cluster is found in many sulfate reducing and sulfur-oxidizing bacteria. The QmoABC complex is essential for sulfate reduction, but electron transfer between QmoABC and AprAB has not been reported. In this work we provide the first direct evidence that QmoABC and AprAB interact in Desulfovibrio spp., using co-immunoprecipitation, cross-linking Far-Western blot, tag-affinity purification, and surface plasmon resonance studies. This showed that the QmoABC–AprAB complex has a strong steady-state affinity (KD = 90 ± 3 nM), but has a transient character due to a fast dissociation rate. Far-Western blot identified QmoA as the Qmo subunit most involved in the interaction. Nevertheless, electron transfer from menaquinol analogs to APS through anaerobically purified QmoABC and AprAB could not be detected. We propose that this reaction requires the involvement of a third partner to allow electron flow driven by a reverse electron bifurcation process, i.e., electron confurcation. This process is deemed essential to allow coupling of APS reduction to chemiosmotic energy conservation. PMID:22536198

  14. Maltose and Maltodextrin Utilization by Listeria monocytogenes Depend on an Inducible ABC Transporter which Is Repressed by Glucose

    PubMed Central

    Gopal, Shubha; Berg, Daniela; Hagen, Nicole; Schriefer, Eva-Maria; Stoll, Regina; Goebel, Werner; Kreft, Jürgen

    2010-01-01

    Background In the environment as well as in the vertebrate intestine, Listeriae have access to complex carbohydrates like maltodextrins. Bacterial exploitation of such compounds requires specific uptake and utilization systems. Methodology/Principal Findings We could show that Listeria monocytogenes and other Listeria species contain genes/gene products with high homology to the maltodextrin ABC transporter and utilization system of B. subtilis. Mutant construction and growth tests revealed that the L. monocytogenes gene cluster was required for the efficient utilization of maltodextrins as well as maltose. The gene for the ATP binding protein of the transporter was located distant from the cluster. Transcription analyses demonstrated that the system was induced by maltose/maltodextrins and repressed by glucose. Its induction was dependent on a LacI type transcriptional regulator. Repression by glucose was independent of the catabolite control protein CcpA, but was relieved in a mutant defective for Hpr kinase/phosphorylase. Conclusions/Significance The data obtained show that in L. monocytogenes the uptake of maltodextrin and, in contrast to B. subtilis, also maltose is exclusively mediated by an ABC transporter. Furthermore, the results suggest that glucose repression of the uptake system possibly is by inducer exclusion, a mechanism not described so far in this organism. PMID:20436965

  15. The contribution of methionine to the stability of the Escherichia coli MetNIQ ABC transporter - substrate binding protein complex

    PubMed Central

    Nguyen, Phong T.; Li, Qi Wen; Kadaba, Neena S.; Lai, Jeffrey Y.; Yang, Janet G.; Rees, Douglas C.

    2015-01-01

    Despite the ubiquitous role of ATP Binding Cassette (ABC) importers in nutrient uptake, only the E. coli maltose and vitamin B12 ABC transporters have been structurally characterized in multiple conformations relevant to the alternating access transport mechanism. To complement our previous structure determination of the E. coli MetNI methionine importer in the inward facing conformation (Kadaba et al. (2008) Science 321, 250–253), we have explored conditions stabilizing the outward facing conformation. Using two variants, the Walker B E166Q mutation with ATP+EDTA to stabilize MetNI in the ATP-bound conformation and the N229A variant of the binding protein MetQ, shown in this work to disrupt methionine binding, a high affinity MetNIQ complex was formed with a dissociation constant measured to be 27 nM. Using wild type MetQ containing a co-purified methionine (for which the crystal structure is reported at 1.6 Å resolution), the dissociation constant for complex formation with MetNI is measured to be ~40-fold weaker, indicating that complex formation lowers the affinity of MetQ for methionine by this amount. Preparation of a stable MetNIQ complex is an essential step towards the crystallographic analysis of the outward facing conformation, a key intermediate in the uptake of methionine by this transport system. PMID:25803078

  16. 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. PMID:26235029

  17. Monitoring Transport Across Modified Nanoporous Alumina Membranes

    PubMed Central

    Penumetcha, Sai S.; Kona, Ravikanth; Hardin, Jonathan L.; Molder, Andrew L.; Steinle, Erich D.

    2007-01-01

    This paper describes the use of several characterization methods to examine alumina nanotubule membranes that have been modified with specific silanes. The function of these silanes is to alter the transport properties through the membrane by changing the local environment inside the alumina nanotube. The presence of alkyl groups, either long (C18) or short and branched (isopropyl) hydrocarbon chains, on these silanes significantly decreases the rate of transport of permeant molecules through membranes containing alumina nanotubes as monitored via absorbance spectroscopy. The presence of an ionic surfactant can alter the polarity of these modified nanotubes, which correlates to an increased transport of ions. Fluorescent spectroscopy is also utilized to enhance the sensitivity of detecting these permeant molecules. Confirmation of the alkylsilane attachment to the alumina membrane is achieved with traditional infrared spectroscopy, which can also examine the lifetime of the modified membrane. The physical parameters of these silane-modified porous alumina membranes are studied via scanning electron microscopy. The alumina nanotubes are not physically closed off or capped by the silanes that are attached to the alumina surfaces.

  18. Actinide transport across cell membranes.

    PubMed

    Bulman, R A; Griffin, R J

    1980-01-01

    Protactinium uptake into the normal liver does not exceed 3%, but when the phospholipid levels in the liver are elevated by administration of thioacetamide this uptake increases to 31%. Phosphatidic acid, which is absent from the normal liver, has been shown to extract protactinium into organic solvents. However, phosphatidylserine, a component of normal liver cell membranes, does not extract protactinium. It might be conjectured that this is why so little protactinium is taken up by the normal liver. The hypothesis is advanced that phosphatidylserine, which is known to complex plutonium, americium and curium, may regulate the uptake of these elements by liver. PMID:7373293

  19. The C-terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis

    PubMed Central

    AlKhatib, Zainab; Lagedroste, Marcel; Zaschke, Julia; Wagner, Manuel; Abts, André; Fey, Iris; Kleinschrodt, Diana; Smits, Sander H J

    2014-01-01

    The lantibiotic nisin is a small 3.4 kDa antimicrobial peptide, which acts against Gram-positive bacteria in the nmol/L range. Nisin is produced and secreted by several Lactococcus lactis strains to ensure advantages against other bacteria in their habitat. Nisin contains five specific lanthionine rings of which the first two are important for Lipid II binding and the last two are crucial for the pore formation in the membrane. To gain immunity against nisin, the producing strain is expressing an ABC transporter called NisFEG, which expels nisin from the membrane. As a result six to eightfold more nisin is needed to affect the cells. The hydrolysis of ATP by NisFEG is required for this immunity as shown by a mutant, where the ATP hydrolysis is disrupted (NisFH181AEG). Furthermore, NisFEG recognizes the C-terminus of nisin, since deletion of the last six amino acids as well as of the last ring lowered the fold of immunity displayed by NisFEG. PMID:25176038

  20. The C-terminus of nisin is important for the ABC transporter NisFEG to confer immunity in Lactococcus lactis.

    PubMed

    AlKhatib, Zainab; Lagedroste, Marcel; Zaschke, Julia; Wagner, Manuel; Abts, André; Fey, Iris; Kleinschrodt, Diana; Smits, Sander H J

    2014-10-01

    The lantibiotic nisin is a small 3.4 kDa antimicrobial peptide, which acts against Gram-positive bacteria in the nmol/L range. Nisin is produced and secreted by several Lactococcus lactis strains to ensure advantages against other bacteria in their habitat. Nisin contains five specific lanthionine rings of which the first two are important for Lipid II binding and the last two are crucial for the pore formation in the membrane. To gain immunity against nisin, the producing strain is expressing an ABC transporter called NisFEG, which expels nisin from the membrane. As a result six to eightfold more nisin is needed to affect the cells. The hydrolysis of ATP by NisFEG is required for this immunity as shown by a mutant, where the ATP hydrolysis is disrupted (NisFH181A EG). Furthermore, NisFEG recognizes the C-terminus of nisin, since deletion of the last six amino acids as well as of the last ring lowered the fold of immunity displayed by NisFEG. PMID:25176038

  1. The ABC transporter YejABEF is required for resistance to antimicrobial peptides and the virulence of Brucella melitensis

    PubMed Central

    Wang, Zhen; Bie, Pengfei; Cheng, Jie; Lu, Lin; Cui, Buyun; Wu, Qingmin

    2016-01-01

    The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens. PMID:27550726

  2. The ABC transporter YejABEF is required for resistance to antimicrobial peptides and the virulence of Brucella melitensis.

    PubMed

    Wang, Zhen; Bie, Pengfei; Cheng, Jie; Lu, Lin; Cui, Buyun; Wu, Qingmin

    2016-01-01

    The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens. PMID:27550726

  3. 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. PMID:27284059

  4. The non-ABC drug transporter RLIP76 (RALBP-1) plays a major role in the mechanisms of drug resistance.

    PubMed

    Awasthi, Yogesh C; Sharma, Rajendra; Yadav, Sushma; Dwivedi, Seema; Sharma, Abha; Awasthi, Sanjay

    2007-05-01

    RLIP76 or Ral binding protein (RalBP-1) was initially cloned as a Ral-effector that was proposed as a link between Ral and Ras pathways. This protein is encoded in humans on chromosome 18p11.3 by a gene with 11 exons and 9 introns and is found ubiquitously from drosophila to humans. RLIP76 displays inhibitory GTPase activity toward Rho/Rac class G-protein cdc42 which is involved in regulation of cytoskeletal organization, lamellipodia, cell migration and apoptosis via Ras. We have recently shown that RLIP76 is also a multispecific transporter of chemotherapeutic agents and glutathione conjugates (GS-E). In human cells RLIP76 accounts for more than two third of the transport activity for GS-E and drugs as opposed to the ABC-transporters including MRP1, which account for less than one third of this activity. Evidence is mounting that RLIP76 is a stress-responsive multi-specific, non-ABC transporter which represents an entirely novel link between stress-inducible G-protein signaling, receptor tyrosine-kinase signaling, endocytosis, heat-shock and stress defense pathways, and transport mediated drug-resistance. The expression of RLIP76 is significantly greater in human cancer cells of diverse origin as compared to the non-malignant cells. Inhibition of RLIP76, using antibodies towards a cell surface epitope, or depletion of RLIP76 using either siRNA or anti-sense phosphorothioate oligonucleotides preferentially causes apoptosis in malignant cells. Administration of RLIP76 antibodies, siRNA, or anti-sense oligonucleotides to mice bearing syngeneic B16 mouse melanoma tumors causes rapid and complete regression of tumors. Studies summarized in this review strongly suggest that RLIP76 is a logical target for clinical intervention of not only multi-drug resistance but also for diseases resulting from oxidative stress. PMID:17504221

  5. The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory, and improved activity.

    PubMed

    Livnat-Levanon, Nurit; I Gilson, Amy; Ben-Tal, Nir; Lewinson, Oded

    2016-01-01

    ABC transporters comprise a large and ubiquitous family of proteins. From bacteria to man they translocate solutes at the expense of ATP hydrolysis. Unlike other enzymes that use ATP as an energy source, ABC transporters are notorious for having high levels of basal ATPase activity: they hydrolyze ATP also in the absence of their substrate. It is unknown what are the effects of such prolonged and constant activity on the stability and function of ABC transporters or any other enzyme. Here we report that prolonged ATP hydrolysis is beneficial to the ABC transporter BtuC2D2. Using ATPase assays, surface plasmon resonance interaction experiments, and transport assays we observe that the constantly active transporter remains stable and functional for much longer than the idle one. Remarkably, during extended activity the transporter undergoes a slow conformational change (hysteresis) and gradually attains a hyperactive state in which it is more active than it was to begin with. This phenomenon is different from stabilization of enzymes by ligand binding: the hyperactive state is only reached through ATP hydrolysis, and not ATP binding. BtuC2D2 displays a strong conformational memory for this excited state, and takes hours to return to its basal state after catalysis terminates. PMID:26905293

  6. The uncoupled ATPase activity of the ABC transporter BtuC2D2 leads to a hysteretic conformational change, conformational memory, and improved activity

    PubMed Central

    Livnat-Levanon, Nurit; I. Gilson, Amy; Ben-Tal, Nir; Lewinson, Oded

    2016-01-01

    ABC transporters comprise a large and ubiquitous family of proteins. From bacteria to man they translocate solutes at the expense of ATP hydrolysis. Unlike other enzymes that use ATP as an energy source, ABC transporters are notorious for having high levels of basal ATPase activity: they hydrolyze ATP also in the absence of their substrate. It is unknown what are the effects of such prolonged and constant activity on the stability and function of ABC transporters or any other enzyme. Here we report that prolonged ATP hydrolysis is beneficial to the ABC transporter BtuC2D2. Using ATPase assays, surface plasmon resonance interaction experiments, and transport assays we observe that the constantly active transporter remains stable and functional for much longer than the idle one. Remarkably, during extended activity the transporter undergoes a slow conformational change (hysteresis) and gradually attains a hyperactive state in which it is more active than it was to begin with. This phenomenon is different from stabilization of enzymes by ligand binding: the hyperactive state is only reached through ATP hydrolysis, and not ATP binding. BtuC2D2 displays a strong conformational memory for this excited state, and takes hours to return to its basal state after catalysis terminates. PMID:26905293

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

    PubMed Central

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

    2009-01-01

    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. PMID:16554808

  9. 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. PMID:26517918

  10. The ABC-transporter AtmA is involved in nickel and cobalt resistance of Cupriavidus metallidurans strain CH34.

    PubMed

    Mikolay, André; Nies, Dietrich H

    2009-08-01

    Cupriavidus metallidurans CH34 genome contains an ortholog of Atm1p named AtmA (Rmet_0391, YP_582546). In Saccharomyces cerevisiae, the ABC-type transport system Atm1p is involved in export of iron-sulfur clusters from mitochondria into the cytoplasm for assembly of cytoplasmic iron-sulfur containing proteins. An atmA mutant of C. metallidurans was sensitive to nickel and cobalt but not iron cations. AtmA increased also resistance to these cations in Escherichia coli strains that carry deletions of the genes for other nickel and cobalt transport systems. In C. metallidurans, atmA expression was not significantly induced by nickel and cobalt, but repressed by zinc. AtmA was purified as a 70 kDa protein after expression in E. coli. ATPase activity of AtmA was stimulated by nickel and cobalt. PMID:19132541

  11. Hydrogen transport in composite inorganic membranes

    SciTech Connect

    Gabitto, Jorge; Tsouris, Costas

    2008-01-01

    A theoretical model simulating hydrogen transport through composite inorganic membranes is proposed. This model simulates operation of membranes made of three or more porous or metallic layers. Transport through Pd-alloy metallic layers is simulated using a comprehensive model proposed by Ward and Dao. The model accounts for external mass transfer, surface adsorption and desorption, transport to and from the bulk metal, and diffusion within the metal. Transport through porous ceramic layers is simulated following Burggraaf, who proposed an expression that combines viscous flow, Knudsen diffusion, and transition flow through porous media of complex geometrical structure. The model can also use experimentally determined permeance data when available. The theoretical model has been computationally implemented. Computations show very good agreement with experimental data available in the literature. The proposed model predicts hydrogen fluxes through composite membranes of several layers for standard operating conditions. The model can also predict which of the several layers used in manufacturing the membrane is controlling the total hydrogen flux. This information can be used to determine optimal thickness values for metallic and porous layers.

  12. Transport processes of the legume symbiosome membrane

    PubMed Central

    Clarke, Victoria C.; Loughlin, Patrick C.; Day, David A.; Smith, Penelope M. C.

    2014-01-01

    The symbiosome membrane (SM) is a physical barrier between the host plant and nitrogen-fixing bacteria in the legume:rhizobia symbiosis, and represents a regulated interface for the movement of solutes between the symbionts that is under plant control. The primary nutrient exchange across the SM is the transport of a carbon energy source from plant to bacteroid in exchange for fixed nitrogen. At a biochemical level two channels have been implicated in movement of fixed nitrogen across the SM and a uniporter that transports monovalent dicarboxylate ions has been characterized that would transport fixed carbon. The aquaporin NOD26 may provide a channel for ammonia, but the genes encoding the other transporters have not been identified. Transport of several other solutes, including calcium and potassium, have been demonstrated in isolated symbiosomes, and genes encoding transport systems for the movement of iron, nitrate, sulfate, and zinc in nodules have been identified. However, definitively matching transport activities with these genes has proved difficult and many further transport processes are expected on the SM to facilitate the movement of nutrients between the symbionts. Recently, work detailing the SM proteome in soybean has been completed, contributing significantly to the database of known SM proteins. This represents a valuable resource for the identification of transporter protein candidates, some of which may correspond to transport processes previously described, or to novel transport systems in the symbiosis. Putative transporters identified from the proteome include homologs of transporters of sulfate, calcium, peptides, and various metal ions. Here we review current knowledge of transport processes of the SM and discuss the requirements for additional transport routes of other nutrients exchanged in the symbiosis, with a focus on transport systems identified through the soybean SM proteome. PMID:25566274

  13. Advanced Hydrogen Transport Membrane for Coal Gasification

    SciTech Connect

    Schwartz, Joseph; Porter, Jason; Patki, Neil; Kelley, Madison; Stanislowski, Josh; Tolbert, Scott; Way, J. Douglas; Makuch, David

    2015-12-23

    A pilot-scale hydrogen transport membrane (HTM) separator was built that incorporated 98 membranes that were each 24 inches long. This separator used an advanced design to minimize the impact of concentration polarization and separated over 1000 scfh of hydrogen from a hydrogen-nitrogen feed of 5000 scfh that contained 30% hydrogen. This mixture was chosen because it was representative of the hydrogen concentration expected in coal gasification. When tested with an operating gasifier, the hydrogen concentration was lower and contaminants in the syngas adversely impacted membrane performance. All 98 membranes survived the test, but flux was lower than expected. Improved ceramic substrates were produced that have small surface pores to enable membrane production and large pores in the bulk of the substrate to allow high flux. Pd-Au was chosen as the membrane alloy because of its resistance to sulfur contamination and good flux. Processes were developed to produce a large quantity of long membranes for use in the demonstration test.

  14. Recent Advances in Synthetic Membrane Transporters

    PubMed Central

    McNALLY, BETH A.; LEEVY, W. MATTHEW; SMITH, BRADLEY D.

    2010-01-01

    It is 25 years since the first report of a synthetic ion channel transporter. Today, dozens of molecular and supramolecular designs have been developed to facilitate ion and small molecule transport across a bilayer membrane. Presented here is a concise summary of the advances made over the past four years. The transporters are grouped into three mechanistic classes: mobile carrier, monomeric channel, and self-assembled pore. Common building blocks are crown ethers, steroids, cyclodextrins, peptides, curcubiturils, and calixarenes. The eventual goal is to produce functional supramolecular devices such as sensors, enzyme assays, and lead candidates for pharmaceutical development. PMID:20376284

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

    PubMed

    Adewoye, L O; Worobec, E A

    2000-08-01

    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. PMID:10940570

  16. Inward facing conformations of the MetNI methionine ABC transporter: Implications for the mechanism of transinhibition

    PubMed Central

    Johnson, Eric; Nguyen, Phong T; Yeates, Todd O; Rees, Douglas C

    2012-01-01

    Two new crystal structures of the Escherichia coli high affinity methionine uptake ATP Binding Cassette (ABC) transporter MetNI, purified in the detergents cyclohexyl-pentyl-β-d-maltoside (CY5) and n-decyl-β-d-maltopyranoside (DM), have been solved in inward facing conformations to resolutions of 2.9 and 4.0 Å, respectively. Compared to the previously reported 3.7 Å resolution structure of MetNI purified in n-dodecyl-β-d-maltopyranoside (DDM), the higher resolution of the CY5 data enabled significant improvements to the structural model in several regions, including corrections to the sequence registry, and identification of ADP in the nucleotide binding site. CY5 crystals soaked with selenomethionine established details of the methionine binding site in the C2 regulatory domain of the ABC subunit, including the displacement of the side chain of MetN residue methionine 301 by the exogenous ligand. When compared to the CY5 or DDM structures, the DM structure exhibits a significant repositioning of the dimeric C2 domains, including an unexpected register shift in the intermolecular β-sheet hydrogen bonding between monomers, and a narrowing of the nucleotide binding space. The immediate proximity of the exogenous methionine binding site to the conformationally variable dimeric interface provides an indication of how methionine binding to the regulatory domains might mediate the phenomenon of transinhibition. PMID:22095702

  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

    2016-01-01

    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

  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. Lateral gene transfer of an ABC transporter complex between major constituents of the human gut microbiome

    PubMed Central

    2012-01-01

    Background Several links have been established between the human gut microbiome and conditions such as obesity and inflammatory bowel syndrome. This highlights the importance of understanding what properties of the gut microbiome can affect the health of the human host. Studies have been undertaken to determine the species composition of this microbiome and infer functional profiles associated with such host properties. However, lateral gene transfer (LGT) between community members may result in misleading taxonomic attributions for the recipient organisms, thus making species-function links difficult to establish. Results We identified a peptides/nickel transport complex whose components differed in abundance based upon levels of host obesity, and assigned the encoded proteins to members of the microbial community. Each protein was assigned to several distinct taxonomic groups, with moderate levels of agreement observed among different proteins in the complex. Phylogenetic trees of these proteins produced clusters that differed greatly from taxonomic attributions and indicated that habitat-directed LGT of this complex is likely to have occurred, though not always between the same partners. Conclusions These findings demonstrate that certain membrane transport systems may be an important factor within an obese-associated gut microbiome and that such complexes may be acquired several times by different strains of the same species. Additionally, an example of individual proteins from different organisms being transferred into one operon was observed, potentially demonstrating a functional complex despite the donors of the subunits being taxonomically disparate. Our results also highlight the potential impact of habitat-directed LGT on the resident microbiota. PMID:23116195

  20. Structural basis for the hydrolysis of ATP by a nucleotide binding subunit of an amino acid ABC transporter from Thermus thermophilus.

    PubMed

    Devi, Seenivasan Karthiga; Chichili, Vishnu Priyanka Reddy; Jeyakanthan, J; Velmurugan, D; Sivaraman, J

    2015-06-01

    ATP-binding cassette (ABC) transporters are a major family of small molecule transporter proteins, and their deregulation is associated with several diseases, including cancer. Here, we report the crystal structure of the nucleotide binding domain (NBD) of an amino acid ABC transporter from Thermus thermophilus (TTHA1159) in its apo form and as a complex with ADP along with functional studies. TTHA1159 is a putative arginine ABC transporter. The apo-TTHA1159 was crystallized in dimeric form, a hitherto unreported form of an apo NBD. Structural comparison of the apo and ADP-Mg(2+) complexes revealed that Phe14 of TTHA1159 undergoes a significant conformational change to accommodate ADP, and that the bound ADP interacts with the P-loop (Gly40-Thr45). Modeling of ATP-Mg(2+):TTHA1159 complex revealed that Gln86 and Glu164 are involved in water-mediated hydrogen bonding contacts and Asp163 in Mg(2+) ion-mediated hydrogen bonding contacts with the γ-phosphate of ATP, consistent with the findings of other ABC transporters. Mutational studies confirmed the necessity of each of these residues, and a comparison of the apo/ADP Mg(2+):TTHA1159 with its ATP-complex model suggests the likelihood of a key conformational change to the Gln86 side chain for ATP hydrolysis. PMID:25916755

  1. 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

  2. Mechanistic picture for conformational transition of a membrane transporter at atomic resolution.

    PubMed

    Moradi, Mahmoud; Tajkhorshid, Emad

    2013-11-19

    During their transport cycle, ATP-binding cassette (ABC) transporters undergo large-scale conformational changes between inward- and outward-facing states. Using an approach based on designing system-specific reaction coordinates and using nonequilibrium work relations, we have performed extensive all-atom molecular dynamics simulations in the presence of explicit membrane/solvent to sample a large number of mechanistically distinct pathways for the conformational transition of MsbA, a bacterial ABC exporter whose structure has been solved in multiple functional states. The computational approach developed here is based on (i) extensive exploration of system-specific biasing protocols (e.g., using collective variables designed based on available low-resolution crystal structures) and (ii) using nonequilibrium work relations for comparing the relevance of the transition pathways. The most relevant transition pathway identified using this approach involves several distinct stages reflecting the complex nature of the structural changes associated with the function of the protein. The opening of the cytoplasmic gate during the outward- to inward-facing transition of apo MsbA is found to be disfavored when the periplasmic gate is open and facilitated by a twisting motion of the nucleotide-binding domains that involves a dramatic change in their relative orientation. These results highlight the cooperativity between the transmembrane and the nucleotide-binding domains in the conformational transition of ABC exporters. The approach introduced here provides a framework to study large-scale conformational changes of other membrane transporters whose computational investigation at an atomic resolution may not be currently feasible using conventional methods. PMID:24191018

  3. A Putative ABC Transporter Permease Is Necessary for Resistance to Acidified Nitrite and EDTA in Pseudomonas aeruginosa under Aerobic and Anaerobic Planktonic and Biofilm Conditions

    PubMed Central

    McDaniel, Cameron; Su, Shengchang; Panmanee, Warunya; Lau, Gee W.; Browne, Tristan; Cox, Kevin; Paul, Andrew T.; Ko, Seung-Hyun B.; Mortensen, Joel E.; Lam, Joseph S.; Muruve, Daniel A.; Hassett, Daniel J.

    2016-01-01

    Pseudomonas aeruginosa (PA) is an important airway pathogen of cystic fibrosis and chronic obstructive disease patients. Multiply drug resistant PA is becoming increasing prevalent and new strategies are needed to combat such insidious organisms. We have previously shown that a mucoid, mucA22 mutant PA is exquisitely sensitive to acidified nitrite (A-NO2−, pH 6.5) at concentrations that are well tolerated in humans. Here, we used a transposon mutagenesis approach to identify PA mutants that are hypersensitive to A-NO2−. Among greater than 10,000 mutants screened, we focused on PA4455, in which the transposon was found to disrupt the production of a putative cytoplasmic membrane-spanning ABC transporter permease. The PA4455 mutant was not only highly sensitive to A-NO2−, but also the membrane perturbing agent, EDTA and the antibiotics doxycycline, tigecycline, colistin, and chloramphenicol, respectively. Treatment of bacteria with A-NO2− plus EDTA, however, had the most dramatic and synergistic effect, with virtually all bacteria killed by 10 mM A-NO2−, and EDTA (1 mM, aerobic, anaerobic). Most importantly, the PA4455 mutant was also sensitive to A-NO2− in biofilms. A-NO2− sensitivity and an anaerobic growth defect was also noted in two mutants (rmlC and wbpM) that are defective in B-band LPS synthesis, potentially indicating a membrane defect in the PA4455 mutant. Finally, this study describes a gene, PA4455, that when mutated, allows for dramatic sensitivity to the potential therapeutic agent, A-NO2− as well as EDTA. Furthermore, the synergy between the two compounds could offer future benefits against antibiotic resistant PA strains. PMID:27064218

  4. Molecular Transport Studies Through Unsupported Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Rock, William; Parekh, Sapun; Bonn, Mischa

    2014-03-01

    Dendrimers, spherical polymeric nanoparticles made from branched monomers around a central core, show great promise as drug delivery vehicles. Dendrimer size, core contents, and surface functionality can be synthetically tuned, providing unprecedented versatility. Polyamidoamine (PAMAM) dendrimers have been shown to enter cells; however, questions remain about their biophysical interactions with the cell membrane, specifically about the presence and size of transient pores. We monitor dendrimer-lipid bilayer interactions using unsupported black lipid membranes (BLMs) as model cell membranes. Custom bilayer slides contain two vertically stacked aqueous chambers separated by a 25 μm Teflon sheet with a 120 μm aperture where the bilayer is formed. We vary the composition of model membranes (cholesterol content and lipid phase) to create biomimetic systems and study the interaction of PAMAM G6 and G3 dendrimers with these bilayers. Dendrimers, dextran cargo, and bilayers are monitored and quantified using time-lapse fluorescence imaging. Electrical capacitance measurements are simultaneously recorded to determine if the membrane is porous, and the pore size is deduced by monitoring transport of fluorescent dextrans of increasing molecular weight. These experiments shed light on the importance of cholesterol content and lipid phase on the interaction of dendrimer nanoparticles with membranes.

  5. Differential Regulation of Iron- and Manganese-Specific MtsABC and Heme-Specific HtsABC Transporters by the Metalloregulator MtsR of Group A Streptococcus

    PubMed Central

    Hanks, Tracey S.; Liu, Mengyao; McClure, Michael J.; Fukumura, Maki; Duffy, Angela; Lei, Benfang

    2006-01-01

    The genome of the human pathogen group A Streptococcus (GAS) encodes the transporters MtsABC, FtsABCD, and HtsABC to take up ferric and manganese ions, ferric ferrichrome, and heme, respectively. The GAS genome also encodes two metalloregulators PerR and MtsR. To understand the regulation of the expression of these transporters, the mtsR and perR deletion mutants of a GAS serotype M1 strain were generated, and the effects of the deletions and Fe3+, Mn2+, and Zn2+ on the expression of mtsA, htsA, and ftsB were examined. Mn2+ dramatically depresses mtsA transcription and levels of the MtsA protein but does not downregulate the expression of htsA and ftsB. Fe3+ decreases the expression of mtsA and htsA but has no effect on ftsB expression. Zn2+ has no effect on the expression of all three genes. The deletion of mtsR abolishes the Mn2+- and Fe3+-induced depression of mtsA expression and the Fe3+-dependent decrease in htsA expression. The deletion of mtsR does not significantly alter GAS virulence in a mouse model of subcutaneous infection. The deletion of perR does not affect the expression of the genes in response to the metal ions. MtsR binds to the mts promoter region in the presence of Mn2+ or Fe2+. The results indicate that MtsR differentially regulates the expression of mtsABC and htsABC. PMID:16926405

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

    PubMed Central

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

    2016-01-01

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

  7. Cell differentiation and infectivity of Leishmania mexicana are inhibited in a strain resistant to an ABC-transporter blocker.

    PubMed

    Silva, N; Camacho, N; Figarella, K; Ponte-Sucre, A

    2004-06-01

    We analysed whether markers of cell differentiation and infectivity differed when compared to the parental sensitive strain [NR(Gs)] in an in vitro selected Leishmania strain [NR(Gr)] resistant to Glibenclamide, an ATP-binding-cassette (ABC)-transporter blocker. The data show that the cell body area was larger in NR(Gr) compared to NR(Gs) and that functional characters associated with an infective metacyclic phenotype, such as resistance to the lytic effect of the alternative complement pathway and expression of the Meta-1 protein, were reduced. The infectivity of NR(Gr) to J774.1 macrophages was also significantly reduced. These results suggest that resistance in Leishmania against Glibenclamide, a general blocker of P-glycoproteins, could produce functional modifications that may be relevant for Leishmania differentiation, infectivity and survival. PMID:15206465

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

    PubMed

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

    2010-09-01

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

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

    SciTech Connect

    Fuchs, Dominik; Daniel, Volker; Sadeghi, Mahmoud; Opelz, Gerhard; Naujokat, Cord

    2010-04-16

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

  10. The expression of superoxide dismutase (SOD) and a putative ABC transporter permease is inversely correlated during biofilm formation in Listeria monocytogenes 4b G

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC_transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant delta ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  12. Relative Rates of Amino Acid Import via the ABC Transporter GlnPQ Determine the Growth Performance of Lactococcus lactis

    PubMed Central

    Fulyani, Faizah; Schuurman-Wolters, Gea K.; Slotboom, Dirk-Jan

    2015-01-01

    ABSTRACT The GlnPQ transporter from Lactococcus lactis has the remarkable feature of having two substrate-binding domains (SBDs) fused to the N terminus of the transmembrane domain (TMD), and thus four SBDs are present in the homodimeric complex. Although X-ray structures and ligand binding data are available for both SBDs, little is known of how different amino acids compete with each other for transport via GlnPQ. Here we show GlnPQ has a broader substrate specificity than previously thought, with the ability to take up asparagine, glutamine, and glutamic acid, albeit via different routes and with different affinities. Asparagine and glutamine compete with each other at the level of binding to SBD1 and SBD2 (with differences in dissociation constant), but at the same time SBD1 and SBD2 compete with each other at the level of interaction with the translocator domain (with differences in affinity constant and rate of transport). Although glutamine transport via SBD1 is outcompeted by physiological concentrations of asparagine, SBD2 ensures high rates of import of the essential amino acid glutamine. Taken together, this study demonstrates that even in the presence of competing asparagine concentrations, GlnPQ has a high capacity to transport glutamine, which matches the high needs of the cell for glutamine and glutamate. IMPORTANCE GlnPQ is an ATP-binding cassette (ABC) transporter for glutamine, glutamic acid, and asparagine. The system is essential in various Gram-positive bacteria, including L. lactis and several pathogens. Here we show how the amino acids compete with each other for binding to the multiple SBDs of GlnPQ and how these SBDs compete with each other for substrate delivery to the transporter. Overall, our results show that GlnPQ has evolved to transport diverse substrates via different paths and to optimally acquire the abundant and essential amino acid glutamine. PMID:26553850

  13. Membrane Transport in Yeast, An Introduction.

    PubMed

    Kschischo, Maik; Ramos, José; Sychrová, Hana

    2016-01-01

    Research on membrane transport has made continuous progress in the last decades and remains an active field of scientific investigation. In the case of yeast, most of the research has been conducted for the model organism Saccharomyces cerevisiae, but also the so-called non-conventional yeasts are being studied, especially because of their peculiarities and, in some cases, specific transport systems. This book is based on the experience of several experts summarizing the current knowledge about important substrate transport processes in yeast. Each chapter provides both a general overview of the main transport characteristics of a specific substrate or group of substrates and the unique details that only an expert working in the field is able to transmit to the reader. PMID:26721268

  14. Mutational Analysis of Cvab, an ABC Transporter Involved in the Secretion of Active Colicin V

    PubMed Central

    Tai, Phang C.

    2012-01-01

    CvaB is the central membrane transporter of the colicin V secretion system that belongs to an ATP-binding cassette superfamily. Previous data showed that the N-terminal and C-terminal domains of CvaB are essential for the function of CvaB. N-terminal domain of CvaB possesses Ca2+-dependent cysteine proteolytic activity, and two critical residues, Cys32 and His105, have been identified. In this study, we also identify Asp121 as being the third residue of the putative catalytic triad within the active site of the enzyme. The Asp121 mutants lose both their colicin V secretion activity and N-terminal proteolytic activity. The adjacent residue Pro122 also appears to play a critical role in the colicin V secretion. However, the reversal of the two residues D121P - P122D results in loss of activity. Based on molecular modeling and protein sequence alignment, several residues adjacent to the critical residues, Cys32 and His105, were also examined and characterized. Site-directed mutagenesis of Trp101, Asp102, Val108, Leu76, Gly77, and Gln26 indicate that the neighboring residues around the catalytic triad affect colicin V secretion. Several mutated CvaB proteins with defective secretion were also tested, including Asp121 and Pro122, and were found to be structurally stable. These results indicate that the residues surrounding the identified catalytic triad are functionally involved in the secretion of biologically active colicin V. PMID:22539970

  15. ABC transporter PEN3/PDR8/ABCG36 interacts with calmodulin that, like PEN3, is required for Arabidopsis nonhost resistance.

    PubMed

    Campe, Ruth; Langenbach, Caspar; Leissing, Franz; Popescu, George V; Popescu, Sorina C; Goellner, Katharina; Beckers, Gerold J M; Conrath, Uwe

    2016-01-01

    Nonhost resistance (NHR) is the most prevalent form of plant immunity. In Arabidopsis, NHR requires membrane-localized ATP-binding cassette (ABC) transporter PENETRATION (PEN) 3. Upon perception of pathogen-associated molecular patterns, PEN3 becomes phosphorylated, suggestive of PEN3 regulation by post-translational modification. Here, we investigated the PEN3 protein interaction network. We probed the Arabidopsis protein microarray AtPMA-5000 with the N-terminal cytoplasmic domain of PEN3. Several of the proteins identified to interact with PEN3 in vitro represent cellular Ca(2+) sensors, including calmodulin (CaM) 3, CaM7 and several CaM-like proteins, pointing to the importance of Ca(2+) sensing to PEN3-mediated NHR. We demonstrated co-localization of PEN3 and CaM7, and we confirmed PEN3-CaM interaction in vitro and in vivo by PEN3 pull-down with CaM Sepharose, CaM overlay assay and bimolecular fluorescence complementation. We also show that just like in pen3, NHR to the nonadapted fungal pathogens Phakopsora pachyrhizi and Blumeria graminis f.sp. hordei is compromised in the Arabidopsis cam7 and pen3 cam7 mutants. Our study discloses CaM7 as a PEN3-interacting protein crucial to Arabidopsis NHR and emphasizes the importance of Ca(2+) sensing to plant immunity. PMID:26315018

  16. The ABC of ABCS: a phylogenetic and functional classification of ABC systems in living organisms.

    PubMed

    Dassa, E; Bouige, P

    2001-01-01

    ATP binding cassette (ABC) systems constitute one of the most abundant superfamilies of proteins. They are involved not only in the transport of a wide variety of substances, but also in many cellular processes and in their regulation. In this paper, we made a comparative analysis of the properties of ABC systems and we provide a phylogenetic and functional classification. This analysis will be helpful to accurately annotate ABC systems discovered during the sequencing of the genome of living organisms and to identify the partners of the ABC ATPases. PMID:11421270

  17. The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

    PubMed Central

    Kemner, J M; Liang, X; Nester, E W

    1997-01-01

    The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose. PMID:9079938

  18. Morphology and transport in ionic membranes

    NASA Astrophysics Data System (ADS)

    Disabb-Miller, Melanie Lisa

    Ion-containing polymers for fuel cell membranes have been studied to determine the chemical structure and ion content relationship to membrane water uptake, conductivity, and morphology. Random and block copolymer proton exchange membranes (PEMs) and anion exchange membranes (AEMs) with unique properties, such as diblock and triblock copolymers, superacidic moieties, and charge-delocalized polymer-tethered Ru-complex based cations, were investigated, and new metrics were developed to analyze fundamental ion transport behavior in these polymers. The morphology of the polymer systems was examined using small angle x-ray scattering (SAXS), small angle neutron scattering (SANS), and transmission electron microscopy (TEM). By studying a number of different ion-conducting systems using multiple techniques and deep analysis of structure-property relationships, a more complete picture of the property landscape of these materials was developed. Model diblock and unique triblock copolymer systems with center-functionalized blocks based on poly(styrene), PS, and poly(hexyl methacrylate), PHMA, were synthesized via atom transfer radical polymerization (ATRP). The PS block was functionalized for backbone-independent comparisons of PEM and AEM water uptake and conductivity to provide insight in how the properties of PEMs and AEMs compare and aid in further AEM development. The ratio of the mobile ion diffusion coefficients and dilute solution ion diffusivity (D/D0) was developed as a new metric, allowing for accurate comparison of polymer systems with different ion moieties and contents. Subsequently, it was determined that block copolymer PEMs and AEMs demonstrate the same barriers to ion transport if the mobility of the charge carrier is considered.

  19. Caught in the act: ATP hydrolysis of an ABC-multidrug transporter followed by real-time magic angle spinning NMR.

    PubMed

    Hellmich, Ute A; Haase, Winfried; Velamakanni, Saroj; van Veen, Hendrik W; Glaubitz, Clemens

    2008-10-15

    The ATP binding cassette (ABC) transporter LmrA from Lactococcus lactis transports cytotoxic molecules at the expense of ATP. Molecular and kinetic details of LmrA can be assessed by solid-state nuclear magnetic resonance (ssNMR), if functional reconstitution at a high protein-lipid ratio can be achieved and the kinetic rate constants are small enough. In order to follow ATP hydrolysis directly by 31P-magic angle spinning (MAS) nuclear magnetic resonance (NMR), we generated such conditions by reconstituting LmrA-dK388, a mutant with slower ATP turnover rate, at a protein-lipid ration of 1:150. By analysing time-resolved 31P spectra, protein activity has been directly assessed. These data demonstrate the general possibility to perform ssNMR studies on a fully active full length ABC transporter and also form the foundation for further kinetic studies on LmrA by NMR. PMID:18817774

  20. Proton transport via the membrane surface.

    PubMed Central

    Georgievskii, Yuri; Medvedev, Emile S; Stuchebrukhov, Alexei A

    2002-01-01

    Some proton pumps, such as cytochrome c oxidase (C(c)O), translocate protons across biological membranes at a rate that considerably exceeds the rate of proton transport to the entrance of the proton-conducting channel via bulk diffusion. This effect is usually ascribed to a proton-collecting antenna surrounding the channel entrance. In this paper, we consider a realistic phenomenological model of such an antenna. In our model, a homogeneous membrane surface, which can mediate proton diffusion toward the channel entrance, is populated with protolytic groups that are in dynamic equilibrium with the solution. Equations that describe coupled surface-bulk proton diffusion are derived and analyzed. A general expression for the rate constant of proton transport via such a coupled surface-bulk diffusion mechanism is obtained. A rigorous criterion is formulated of when proton diffusion along the surface enhances the transport. The enhancement factor is found to depend on the ratio of the surface and bulk diffusional constants, pK(a) values of surface protolytic groups, and their concentration. A capture radius for a proton on the surface and an effective size of the antenna are found. The theory also predicts the effective distance that a proton can migrate on the membrane surface between a source (such as CcO) and a sink (such as ATP synthase) without fully equilibrating with the bulk. In pure aqueous solutions, protons can travel over long distances (microns). In buffered solutions, the travel distance is much shorter (nanometers); still the enhancement effect of the surface diffusion on the proton flow to a target on the surface can be tens to hundreds at physiological buffer concentrations. These results are discussed in a general context of chemiosmotic theory. PMID:12023208

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

    PubMed

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

    2016-04-29

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

  2. A two-gene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore.

    PubMed

    Cheng, J; Guffanti, A A; Krulwich, T A

    1997-03-01

    A transposition mutant of Bacillus subtilis (designated JC901) that was isolated on the basis of growth inhibition by Na+ at elevated pH, was deficient in energy-dependent Na+ extrusion. The capacity of the mutant JC901 for Na(+)-dependent pH homeostasis was unaffected relative to the wild-type strain, as assessed by regulation of cytoplasmic pH after an alkaline shift. The site of transposition was near the 3'-terminal end of a gene, natB, predicted to encode a membrane protein, NatB. NatB possesses six putative membrane-spanning regions at its C-terminus, and exhibits modest sequence similarity to regions of eukaryotic Na+/H+ exchangers. Sequence and Northern blot analyses suggested that natB forms an operon with an upstream gene, natA. The predicted product of natA is a member of the family of ATP-binding proteins that are components of transport systems of the ATP-binding cassette (ABC) or traffic ATPase type. Expression of the lacZ gene that was under control of the promoter for natB indicated that expression of the operon was induced by ethanol and the protonophore carbonylcyanide p-chlorophenylhydrazone (CCCP), and more modestly, by Na+, and K+, but not by choline or a high concentration of sucrose. Restoration of the natAB genes, cloned in a recombinant plasmid (pJY1), complemented the Na(+)-sensitive phenotype of the mutant JC901 at elevated pH and significantly increased the resistance of the mutant to growth inhibition by ethanol and CCCP at pH 7; ethanol was not excluded, however, from the cells expressing natAB, so ethanol-resistance does not result from NatAB-dependent ethanol efflux. Transformation of the mutant with pJY1 did markedly enhance the capacity for Na+ efflux, which was further stimulated by CCCP. In the absence of CCCP, NatAB-mediated Na+ efflux was stimulated by K+. Concomitant NatAB-dependent K+ uptake occurred, as monitored by 86Rb+ uptake; this uptake was inhibited by CCCP and is thus secondary to the primary, electrogenic Na+ efflux

  3. 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. PMID:27257556

  4. 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

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

    PubMed Central

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

    2016-01-01

    Exposure to high sugar diet (HSD) is an experimental model of insulin resistance (IR) and type 2 diabetes (T2D) in mammals and insects. In Drosophila, HSD-induced IR delays emergence of pupae from larvae and eclosion of imago from pupae. Understanding of mechanisms of IR/T2D is essential for refining T2D prevention and treatment strategies. Dysregulation of tryptophan (Trp)-kynurenine (Kyn) pathway was suggested as one of the mechanisms of IR/T2D development. Rate-limiting enzyme of Trp-Kyn pathway in Drosophila is Trp 2,3-dioxygenase (TDO), an evolutionary conserved ortholog of human TDO. We previously reported attenuation of HSD-induced IR in vermilion mutants with inactive TDO. Conversion of Trp to Kyn is regulated not only by TDO activity but by intracellular Trp transport via ATP-binding cassette (ABC) transporter encoded by white gene in Drosophila. In order to evaluate the possible impact of deficient intracellular Trp transport on the inducement of IR by HSD, we compared the effect of HSD on pre-imago development in wild type flies, Canton-Special (C-S), and C-S flies containing white gene, white (C-S). Presence of white gene attenuated (by 50%) HSD-induced delay of pupae emergence from larvae and female and male imago eclosion from pupae. Present study together with our earlier report reveals that both decreased TDO activity (due to vermilion gene mutation) or deficient Trp transport into cell without affecting TDO levels (due to white gene mutation) attenuate HSD-induced development of IR in Drosophila model of T2D. Our data provide further support for hypothesis that dysregulation of Trp-Kyn pathway is one of the pathophysiological mechanisms and potential target for early diagnosis, prevention and treatment of IR/T2D. PMID:27375855

  6. Shared Molecular Mechanisms of Membrane Transporters.

    PubMed

    Drew, David; Boudker, Olga

    2016-06-01

    The determination of the crystal structures of small-molecule transporters has shed light on the conformational changes that take place during structural isomerization from outward- to inward-facing states. Rather than using a simple rocking movement of two bundles around a central substrate-binding site, it has become clear that even the most simplistic transporters utilize rearrangements of nonrigid bodies. In the most dramatic cases, one bundle is fixed while the other, structurally divergent, bundle carries the substrate some 18 Å across the membrane, which in this review is termed an elevator alternating-access mechanism. Here, we compare and contrast rocker-switch, rocking-bundle, and elevator alternating-access mechanisms to highlight shared features and novel refinements to the basic alternating-access model. PMID:27023848

  7. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein.

    PubMed

    Tay, Wee Tek; Mahon, Rod J; Heckel, David G; Walsh, Thomas K; Downes, Sharon; James, William J; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K; Gordon, Karl H J

    2015-11-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  8. Insect Resistance to Bacillus thuringiensis Toxin Cry2Ab Is Conferred by Mutations in an ABC Transporter Subfamily A Protein

    PubMed Central

    Tay, Wee Tek; Mahon, Rod J.; Heckel, David G.; Walsh, Thomas K.; Downes, Sharon; James, William J.; Lee, Sui-Fai; Reineke, Annette; Williams, Adam K.; Gordon, Karl H. J.

    2015-01-01

    The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode

  9. Osmotic water transport through carbon nanotube membranes

    PubMed Central

    Kalra, Amrit; Garde, Shekhar; Hummer, Gerhard

    2003-01-01

    We use molecular dynamics simulations to study osmotically driven transport of water molecules through hexagonally packed carbon nanotube membranes. Our simulation setup comprises two such semipermeable membranes separating compartments of pure water and salt solution. The osmotic force drives water flow from the pure-water to the salt-solution compartment. Monitoring the flow at molecular resolution reveals several distinct features of nanoscale flows. In particular, thermal fluctuations become significant at the nanoscopic length scales, and as a result, the flow is stochastic in nature. Further, the flow appears frictionless and is limited primarily by the barriers at the entry and exit of the nanotube pore. The observed flow rates are high (5.8 water molecules per nanosecond and nanotube), comparable to those through the transmembrane protein aquaporin-1, and are practically independent of the length of the nanotube, in contrast to predictions of macroscopic hydrodynamics. All of these distinct characteristics of nanoscopic water flow can be modeled quantitatively by a 1D continuous-time random walk. At long times, the pure-water compartment is drained, and the net flow of water is interrupted by the formation of structured solvation layers of water sandwiched between two nanotube membranes. Structural and thermodynamic aspects of confined water monolayers are studied. PMID:12878724

  10. The N-Terminal Extension Domain of the C. elegans Half-Molecule ABC Transporter, HMT-1, Is Required for Protein-Protein Interactions and Function

    PubMed Central

    Kim, Sungjin; Selote, Devarshi S.; Vatamaniuk, Olena K.

    2010-01-01

    Background Members of the HMT-1 (heavy metal tolerance factor 1) subfamily of the ATP-binding cassette (ABC) transporter superfamily detoxify heavy metals and have unique topology: they are half-molecule ABC transporters that, in addition to a single transmembrane domain (TMD1) and a single nucleotide-binding domain (NBD1), possess a hydrophobic NH2-terminal extension (NTE). These structural features distinguish HMTs from other ABC transporters in different species including Drosophila and humans. Functional ABC transporters, however, are comprised of at least four-domains (two TMDs and two NDBs) formed from either a single polypeptide or by the association of two or four separate subunits. Whether HMTs act as oligomers and what role the NTE domain plays in their function have not been determined. Methodology/Principal Findings In this study, we examined the oligomeric status of Caenorhabditis elegans HMT-1 and the functional significance of its NTE using gel-filtration chromatography in combination with the mating-based split-ubiquitin yeast two-hybrid system (mbSUS) and functional in vivo assays. We found that HMT-1 exists in a protein complex in C. elegans. Studies in S. cerevisiae showed that HMT-1 at a minimum homodimerizes and that oligomerization is essential for HMT-1 to confer cadmium tolerance. We also established that the NTE domain plays an important structural and functional role: it is essential for HMT-1 oligomerization and Cd-detoxification function. However, the NTE itself was not sufficient for oligomerization suggesting that multiple structural features of HMT-1 must associate to form a functional transporter. Conclusions The prominence of heavy metals as environmental toxins and the remarkable conservation of HMT-1 structural architecture and function in different species reinforce the value of continued studies of HMT-1 in model systems for identifying functional domains in HMT1 of humans. PMID:20886084

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

    PubMed Central

    Paul, Sanjoy; Diekema, Daniel

    2013-01-01

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

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

    SciTech Connect

    Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C.; Pinkett, Heather W.

    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. The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus.

  13. Classification of a Haemophilus influenzae ABC transporter HI1470/71 through its cognate molybdate periplasmic binding protein, MolA.

    PubMed

    Tirado-Lee, Leidamarie; Lee, Allen; Rees, Douglas C; Pinkett, Heather W

    2011-11-01

    molA (HI1472) from H. influenzae encodes a periplasmic binding protein (PBP) that delivers substrate to the ABC transporter MolB(2)C(2) (formerly HI1470/71). The structures of MolA with molybdate and tungstate in the binding pocket were solved to 1.6 and 1.7 Å 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 ∼100 μM 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. The presence of two molybdate loci in H. influenzae suggests multiple transport systems for one substrate, with molABC constituting a low-affinity molybdate locus. PMID:22078568

  14. Mapping of the rod photoreceptor ABC transporter (ABCR) to 1p21-p22.1 and identification of novel mutations in Stargardt's disease.

    PubMed

    Nasonkin, I; Illing, M; Koehler, M R; Schmid, M; Molday, R S; Weber, B H

    1998-01-01

    Using a bovine rod photoreceptor cell-specific ATP-binding cassette (ABC) transporter cDNA we have cloned the full-length transcript of the homologous human gene and demonstrate that it is identical to the photoreceptor cell-specific ABC transporter (ABCR) recently shown to be mutated in Stargardt's disease. By fluorescence in situ hybridization we have mapped the ABCR gene to chromosomal band 1p21-p22.1. Mutational analysis of part of the gene in 15 Stargardt's disease patients has identified four disease-causing mutations, of which two represent potential null alleles. This brings the total number of independently identified mutations to 23, providing further evidence that the human ABCR gene is associated with Stargardt's disease. PMID:9490294

  15. Development, Maintenance, and Reversal of Multiple Drug Resistance: At the Crossroads of TFPI1, ABC Transporters, and HIF1α

    PubMed Central

    Arnason, Terra; Harkness, Troy

    2015-01-01

    Early detection and improved therapies for many cancers are enhancing survival rates. Although many cytotoxic therapies are approved for aggressive or metastatic cancer; response rates are low and acquisition of de novo resistance is virtually universal. For decades; chemotherapeutic treatments for cancer have included anthracyclines such as Doxorubicin (DOX); and its use in aggressive tumors appears to remain a viable option; but drug resistance arises against DOX; as for all other classes of compounds. Our recent work suggests the anticoagulant protein Tissue Factor Pathway Inhibitor 1α (TFPI1α) plays a role in driving the development of multiple drug resistance (MDR); but not maintenance; of the MDR state. Other factors; such as the ABC transporter drug efflux pumps MDR-1/P-gp (ABCB1) and BCRP (ABCG2); are required for MDR maintenance; as well as development. The patient population struggling with therapeutic resistance specifically requires novel treatment options to resensitize these tumor cells to therapy. In this review we discuss the development, maintenance, and reversal of MDR as three distinct phases of cancer biology. Possible means to exploit these stages to reverse MDR will be explored. Early molecular detection of MDR cancers before clinical failure has the potential to offer new approaches to fighting MDR cancer. PMID:26501324

  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. 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

    2015-01-01

    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

  18. ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity.

    PubMed

    Randak, Christoph O; Welsh, Michael J

    2005-02-01

    ADP interacts with the nucleotide-binding domains (NBDs) of the cystic fibrosis transmembrane conductance regulator (CFTR) to inhibit its Cl- channel activity. Because CFTR NBD2 has reversible adenylate kinase activity (ATP + AMP<==> ADP + ADP) that gates the channel, we asked whether ADP might inhibit current through this enzymatic activity. In adenylate kinases, binding of the two ADP molecules is cooperative. Consistent with this hypothesis, CFTR current inhibition showed positive cooperativity for ADP. We also found that ADP inhibition of current was attenuated when we prevented adenylate kinase activity with P1,P5-di(adenosine-5') pentaphosphate. Additional studies suggested that adenylate kinase-dependent inhibition involved phosphotransfer between two nucleotide diphosphates. These data indicate that the adenylate kinase reaction at NBD2 contributed to the inhibitory effect of ADP. Finding that ADP inhibits function via an adenylate kinase activity also helps explain the earlier observation that mutations that disrupt adenylate kinase activity also disrupt ADP inhibition. Thus, the results reveal a previously unrecognized mechanism by which ADP inhibits an ABC transporter. PMID:15684079

  19. ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity

    PubMed Central

    Randak, Christoph O.; Welsh, Michael J.

    2005-01-01

    ADP interacts with the nucleotide-binding domains (NBDs) of the cystic fibrosis transmembrane conductance regulator (CFTR) to inhibit its Cl- channel activity. Because CFTR NBD2 has reversible adenylate kinase activity (ATP + AMP ⇆ ADP + ADP) that gates the channel, we asked whether ADP might inhibit current through this enzymatic activity. In adenylate kinases, binding of the two ADP molecules is cooperative. Consistent with this hypothesis, CFTR current inhibition showed positive cooperativity for ADP. We also found that ADP inhibition of current was attenuated when we prevented adenylate kinase activity with P1,P5-di(adenosine-5′) pentaphosphate. Additional studies suggested that adenylate kinase-dependent inhibition involved phosphotransfer between two nucleotide diphosphates. These data indicate that the adenylate kinase reaction at NBD2 contributed to the inhibitory effect of ADP. Finding that ADP inhibits function via an adenylate kinase activity also helps explain the earlier observation that mutations that disrupt adenylate kinase activity also disrupt ADP inhibition. Thus, the results reveal a previously unrecognized mechanism by which ADP inhibits an ABC transporter. PMID:15684079

  20. Application of Human Placental Villous Tissue Explants to Study ABC Transporter Mediated Efflux of 2,4-Dinitrophenyl-S-Glutathione

    PubMed Central

    Vaidya, Soniya S.; Walsh, Scott W.; Gerk, Phillip M.

    2011-01-01

    Objective The purpose of this study was to characterize the human term placental villous tissue explant culture model as a tool to study the formation and efflux of 1-chloro-2,4-dinitrobenzene (CDNB) conjugate 2,4-dinitrophenyl-S-glutathione (DNP-SG) as a model system for phase II metabolism and ATP-binding cassette (ABC) transporter-mediated cellular efflux. Methods Placental tissue samples were obtained after cesarean section following normal pregnancies (n=9). Cultured villous tissue was monitored up to 48 h to study the effect of time in culture on biochemical parameters, formation and efflux of DNP-SG in the absence or presence of ATPase inhibitor sodium orthovanadate and the protein expression of ABC transporters - multidrug resistance associated protein 2 (MRP2), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and enzyme glutathione-S-transferase isoform P1-1 (GSTP1-1). Results Villous tissue structure, tissue viability and expression of BCRP, GSTP1-1 remained unchanged, while expression of MRP2, P-gp and total tissue glutathione decreased with time in culture. Tissue integrity was unchanged up to 24 h but declined at 48 h. However, DNP-SG formation, DNP-SG efflux, and the extent of inhibition of DNP-SG efflux by sodium orthovanadate showed only minor changes through 48 h. Sodium orthovanadate decreased DNP-SG efflux, consistent with inhibition of apical ABC transporters. Conclusion The results support the use of the cultured human term placental villous tissue explants model to study coordinated function of GSTP1-1 and apical ABC transporters in the formation and efflux of the model substrate DNP-SG. PMID:21342117

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

    PubMed

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

    2015-10-16

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

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

    PubMed

    Aszalos, Adorjan

    2008-12-01

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

  3. Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

    PubMed Central

    Yanagisawa, Tatsuo; Kim, Kwang Sik; Yokoyama, Shigeyuki; Ohnishi, Makoto

    2015-01-01

    We previously reported that Neisseria meningitidis internalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellular l-glutamate via the GltT-GltM l-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltT ΔgltM invasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(−S)]. The alleviation disappeared again in AM(−S) supplemented with 500 μM glutamate. Glutamate uptake by the ΔgltT ΔgltM mutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced when N. meningitidis formed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltT ΔgltM mutant was much lower than that within the wild-type N. meningitidis strain only upon HBMEC infection and was correlated with intracellular survival. Considering that the l-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells, l-glutamate uptake via GltT-GltM plays multiple roles in N. meningitidis internalization into HBMEC. PMID:26099588

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. Facilitative plasma membrane transporters function during ER transit

    PubMed Central

    Takanaga, Hitomi; Frommer, Wolf B.

    2010-01-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na+-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.—Takanaga, H., Frommer, W. B. Facilitative plasma membrane transporters function during ER transit. PMID:20354141

  6. Membrane transporters in self resistance of Cercospora nicotianae to the photoactivated toxin cercosporin.

    PubMed

    Beseli, Aydin; Amnuaykanjanasin, Alongkorn; Herrero, Sonia; Thomas, Elizabeth; Daub, Margaret E

    2015-11-01

    The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene (CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants. PMID:25862648

  7. Compartment-specific roles of ABC transporters define differential topotecan distribution in brain parenchyma and cerebrospinal fluid

    PubMed Central

    Shen, Jun; Carcaboso, Angel M.; Hubbard, K. Elaine; Tagen, Michael; Wynn, Henry G.; Panetta, John C.; Waters, Christopher M.; Elmeliegy, Mohamed A.; Stewart, Clinton F.

    2009-01-01

    Topotecan is a substrate of the ABC transporters P-glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). To define the role of these transporters in topotecan penetration into the ventricular cerebrospinal fluid (vCSF) and brain parenchymal extracellular fluid (ECF) compartments we performed intracerebral microdialysis on transporter-deficient mice after an intravenous dose of topotecan (4 mg/kg). vCSF penetration of unbound topotecan lactone was measured as the ratio of vCSF-to-plasma area under the concentration-time curves (AUCs). The mean (±SD) ratios for wild-type, Mdr1a/b−/−, Bcrp1−/− and Mdr1a/b−/−Bcrp1−/− mice were 3.07±0.09, 2.57±0.17, 1.63±0.12 and 0.86±0.05, respectively. In contrast, the ECF-to-plasma ratios for wild-type, Bcrp1−/− and Mdr1a/b−/−Bcrp1−/− mice were 0.36±0.06, 0.42±0.06 and 0.88±0.07. Topotecan lactone was below detectable limits in the ECF of Mdr1a/b−/−mice. When gefitinib (200 mg/kg) was pre-administered to inhibit Bcrp1 and P-gp, the vCSF-to-plasma ratio decreased to 1.29±0.09 in wild-type mice and increased to 1.13±0.13 in Mdr1a/b−/−Bcrp1−/− mice, whereas the ECF-to-plasma ratio increased to 0.74±0.14 in wild-type and 1.07±0.03 in Mdr1a/b−/−Bcrp1−/− mice. Preferential active transport of topotecan lactone over topotecan carboxylate was shown in vivo by vCSF lactone-to-carboxylate AUC ratios for wild-type, Mdr1a/b−/−, Bcrp1−/− and Mdr1a/b−/−Bcrp1−/− mice of 5.69±0.83, 3.85±0.64, 3.61±0.46 and 0.78±0.19. Our results suggest that Bcrp1 and P-gp transport topotecan into vCSF and out of brain parenchyma through the blood-brain barrier. These findings may help to improve pharmacological strategies to treat brain tumors. PMID:19567673

  8. Subnanometer resolution cryo-EM structure of a nucleotide free heterodimeric ABC exporter

    PubMed Central

    Kim, JungMin; Wu, Shenping; Tomasiak, Thomas; Mergel, Claudia; Winter, Michael B.; Stiller, Sebastian B.; Robles-Colmanares, Yaneth; Stroud, Robert M.; Tampé, Robert; Craik, Charles S.; Cheng, Yifan

    2015-01-01

    ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide binding domains (NBDs)1,2. ABC exporters are present in both prokaryotes and eukaryotes with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases3,4. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus homologous to various multidrug transporters and containing one degenerate site with a non-catalytic residue next to the Walker B motif5. Here we report a subnanometer resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single particle electron cryomicroscopy (cryo-EM). The reconstructions clearly resolved characteristic features of ABC transporters, including helices in the transmembrane domain (TMD) and NBDs. A cavity in the TMD is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two NBDs remain in contact via their C-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two NBDs during the transition from the inward facing to outward facing conformations. PMID:25363761

  9. Fabrication of catalyzed ion transport membrane systems

    DOEpatents

    Carolan, Michael Francis; Kibby, Charles Leonard

    2013-06-04

    Process for fabricating a catalyzed ion transport membrane (ITM). In one embodiment, an uncatalyzed ITM is (a) contacted with a non-reducing gaseous stream while heating to a temperature and for a time period sufficient to provide an ITM possessing anion mobility; (b) contacted with a reducing gaseous stream for a time period sufficient to provide an ITM having anion mobility and essentially constant oxygen stoichiometry; (c) cooled while contacting the ITM with the reducing gaseous stream to provide an ITM having essentially constant oxygen stoichiometry and no anion mobility; and (d) treated by applying catalyst to at least one of (1) a porous mixed conducting multicomponent metallic oxide (MCMO) layer contiguous with a first side of a dense layer of MCMO and (2) a second side of the dense MCMO layer. In another embodiment, these steps are carried out in the alternative order of (a), (d), (b), and (c).

  10. Urea transport through composite polyallylamine membranes

    NASA Technical Reports Server (NTRS)

    Ballou, E. V.; Kubo, L. Y.; Spitze, L. A.; Wydeven, T.; Clark, J. A.

    1977-01-01

    Polyallylamine composite reverse osmosis membranes were prepared by plasma polymerization and deposition onto small-pored cellulose acetate/cellulose nitrate films. The polyallylamine coated the porous substrate with a thin uniform polymer film which exhibited water permeability and urea rejection, of interest because of the potential application of reverse osmosis to urine purification in closed environmental systems. The flux of C-14 labeled urea was studied under the influence of osmotic gradients provided by sodium chloride solutions. The urea flux was found to be enhanced by an osmotic pressure gradient in the same direction and diminished, but not prevented, by an opposing osmotic pressure gradient. Consideration is given to the mechanism of the urea transport, as well as to the influence of concentration polarization on the experimental results. The minimization of coupled flow in pores of a critical size range is apparently necessary to improve urea rejection.

  11. Substrate specificities and expression patterns reflect the evolutionary divergence of maltose ABC transporters in Thermotoga maritima.

    PubMed

    Nanavati, Dhaval M; Nguyen, Tu N; Noll, Kenneth M

    2005-03-01

    Duplication of transporter genes is apparent in the genome sequence of the hyperthermophilic bacterium Thermotoga maritima. The physiological impacts of these duplications are not well understood, so we used the bacterium's two putative maltose transporters to begin a study of the evolutionary relationship between a transporter's function and the control of expression of its genes. We show that the substrate binding proteins encoded by these operons, MalE1 and MalE2, have different substrate specificities and affinities and that they are expressed under different growth conditions. MalE1 binds maltose (dissociation constant [KD], 24 +/- 1 microM), maltotriose (KD, 8 +/- 0.5 nM), and beta-(1-->4)-mannotetraose (KD, 38 +/- 1 microM). In contrast, MalE2 binds maltose (KD, 8.4 +/- 1 microM), maltotriose (KD, 11.5 +/- 1.5 microM), and trehalose (KD, 9.5 +/- 1.0 microM) confirming the findings of Wassenberg et al. (J. Mol. Biol. 295:279-288, 2000). Neither protein binds lactose. We examined the expression of these operons at both the transcriptional and translational levels and found that MalE1 is expressed in cells grown on lactose or guar gum and that MalE2 is highly expressed in starch- and trehalose-grown cells. Evidence is provided that malE1, malF1, and perhaps malG1 are cotranscribed and so constitute an operon. An open reading frame encoding a putative transcriptional regulatory protein adjacent to this operon (TM1200) is also up-regulated in response to growth on lactose. These evolutionarily related transporter operons have diverged both in function and expression to assume apparently different physiological roles. PMID:15743948

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

    PubMed

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

    2011-12-01

    Along with many other mammalian ATP-binding cassette (ABC) transporters, members of the ABCG group are involved in the regulated transport of hydrophobic compounds across cellular membranes. In humans, five ABCG family members have been identified, encoding proteins ranging from 638 to 678 amino acids in length. All five have been the subject of intensive investigation to better understand their physiological roles, expression patterns, interactions with substrates and inhibitors, and regulation at both the transcript and protein level. The principal substrates for at least four of the ABCG proteins are endogenous and dietary lipids, with ABCG1 implicated in particular in the export of cholesterol, and ABCG5 and G8 forming a functional heterodimer responsible for plant sterol elimination from the body. ABCG2 has a much broader substrate specificity and its ability to transport numerous diverse pharmaceuticals has implications for the absorption, distribution, metabolism, excretion and toxicity (ADMETOx) profile of these compounds. ABCG2 is one of at least three so-called multidrug resistant ABC transporters expressed in humans, and its activity is associated with decreased efficacy of anti-cancer agents in several carcinomas. In addition to its role in cancer, ABCG2 also plays a role in the normal physiological transport of urate and haem, the implications of which are described. We summarize here data on all five human ABCG transporters and provide a current perspective on their roles in human health and disease. PMID:21175590

  13. Kidney versus Liver Specification of SLC and ABC Drug Transporters, Tight Junction Molecules, and Biomarkers.

    PubMed

    Martovetsky, Gleb; Bush, Kevin T; Nigam, Sanjay K

    2016-07-01

    The hepatocyte nuclear factors, Hnf1a and Hnf4a, in addition to playing key roles in determining hepatocyte fate, have been implicated as candidate lineage-determining transcription factors in the kidney proximal tubule (PT) [Martovetsky et. al., (2012) Mol Pharmacol 84:808], implying an additional level of regulation that is potentially important in developmental and/or tissue-engineering contexts. Mouse embryonic fibroblasts (MEFs) transduced with Hnf1a and Hnf4a form tight junctions and express multiple PT drug transporters (e.g., Slc22a6/Oat1, Slc47a1/Mate1, Slc22a12/Urat1, Abcg2/Bcrp, Abcc2/Mrp2, Abcc4/Mrp4), nutrient transporters (e.g., Slc34a1/NaPi-2, Slco1a6), and tight junction proteins (occludin, claudin 6, ZO-1/Tjp1, ZO-2/Tjp2). In contrast, the coexpression (with Hnf1a and Hnf4a) of GATA binding protein 4 (Gata4), as well as the forkhead box transcription factors, Foxa2 and Foxa3, in MEFs not only downregulates PT markers but also leads to upregulation of several hepatocyte markers, including albumin, apolipoprotein, and transferrin. A similar result was obtained with primary mouse PT cells. Thus, the presence of Gata4 and Foxa2/Foxa3 appears to alter the effect of Hnf1a and Hnf4a by an as-yet unidentified mechanism, leading toward the generation of more hepatocyte-like cells as opposed to cells exhibiting PT characteristics. The different roles of Hnf4a in the kidney and liver was further supported by reanalysis of ChIP-seq data, which revealed Hnf4a colocalization in the kidney near PT-enriched genes compared with those genes enriched in the liver. These findings provide valuable insight, not only into the developmental, and perhaps organotypic, regulation of drug transporters, drug-metabolizing enzymes, and tight junctions, but also for regenerative medicine strategies aimed at restoring the function of the liver and/or kidney (acute kidney injury, AKI; chronic kidney disease, CKD). PMID:27044799

  14. Analytical Applications of Transport Through Bulk Liquid Membranes.

    PubMed

    Diaconu, Ioana; Ruse, Elena; Aboul-Enein, Hassan Y; Bunaciu, Andrei A

    2016-07-01

    This review discusses the results of research in the use of bulk liquid membranes in separation processes and preconcentration for analytical purposes. It includes some theoretical aspects, definitions, types of liquid membranes, and transport mechanism, as well as advantages of using liquid membranes in laboratory studies. These concepts are necessary to understand fundamental principles of liquid membrane transport. Due to the multiple advantages of liquid membranes several studies present analytical applications of the transport through liquid membranes in separation or preconcentration processes of metallic cations and some organic compounds, such as phenol and phenolic derivatives, organic acids, amino acids, carbohydrates, and drugs. This review presents coupled techniques such as separation through the liquid membrane coupled with flow injection analysis. PMID:26185963

  15. Pseudomonas aeruginosa capability to recruit zinc under conditions of limited metal availability is affected by inactivation of the ZnuABC transporter

    PubMed Central

    D'Orazio, Melania; Mastropasqua, Maria Chiara; Cerasi, Mauro; Pacello, Francesca; Consalvo, Ada; Chirullo, Barbara; Mortensen, Brittany; Skaar, Eric P.; Ciavardelli, Domenico; Pasquali, Paolo; Battistoni, Andrea

    2015-01-01

    The ability of a large number of bacterial pathogens to multiply in the infected host and cause disease is dependent on their ability to express high affinity zinc importers. In many bacteria ZnuABC, a transporter of the ABC family, plays a central role in the process of zinc uptake in zinc poor environments, including the tissues of the infected host. To initiate an investigation into the relevance of the zinc uptake apparatus for Pseudomonas aeruginosa pathogenicity, we have generated a znuA mutant in the PA14 strain. We have found that this mutant strain displays a limited growth defect in zinc depleted media. The znuA mutant strain is more sensitive than the wild type strain to calprotectin-mediated growth inhibition, but both the strains are highly resistant to this zinc sequestering antimicrobial protein. Moreover, intracellular zinc content is not evidently affected by inactivation of the ZnuABC transporter. These findings suggest that P. aeruginosa is equipped with redundant mechanisms for the acquisition of zinc that might favor P. aeruginosa colonization of environments containing low levels of this metal. Nonetheless, deletion of znuA affects alginate production, reduces the activity of extracellular zinc-containing proteases, including LasA, LasB and Protease IV, and decreases the ability of P. aeruginosa to disseminate during systemic infections. These results indicate that efficient zinc acquisition is critical for the expression of various virulence features typical of P. aeruginosa and that ZnuABC also plays an important role in zinc homeostasis in this microorganism. PMID:25751674

  16. Proline transport across the intestinal microvillus membrane may be regulated by membrane physical properties.

    PubMed

    Sadowski, D C; Gibbs, D J; Meddings, J B

    1992-03-23

    There is now abundant evidence that integral membrane protein function may be modulated by the physical properties of membrane lipids. The intestinal brush border membrane represents a membrane system highly specialized for nutrient absorption and, thus, provides an opportunity to study the interaction between integral membrane transport proteins and their lipid environment. We have previously demonstrated that alterations in this environment may modulate the function of the sodium-dependent glucose transporter in terms of its affinity for glucose. In this communication we report that membrane lipid-protein interactions are distinctly different for the proline transport proteins. Maximal transport rates for L-proline by either the neutral brush border or imino transport systems are reduced 10-fold when the surrounding membrane environment is made more fluid over the physiological range that exists along the crypt-villus axis. Furthermore, in microvillus membrane vesicles prepared from enterocytes isolated from along the crypt-villus axis a similar gradient exists in the functional activity of these transport systems. This would imply that either the functional activity of these transporters are regulated by membrane physical properties or that the synthesis and insertion of these proteins is coordinated in concert with membrane physical properties as the enterocyte migrates up the crypt-villus axis. PMID:1567897

  17. Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract

    PubMed Central

    Jones, Megan M.; Johnson, Antoinette; Koszelak-Rosenblum, Mary; Kirkham, Charmaine; Brauer, Aimee L.; Malkowski, Michael G.

    2014-01-01

    Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract. PMID:25156736

  18. Conserved Surface Accessible Nucleoside ABC Transporter Component SP0845 Is Essential for Pneumococcal Virulence and Confers Protection In Vivo

    PubMed Central

    Saxena, Sneha; Khan, Naeem; Dehinwal, Ruchika; Kumar, Ajay; Sehgal, Devinder

    2015-01-01

    Streptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis and meningitis. Surface accessible proteins of S. pneumoniae are being explored for the development of a protein-based vaccine in order to overcome the limitations of existing polysaccharide-based pneumococcal vaccines. To identify a potential vaccine candidate, we resolved surface-associated proteins of S. pneumoniae TIGR4 strain using two-dimensional gel electrophoresis followed by immunoblotting with antisera generated against whole heat-killed TIGR4. Ten immunoreactive spots were identified by mass spectrometric analysis that included a putative lipoprotein SP0845. Analysis of the inferred amino acid sequence of sp0845 homologues from 36 pneumococcal strains indicated that SP0845 was highly conserved (>98% identity) and showed less than 11% identity with any human protein. Our bioinformatic and functional analyses demonstrated that SP0845 is the substrate-binding protein of an ATP-binding cassette (ABC) transporter that is involved in nucleoside uptake with cytidine, uridine, guanosine and inosine as the preferred substrates. Deletion of the gene encoding SP0845 renders pneumococci avirulent suggesting that it is essential for virulence. Immunoblot analysis suggested that SP0845 is expressed in in vitro grown pneumococci and during mice infection. Immunofluorescence microscopy and flow cytometry data indicated that SP0845 is surface exposed in encapsulated strains and accessible to antibodies. Subcutaneous immunization with recombinant SP0845 induced high titer antibodies in mice. Hyperimmune sera raised against SP0845 promoted killing of encapsulated pneumococcal strains in a blood bactericidal assay. Immunization with SP0845 protected mice from intraperitoneal challenge with heterologous pneumococcal serotypes. Based on its surface accessibility, role in virulence and ability to elicit protective immunity, we propose that SP0845 may be a potential candidate for a protein

  19. Nanostructured silicon membranes for control of molecular transport.

    PubMed

    Srijanto, Bernadeta R; Retterer, Scott T; Fowlkes, Jason D; Doktycz, Mitchel J

    2010-11-01

    A membrane that allows selective transport of molecular species requires precise engineering on the nanoscale. Membrane permeability can be tuned by controlling the physical structure and surface chemistry of the pores. Here, a combination of electron beam and optical lithography, along with cryogenic deep reactive ion etching, has been used to fabricate silicon membranes that are physically robust, have uniform pore sizes, and are directly integrated into a microfluidic network. Additional reductions in pore size were achieved using plasma enhanced chemical vapor deposition and atomic layer deposition of silicon dioxide to coat membrane surfaces. Cross sectioning of the membranes using focused ion beam milling was used to determine the physical shape of the membrane pores before and after coating. Functional characterization of the membranes was performed by using quantitative fluorescence microscopy to document the transport of molecular species across the membrane. PMID:24932436

  20. Subtle Structural Differences Trigger Inhibitory Activity of Propafenone Analogues at the Two Polyspecific ABC Transporters: P-Glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP).

    PubMed

    Schwarz, Theresa; Montanari, Floriane; Cseke, Anna; Wlcek, Katrin; Visvader, Lene; Palme, Sarah; Chiba, Peter; Kuchler, Karl; Urban, Ernst; Ecker, Gerhard F

    2016-06-20

    The transmembrane ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are widely recognized for their role in cancer multidrug resistance and absorption and distribution of compounds. Furthermore, they are linked to drug-drug interactions and toxicity. Nevertheless, due to their polyspecificity, a molecular understanding of the ligand-transporter interaction, which allows designing of both selective and dual inhibitors, is still in its infancy. This study comprises a combined approach of synthesis, in silico prediction, and in vitro testing to identify molecular features triggering transporter selectivity. Synthesis and testing of a series of 15 propafenone analogues with varied rigidity and basicity of substituents provide first trends for selective and dual inhibitors. Results indicate that both the flexibility of the substituent at the nitrogen atom, as well as the basicity of the nitrogen atom, trigger transporter selectivity. Furthermore, inhibitory activity of compounds at P-gp seems to be much more influenced by logP than those at BCRP. Exploiting these differences further should thus allow designing specific inhibitors for these two polyspecific ABC-transporters. PMID:26970257

  1. Human Ontogeny of Drug Transporters: Review and Recommendations of the Pediatric Transporter Working Group.

    PubMed

    Brouwer, K L R; Aleksunes, L M; Brandys, B; Giacoia, G P; Knipp, G; Lukacova, V; Meibohm, B; Nigam, S K; Rieder, M; de Wildt, S N

    2015-09-01

    The critical importance of membrane-bound transporters in pharmacotherapy is widely recognized, but little is known about drug transporter activity in children. In this white paper, the Pediatric Transporter Working Group presents a systematic review of the ontogeny of clinically relevant membrane transporters (e.g., SLC, ABC superfamilies) in intestine, liver, and kidney. Different developmental patterns for individual transporters emerge, but much remains unknown. Recommendations to increase our understanding of membrane transporters in pediatric pharmacotherapy are presented. PMID:26088472

  2. Water-mediated forces between the nucleotide binding domains generate the power stroke in an ABC transporter

    NASA Astrophysics Data System (ADS)

    Furukawa-Hagiya, Tomoka; Yoshida, Norio; Chiba, Shuntaro; Hayashi, Tomohiko; Furuta, Tadaomi; Sohma, Yoshiro; Sakurai, Minoru

    2014-11-01

    ATP binding cassette proteins shuttle a variety of molecules across cell membranes. The substrate transportation process is initiated by the ATP-driven dimerization of nucleotide binding domains (NBDs). Here, the integral-equation theory of liquids was applied to simulated NBD structures to analyze their dimerization process from the viewpoint of thermodynamics and the water-mediated interaction between the NBDs. It was found that a long-range hydration force of enthalpic origin drives the two NBDs to approach from a large separation. In the subsequent step, the water-mediated attraction of entropic origin brings about a structural adjustment between the two NBDs and their tighter contact.

  3. Membranes for nanometer-scale mass fast transport

    DOEpatents

    Bakajin, Olgica; Holt, Jason; Noy, Aleksandr; Park, Hyung Gyu

    2011-10-18

    Nanoporous membranes comprising single walled, double walled, and multiwalled carbon nanotubes embedded in a matrix material were fabricated for fluid mechanics and mass transfer studies on the nanometer scale and commercial applications. Average pore size can be 2 nm to 20 nm, or seven nm or less, or two nanometers or less. The membrane can be free of large voids spanning the membrane such that transport of material such as gas or liquid occurs exclusively through the tubes. Fast fluid, vapor, and liquid transport are observed. Versatile micromachining methods can be used for membrane fabrication. A single chip can comprise multiple membranes. These membranes are a robust platform for the study of confined molecular transport, with applications in liquid and gas separations and chemical sensing including desalination, dialysis, and fabric formation.

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

    PubMed Central

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

    2014-01-01

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

  5. Proteomic Analysis of the Developmental Trajectory of Human Hepatic Membrane Transporter Proteins in the First Three Months of Life.

    PubMed

    Mooij, Miriam G; van de Steeg, Evita; van Rosmalen, Joost; Windster, Jonathan D; de Koning, Barbara A E; Vaes, Wouter H J; van Groen, Bianca D; Tibboel, Dick; Wortelboer, Heleen M; de Wildt, Saskia N

    2016-07-01

    Human hepatic membrane-embedded transporter proteins are involved in trafficking endogenous and exogenous substrates. Even though impact of transporters on pharmacokinetics is recognized, little is known on maturation of transporter protein expression levels, especially during early life. We aimed to study the protein expression of 10 transporters in liver tissue from fetuses, infants, and adults. Transporter protein expression levels [ATP-binding cassette transporter (ABC)B1, ABCG2, ABCC2, ABCC3, bile salt efflux pump, glucose transporter 1, monocarboxylate transporter 1, organic anion transporter polypeptide (OATP)1B1, OATP2B1, and organic cation/carnitine transporter 2) were quantified using ultraperformance liquid chromatography tandem mass spectrometry in snap-frozen postmortem fetal, infant, and adult liver samples. Protein expression was quantified in isolated crude membrane fractions. The possible association between postnatal and postmenstrual age versus protein expression was studied. We studied 25 liver samples, as follows: 10 fetal [median gestational age 23.2 wk (range 16.4-37.9)], 12 infantile [gestational age at birth 35.1 wk (27.1-41.0), postnatal age 1 wk (0-11.4)], and 3 adult. The relationship of protein expression with age was explored by comparing age groups. Correlating age within the fetal/infant age group suggested four specific protein expression patterns, as follows: stable, low to high, high to low, and low-high-low. The impact of growth and development on human membrane transporter protein expression is transporter-dependent. The suggested age-related differences in transporter protein expression may aid our understanding of normal growth and development, and also may impact the disposition of substrate drugs in neonates and young infants. PMID:27103634

  6. Feed gas contaminant removal in ion transport membrane systems

    DOEpatents

    Underwood, Richard Paul; Makitka, III, Alexander; Carolan, Michael Francis

    2012-04-03

    An oxygen ion transport membrane process wherein a heated oxygen-containing gas having one or more contaminants is contacted with a reactive solid material to remove the one or more contaminants. The reactive solid material is provided as a deposit on a support. The one or more contaminant compounds in the heated oxygen-containing gas react with the reactive solid material. The contaminant-depleted oxygen-containing gas is contacted with a membrane, and oxygen is transported through the membrane to provide transported oxygen.

  7. Residues in the pathway through a membrane transporter.

    PubMed Central

    Yan, R T; Maloney, P C

    1995-01-01

    The structure of solute transporters is understood largely from analysis of their amino acid sequences, and more direct information is greatly needed. Here we report work that applies cysteine scanning mutagenesis to describe structure-function relations in UhpT, a bacterial membrane transporter. By using an impermeant SH-reactive agent to probe single-cysteine variants, we show that UhpT transmembrane segment 7 spans the membrane as an alpha-helix and that the central portion of this helix is exposed to both membrane surfaces, forming part of the translocation pathway through this transporter. Images Fig. 1 Fig. 3 Fig. 4 PMID:7597063

  8. Electrophoretic Transport of Biomolecules through Carbon Nanotube Membranes

    PubMed Central

    Sun, Xinghua; Su, Xin; Wu, Ji; Hinds, Bruce J.

    2013-01-01

    Electrophoretic transport of proteins across electrochemically oxidized multi-walled carbon nanotube (MWCNT) membranes has been investigated. Small charged protein, lysozyme, was successfully pumped across MWCNT membranes by electric field while rejecting larger bovine serum albumin (BSA). Transport of the lysozome was reduced by a factor of about 30 in comparison to bulk mobility and consistent with prediction for hindered transport. Mobilities between 0.33-1.4×10-9 m2/V-s were observed and are approximately 10 fold faster than comparable ordered nanoporous membranes and are consistent with continuum models. For mixtures of BSA and lysozyme, complete rejection of BSA is seen with electrophoretic separations PMID:21338104

  9. Thermodynamics of Ionic Transport through Functionalized Membranes

    NASA Astrophysics Data System (ADS)

    Rathee, Vikramjit; Qu, Siyi; Dilenschneider, Theodore; Phillip, William A.; Whitmer, Jonathan K.

    Through microphase separation of block copolymers, highly porous solid membranes may be assembled. Further functionalization with amine and sulfonic acid groups has demonstrated promise in exquisitely controlling the flux of charged species, and in particular multivalent ions. Using coarse-grained molecular simulations, we explore the essential thermodynamics underlying salt rejection in charge-functionalized membranes, and develop a model capable of linking the performance of these membranes to their molecular character through free energy calculations.

  10. Aquaporin 4-Specific T Cells in Neuromyelitis Optica Exhibit a Th17 Bias and Recognize Clostridium ABC Transporter

    PubMed Central

    Varrin-Doyer, Michel; Spencer, Collin M; Schulze-Topphoff, Ulf; Nelson, Patricia A; Stroud, Robert M; C Cree, Bruce A; Zamvil, Scott S

    2012-01-01

    Objective Aquaporin 4 (AQP4)-specific autoantibodies in neuromyelitis optica (NMO) are immunoglobulin (Ig)G1, a T cell-dependent Ig subclass, indicating that AQP4-specific T cells participate in NMO pathogenesis. Our goal was to identify and characterize AQP4-specific T cells in NMO patients and healthy controls (HC). Methods Peripheral blood T cells from NMO patients and HC were examined for recognition of AQP4 and production of proinflammatory cytokines. Monocytes were evaluated for production of T cell-polarizing cytokines and expression of costimulatory molecules. Results T cells from NMO patients and HC proliferated to intact AQP4 or AQP4 peptides (p11–30, p21–40, p61–80, p131–150, p156–170, p211–230, and p261–280). T cells from NMO patients demonstrated greater proliferation to AQP4 than those from HC, and responded most vigorously to p61–80, a naturally processed immunodominant determinant of intact AQP4. T cells were CD4+, and corresponding to association of NMO with human leukocyte antigen (HLA)-DRB1*0301 and DRB3, AQP4 p61–80-specific T cells were HLA-DR restricted. The T-cell epitope within AQP4 p61–80 was mapped to 63–76, which contains 10 residues with 90% homology to a sequence within Clostridium perfringens adenosine triphosphate-binding cassette (ABC) transporter permease. T cells from NMO patients proliferated to this homologous bacterial sequence, and cross-reactivity between it and self-AQP4 was observed, supporting molecular mimicry. In NMO, AQP4 p61–80-specific T cells exhibited Th17 polarization, and furthermore, monocytes produced more interleukin 6, a Th17-polarizing cytokine, and expressed elevated CD40 and CD80 costimulatory molecules, suggesting innate immunologic dysfunction. Interpretation AQP4-specific T-cell responses are amplified in NMO, exhibit a Th17 bias, and display cross-reactivity to a protein of an indigenous intestinal bacterium, providing new perspectives for investigating NMO pathogenesis. ANN

  11. Nitensidine A, a guanidine alkaloid from Pterogyne nitens, is a novel substrate for human ABC transporter ABCB1.

    PubMed

    Tajima, Yasuhiro; Nakagawa, Hiroshi; Tamura, Ai; Kadioglu, Onat; Satake, Kazuhiro; Mitani, Yuji; Murase, Hayato; Regasini, Luis Octavio; Bolzani, Vanderlan da Silva; Ishikawa, Toshihisa; Fricker, Gert; Efferth, Thomas

    2014-02-15

    The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization

  12. Novel ABC Transporter Gene, vga(C), Located on a Multiresistance Plasmid from a Porcine Methicillin-Resistant Staphylococcus aureus ST398 Strain ▿

    PubMed Central

    Kadlec, Kristina; Schwarz, Stefan

    2009-01-01

    A novel ABC transporter gene, vga(C), was identified on the 14,365-bp multiresistance plasmid pKKS825 in a porcine methicillin (meticillin)-resistant Staphylococcus aureus isolate of sequence type 398. The vga(C) gene encodes a 523-amino-acid protein which confers resistance not only to streptogramin A antibiotics but also to lincosamides and pleuromutilins. Plasmid pKKS825 also carries the resistance genes aadD, tet(L), and dfrK, which may enable the coselection of vga(C) under selective pressure by kanamycin/neomycin, tetracyclines, and trimethoprim. PMID:19470508

  13. 70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH{sub 2}-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins

    SciTech Connect

    Kashiwayama, Yoshinori; Seki, Midori; Yasui, Akina; Murasaki, Yoshiyuki; Morita, Masashi; Yamashita, Yukari; Sakaguchi, Masao; Tanaka, Yoshitaka; Imanaka, Tsuneo

    2009-01-15

    70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH{sub 2}-terminal deletion constructs of P70R. Among the NH{sub 2}-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH{sub 2}-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH{sub 2}-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH{sub 2}-terminal hydrophobic TMS1. Furthermore, attachment of the NH{sub 2}-terminal hydrophilic domain to the NH{sub 2}-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH{sub 2}-terminal region determines the subcellular localization of ABC subfamily D proteins.

  14. Interfacial Water-Transport Effects in Proton-Exchange Membranes

    SciTech Connect

    Kienitz, Brian; Yamada, Haruhiko; Nonoyama, Nobuaki; Weber, Adam

    2009-11-19

    It is well known that the proton-exchange membrane is perhaps the most critical component of a polymer-electrolyte fuel cell. Typical membranes, such as Nafion(R), require hydration to conduct efficiently and are instrumental in cell water management. Recently, evidence has been shown that these membranes might have different interfacial morphology and transport properties than in the bulk. In this paper, experimental data combined with theoretical simulations will be presented that explore the existence and impact of interfacial resistance on water transport for Nafion(R) 21x membranes. A mass-transfer coefficient for the interfacial resistance is calculated from experimental data using different permeation cells. This coefficient is shown to depend exponentially on relative humidity or water activity. The interfacial resistance does not seem to exist for liquid/membrane or membrane/membrane interfaces. The effect of the interfacial resistance is to flatten the water-content profiles within the membrane during operation. Under typical operating conditions, the resistance is on par with the water-transport resistance of the bulk membrane. Thus, the interfacial resistance can be dominant especially in thin, dry membranes and can affect overall fuel-cell performance.

  15. Membrane Transporters as Mediators of Cisplatin Effects and Side Effects

    PubMed Central

    Ciarimboli, Giuliano

    2012-01-01

    Transporters are important mediators of specific cellular uptake and thus, not only for effects, but also for side effects, metabolism, and excretion of many drugs such as cisplatin. Cisplatin is a potent cytostatic drug, whose use is limited by its severe acute and chronic nephro-, oto-, and peripheral neurotoxicity. For this reason, other platinum derivatives, such as carboplatin and oxaliplatin, with less toxicity but still with antitumoral action have been developed. Several transporters, which are expressed on the cell membranes, have been associated with cisplatin transport across the plasma membrane and across the cell: the copper transporter 1 (Ctr1), the copper transporter 2 (Ctr2), the P-type copper-transporting ATPases ATP7A and ATP7B, the organic cation transporter 2 (OCT2), and the multidrug extrusion transporter 1 (MATE1). Some of these transporters are also able to accept other platinum derivatives as substrate. Since membrane transporters display a specific tissue distribution, they can be important molecules that mediate the entry of platinum derivatives in target and also nontarget cells possibly mediating specific effects and side effects of the chemotherapeutic drug. This paper summarizes the literature on toxicities of cisplatin compared to that of carboplatin and oxaliplatin and the interaction of these platinum derivatives with membrane transporters. PMID:24278698

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

    PubMed Central

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

    2015-01-01

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

  17. A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter.

    PubMed

    Smart, Jonathan P; Cliff, Matthew J; Kelly, David J

    2009-11-01

    The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium. PMID:19818021

  18. Transport of Ions Across the Inner Envelope Membrane of Chloroplasts

    SciTech Connect

    McCarty, R. E.

    2004-06-02

    The technical report outlines the results of nine years of research on how ions cross the inner envelope membrane of chloroplasts. The ions include protons, nitrite, calcium and ferrous iron. Bicarbonate transport was also studied.

  19. The quorum-sensing molecule farnesol is a modulator of drug efflux mediated by ABC multidrug transporters and synergizes with drugs in Candida albicans.

    PubMed

    Sharma, Monika; Prasad, Rajendra

    2011-10-01

    Overexpression of the CaCDR1-encoded multidrug efflux pump protein CaCdr1p (Candida drug resistance protein 1), belonging to the ATP binding cassette (ABC) superfamily of transporters, is one of the most prominent contributors of multidrug resistance (MDR) in Candida albicans. Thus, blocking or modulating the function of the drug efflux pumps represents an attractive approach in combating MDR. In the present study, we provide first evidence that the quorum-sensing molecule farnesol (FAR) is a specific modulator of efflux mediated by ABC multidrug transporters, such as CaCdr1p and CaCdr2p of C. albicans and ScPdr5p of Saccharomyces cerevisiae. Interestingly, FAR did not modulate the efflux mediated by the multidrug extrusion pump protein CaMdr1p, belonging to the major facilitator superfamily (MFS). Kinetic data revealed that FAR competitively inhibited rhodamine 6G efflux in CaCdr1p-overexpressing cells, with a simultaneous increase in an apparent K(m) without affecting the V(max) values and the ATPase activity. We also observed that when used in combination, FAR at a nontoxic concentration synergized with the drugs at their respective nonlethal concentrations, as was evident from their <0.5 fractional inhibitory concentration index (FICI) values and from the drop of 14- to 64-fold in the MIC(80) values in the wild-type strain and in azole-resistant clinical isolates of C. albicans. Our biochemical experiments revealed that the synergistic interaction of FAR with the drugs led to reactive oxygen species accumulation, which triggered early apoptosis, and that both could be partly reversed by the addition of an antioxidant. Collectively, FAR modulates drug extrusion mediated exclusively by ABC proteins and is synergistic to fluconazole (FLC), ketoconazole (KTC), miconazole (MCZ), and amphotericin (AMB). PMID:21768514

  20. Structure of an ABC transporter solute-binding protein specific for the amino sugars glucosamine and galactosamine.

    PubMed

    Yadava, Umesh; Vetting, Matthew W; Al Obaidi, Nawar; Carter, Michael S; Gerlt, John A; Almo, Steven C

    2016-06-01

    The uptake of exogenous solutes by prokaryotes is mediated by transport systems embedded in the plasma membrane. In many cases, a solute-binding protein (SBP) is utilized to bind ligands with high affinity and deliver them to the membrane-bound components responsible for translocation into the cytoplasm. In the present study, Avi_5305, an Agrobacterium vitis SBP belonging to Pfam13407, was screened by differential scanning fluorimetry (DSF) and found to be stabilized by D-glucosamine and D-galactosamine. Avi_5305 is the first protein from Pfam13407 shown to be specific for amino sugars, and co-crystallization resulted in structures of Avi_5305 bound to D-glucosamine and D-galactosamine. Typical of Pfam13407, Avi_5305 consists of two α/β domains linked through a hinge region, with the ligand-binding site located in a cleft between the two domains. Comparisons with Escherichia coli ribose-binding protein suggest that a cation-π interaction with Tyr168 provides the specificity for D-glucosamine/D-galactosamine over D-glucose/D-galactose. PMID:27303900

  1. Ionic transport properties of template-synthesized gold nanotube membranes

    NASA Astrophysics Data System (ADS)

    Gao, Peng

    Ionic transport in nanotubes exhibits unique properties due to the strong interactions between ions and the nanotube surface. The main objective of my research is to explore and regulate the ionic transport in gold nanotube membranes. Chapter 1 overviews a versatile method of fabricating nanostructured materials, called the template synthesis. Important parameters of the template synthesis are introduced such as templates and deposition methods. The template synthesis method is used to prepare membranes used in this dissertation. Chapter 2 describes a method to increase the ionic conductivity in membranes containing gold nanotubes with small diameter (4 nm). The gold nanotube membrane is prepared by the electroless plating of gold in a commercially available polycarbonate membrane. Voltages are applied to the gold nanotube membrane and fixed charges are injected on the gold nanotube walls. We show that ionic conductivity of the gold nanotube membrane can be enhanced in aqueous potassium chloride (KCl) solution at negative applied voltages. When the most negative voltage (-0.8 V vs. Ag/AgCl) is applied to the membrane, the ionic conductivity of the solution inside the gold nanotube (94 mS.cm-1) is comparable to that of 1 M aqueous KCl, over two orders of magnitude higher than that of the 0.01 M KCl contacting the membrane. Chapter 3 explores another important transport property of the gold nanotube membrane -- ion permselectivity. When the permselective membrane separates two electrolyte solutions at different concentrations, a membrane potential is developed and measured by the potentiometric method. Surface charge density and the ion mobilities are estimated by fitting the experimental data with a pre-existing model. The surface charge density of the gold nanotube membrane in this research is estimated to be 2 muC/cm2. Chapter 4 describes voltage-controlled ionic transport in a gold/polypyrrole membrane doped with sodium dodecylbenzene sulfonate (DBS). Polypyrrole

  2. Development of active-transport membrane devices

    SciTech Connect

    Laciak, D.V.

    1994-07-01

    This report introduces the concept of Air Products` AT membranes for the separation of NH{sub 3} and CO{sub 2} from process gas streams and presents results from the first year fabrication concept development studies.

  3. Transport proteins of the plant plasma membrane

    NASA Technical Reports Server (NTRS)

    Assmann, S. M.; Haubrick, L. L.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Recently developed molecular and genetic approaches have enabled the identification and functional characterization of novel genes encoding ion channels, ion carriers, and water channels of the plant plasma membrane.

  4. Role of plasma membrane transporters in muscle metabolism.

    PubMed Central

    Zorzano, A; Fandos, C; Palacín, M

    2000-01-01

    Muscle plays a major role in metabolism. Thus it is a major glucose-utilizing tissue in the absorptive state, and changes in muscle insulin-stimulated glucose uptake alter whole-body glucose disposal. In some conditions, muscle preferentially uses lipid substrates, such as fatty acids or ketone bodies. Furthermore, muscle is the main reservoir of amino acids and protein. The activity of many different plasma membrane transporters, such as glucose carriers and transporters of carnitine, creatine and amino acids, play a crucial role in muscle metabolism by catalysing the influx or the efflux of substrates across the cell surface. In some cases, the membrane transport process is subjected to intense regulatory control and may become a potential pharmacological target, as is the case with the glucose transporter GLUT4. The goal of this review is the molecular characterization of muscle membrane transporter proteins, as well as the analysis of their possible regulatory role. PMID:10903126

  5. Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

    PubMed Central

    Pizeta Semighini, Camile; Marins, Mozart; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2002-01-01

    The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background. PMID:11872487

  6. Water and Molecular Transport across Nanopores in Monolayer Graphene Membranes

    NASA Astrophysics Data System (ADS)

    Jang, Doojoon; O'Hern, Sean; Kidambi, Piran; Boutilier, Michael; Song, Yi; Idrobo, Juan-Carlos; Kong, Jing; Laoui, Tahar; Karnik, Rohit

    2015-11-01

    Graphene's atomic thickness and high tensile strength allow it to outstand as backbone material for next-generation high flux separation membrane. Molecular dynamics simulations predicted that a single-layer graphene membrane could exhibit high permeability and selectivity for water over ions/molecules, qualifying as novel water desalination membranes. However, experimental investigation of water and molecular transport across graphene nanopores had remained barely explored due to the presence of intrinsic defects and tears in graphene. We introduce two-step methods to seal leakage across centimeter scale single-layer graphene membranes create sub-nanometer pores using ion irradiation and oxidative etching. Pore creation parameters were varied to explore the effects of created pore structures on water and molecular transport driven by forward osmosis. The results demonstrate the potential of nanoporous graphene as a reliable platform for high flux nanofiltration membranes.

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

    PubMed Central

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

    2013-01-01

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

  8. Simulating and Modeling Transport Through Atomically Thin Membranes

    NASA Astrophysics Data System (ADS)

    Ostrowski, Joseph; Eaves, Joel

    2014-03-01

    The world is running out of clean portable water. The efficacy of water desalination technologies using porous materials is a balance between membrane selectivity and solute throughput. These properties are just starting to be understood on the nanoscale, but in the limit of atomically thin membranes it is unclear whether one can apply typical continuous time random walk models. Depending on the size of the pore and thickness of the membrane, mass transport can range from single stochastic passage events to continuous flow describable by the usual hydrodynamic equations. We present a study of mass transport through membranes of various pore geometries using reverse nonequilibrium simulations, and analyze transport rates using stochastic master equations.

  9. Hydroxide Solvation and Transport in Anion Exchange Membranes.

    PubMed

    Chen, Chen; Tse, Ying-Lung Steve; Lindberg, Gerrick E; Knight, Chris; Voth, Gregory A

    2016-01-27

    Understanding hydroxide solvation and transport in anion exchange membranes (AEMs) can provide important insight into the design principles of these new membranes. To accurately model hydroxide solvation and transport, we developed a new multiscale reactive molecular dynamics model for hydroxide in aqueous solution, which was then subsequently modified for an AEM material. With this model, we investigated the hydroxide solvation structure and transport mechanism in the membrane. We found that a relatively even separation of the rigid side chains produces a continuous overlapping region for hydroxide transport that is made up of the first hydration shell of the tethered cationic groups. Our results show that hydroxide has a significant preference for this overlapping region, transporting through it and between the AEM side chains with substantial contributions from both vehicular (standard diffusion) and Grotthuss (proton hopping) mechanisms. Comparison of the AEM with common proton exchange membranes (PEMs) showed that the excess charge is less delocalized in the AEM than the PEMs, which is correlated with a higher free energy barrier for proton transfer reactions. The vehicular mechanism also contributes considerably more than the Grotthuss mechanism for hydroxide transport in the AEM, while our previous studies of PEM systems showed a larger contribution from the Grotthuss mechanism than the vehicular mechanism for proton transport. The activation energy barrier for hydroxide diffusion in the AEM is greater than that for proton diffusion in PEMs, implying a more significant enhancement of ion transport in the AEM at elevated temperatures. PMID:26716727

  10. Transport in Polymer-Electrolyte Membranes I. Physical Model

    SciTech Connect

    Weber, Adam Z.; Newman, John

    2003-06-02

    In this paper, a physical model is developed that is semiphenomenological and takes into account Schroeder's paradox. Using the wealth of knowledge contained in the literature regarding polymer-electrolyte membranes as a basis, a novel approach is taken in tying together all of the data into a single coherent theory. This approach involves describing the structural changes of the membrane due to water content, and casting this in terms of capillary phenomena. By treating the membrane in this fashion, Schroeder's paradox can be elucidated. Along with the structural changes, two different transport mechanisms are presented and discussed. These mechanisms, along with the membrane's structural changes, comprise the complete physical model of the membrane. The model is shown to agree qualitatively with different membranes and different membrane forms, and is applicable to modeling perfluorinated sulfonic acid and similar membranes. It is also the first physically based comprehensive model of transport in a membrane that includes a physical description of Schroeder's paradox, and it bridges the gap between the two types of macroscopic models currently in the literature.

  11. The ABC transporter AnrAB contributes to the innate resistance of Listeria monocytogenes to nisin, bacitracin, and various beta-lactam antibiotics.

    PubMed

    Collins, Barry; Curtis, Nicola; Cotter, Paul D; Hill, Colin; Ross, R Paul

    2010-10-01

    A mariner transposon bank was used to identify loci that contribute to the innate resistance of Listeria monocytogenes to the lantibiotic nisin. In addition to highlighting the importance of a number of loci previously associated with nisin resistance (mprF, virRS, and telA), a nisin-sensitive phenotype was associated with the disruption of anrB (lmo2115), a gene encoding the permease component of an ABC transporter. The contribution of anrB to nisin resistance was confirmed by the creation of nonpolar deletion mutants. The loss of this putative multidrug resistance transporter also greatly enhanced sensitivity to bacitracin, gallidermin, and a selection of β-lactam antibiotics. A comparison of the relative antimicrobial sensitivities of a number of mutants established the ΔanrB strain as being one of the most bacitracin-sensitive L. monocytogenes strains identified to date. PMID:20643901

  12. Cholesterol transport through lysosome-peroxisome membrane contacts.

    PubMed

    Chu, Bei-Bei; Liao, Ya-Cheng; Qi, Wei; Xie, Chang; Du, Ximing; Wang, Jiang; Yang, Hongyuan; Miao, Hong-Hua; Li, Bo-Liang; Song, Bao-Liang

    2015-04-01

    Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases. PMID:25860611

  13. An ABC Transporter Mutation Alters Root Exudation of Phytochemicals That Provoke an Overhaul of Natural Soil Microbiota1[C][W][OA

    PubMed Central

    Badri, Dayakar V.; Quintana, Naira; El Kassis, Elie G.; Kim, Hye Kyong; Choi, Young Hae; Sugiyama, Akifumi; Verpoorte, Robert; Martinoia, Enrico; Manter, Daniel K.; Vivanco, Jorge M.

    2009-01-01

    Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATP-binding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis (Arabidopsis thaliana) ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Columbia-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some sugar transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with Columbia-0 and abcg30 cultured soils determined by pyrosequencing revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community of organisms as well. PMID:19854857

  14. Current topics in membranes and transport

    SciTech Connect

    Kleinzeller, A.

    1987-01-01

    This book contains 10 chapters. Some of the chapter titles are: Expression of the Oxytocin and Vasopressin Genes; Steroid Effects on Excitable Membranes: The Secretory Vesicle in Processing and Secretion of Neuropeptides: and Steroid Hormone Influences on Cyclic AMP-Generating Systems.

  15. Novel macrocyclic carriers for proton-coupled liquid membrane transport

    SciTech Connect

    Lamb, J.D.

    1991-06-10

    The objective of our research program is to elucidate the chemical principles which are responsible for the cation selectivity and permeability of liquid membranes containing macrocyclic carriers. Several new macrocyclic carriers were synthesized during the last three year period, including selenium-containing macrocycles, new crown-4 structures, and several new crown structures containing nitrogen based heterocycles as substituents in the principal macrocyclic ring. The cation binding properties of these macrocycles were investigated by potentiometric titration, calorimetric titration, solvent extraction, and NMR techniques. In addition, hydrophobic macrocycles were incorporated into dual hollow fiber membrane systems to investigate their membrane performance, especially in the proton-coupled transport mode. It was found that the dual hollow fiber system maintains the cation selectivity and permeability of supported liquid membranes, while enhancing membrane stability. The diffusion limited transport model was expanded to account for membrane solvent effects. Furthermore, Eu{sup 2+} transport was found to be similar to that of strontium and much higher than that of the lanthanides, in supported liquid membrane systems.

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

    PubMed Central

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

    2014-01-01

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

  17. Structure and Water Transport in Nafion Nanocomposite Membranes

    NASA Astrophysics Data System (ADS)

    Davis, Eric; Page, Kirt

    2014-03-01

    Perfluorinated ionomers, specifically Nafion, are the most widely used ion exchange membranes for vanadium redox flow battery applications, where an understanding of the relationship between membrane structure and transport of water/ions is critical to battery performance. In this study, the structure of Nafion/SiO2 nanocomposite membranes, synthesized using sol-gel chemistry, as well as cast directly from Nafion/SiO2 nanoparticle dispersions, was measured using both small-angle neutron scattering (SANS) and ultra-small-angle neutron scattering (USANS). Through contrast match studies of the SiO2 nanoparticles, direct information on the change in the structure of the Nafion membranes and the ion-transport channels within was obtained, where differences in membrane structure was observed between the solution-cast membranes and the membranes synthesized using sol-gel chemistry. Additionally, water sorption and diffusion in these Nafion/SiO2 nanocomposite membranes were measured using in situ time-resolved Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectroscopy and dynamic vapor sorption (DVS).

  18. Hydrogen transport membranes for dehydrogenation reactions

    DOEpatents

    Balachandran; Uthamalingam

    2008-02-12

    A method of converting C.sub.2 and/or higher alkanes to olefins by contacting a feedstock containing C.sub.2 and/or higher alkanes with a first surface of a metal composite membrane of a sintered homogenous mixture of an Al oxide or stabilized or partially stabilized Zr oxide ceramic powder and a metal powder of one or more of Pd, Nb, V, Zr, Ta and/or alloys or mixtures thereof. The alkanes dehydrogenate to olefins by contact with the first surface with substantially only atomic hydrogen from the dehydrogenation of the alkanes passing through the metal composite membrane. Apparatus for effecting the conversion and separation is also disclosed.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Natural polyphenols: Influence on membrane transporters

    PubMed Central

    Hussain, Saad Abdulrahman; Sulaiman, Amal Ajaweed; Alhaddad, Hasan; Alhadidi, Qasim

    2016-01-01

    Accumulated evidence has focused on the use of natural polyphenolic compounds as nutraceuticals since they showed a wide range of bioactivities and exhibited protection against variety of age-related disorders. Polyphenols have variable potencies to interact, and hence alter the activities of various transporter proteins, many of them classified as anion transporting polypeptide-binding cassette transporters like multidrug resistance protein and p-glycoprotein. Some of the efflux transporters are, generally, linked with anticancer and antiviral drug resistance; in this context, polyphenols may be beneficial in modulating drug resistance by increasing the efficacy of anticancer and antiviral drugs. In addition, these effects were implicated to explain the influence of dietary polyphenols on drug efficacy as result of food-drug interactions. However, limited data are available about the influence of these components on uptake transporters. Therefore, the objective of this article is to review the potential efficacies of polyphenols in modulating the functional integrity of uptake transporter proteins, including those terminated the effect of neurotransmitters, and their possible influence in neuropharmacology. PMID:27069731

  1. Natural polyphenols: Influence on membrane transporters.

    PubMed

    Hussain, Saad Abdulrahman; Sulaiman, Amal Ajaweed; Alhaddad, Hasan; Alhadidi, Qasim

    2016-01-01

    Accumulated evidence has focused on the use of natural polyphenolic compounds as nutraceuticals since they showed a wide range of bioactivities and exhibited protection against variety of age-related disorders. Polyphenols have variable potencies to interact, and hence alter the activities of various transporter proteins, many of them classified as anion transporting polypeptide-binding cassette transporters like multidrug resistance protein and p-glycoprotein. Some of the efflux transporters are, generally, linked with anticancer and antiviral drug resistance; in this context, polyphenols may be beneficial in modulating drug resistance by increasing the efficacy of anticancer and antiviral drugs. In addition, these effects were implicated to explain the influence of dietary polyphenols on drug efficacy as result of food-drug interactions. However, limited data are available about the influence of these components on uptake transporters. Therefore, the objective of this article is to review the potential efficacies of polyphenols in modulating the functional integrity of uptake transporter proteins, including those terminated the effect of neurotransmitters, and their possible influence in neuropharmacology. PMID:27069731

  2. Phospholipid flippases: building asymmetric membranes and transport vesicles

    PubMed Central

    Sebastian, Tessy T.; Baldridge, Ryan D.; Xu, Peng; Graham, Todd R.

    2012-01-01

    Phospholipid flippases in the type IV P-type ATPase family (P4-ATPases) are essential components of the Golgi, plasma membrane and endosomal system that play critical roles in membrane biogenesis. These pumps flip phospholipid across the bilayer to create an asymmetric membrane structure with substrate phospholipids, such as phosphatidylserine and phosphatidylethanolamine, enriched within the cytosolic leaflet. The P4-ATPases also help form transport vesicles that bud from Golgi and endosomal membranes, thereby impacting the sorting and localization of many different proteins in the secretory and endocytic pathways. At the organismal level, P4-ATPase deficiencies are linked to liver disease, obesity, diabetes, hearing loss, neurological deficits, immune deficiency and reduced fertility. Here, we review the biochemical, cellular and physiological functions of P4-ATPases, with an emphasis on their roles in vesicle-mediated protein transport. PMID:22234261

  3. Membrane Transporters: Structure, Function and Targets for Drug Design

    NASA Astrophysics Data System (ADS)

    Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

    Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

  4. Membranes with functionalized carbon nanotube pores for selective transport

    DOEpatents

    Bakajin, Olgica; Noy, Aleksandr; Fornasiero, Francesco; Park, Hyung Gyu; Holt, Jason K; Kim, Sangil

    2015-01-27

    Provided herein composition and methods for nanoporous membranes comprising single walled, double walled, or multi-walled carbon nanotubes embedded in a matrix material. Average pore size of the carbon nanotube can be 6 nm or less. These membranes are a robust platform for the study of confined molecular transport, with applications in liquid and gas separations and chemical sensing including desalination, dialysis, and fabric formation.

  5. Subtle Structural Differences Trigger Inhibitory Activity of Propafenone Analogues at the Two Polyspecific ABC Transporters: P‐Glycoprotein (P‐gp) and Breast Cancer Resistance Protein (BCRP)

    PubMed Central

    Schwarz, Theresa; Montanari, Floriane; Cseke, Anna; Wlcek, Katrin; Visvader, Lene; Palme, Sarah; Chiba, Peter; Kuchler, Karl; Urban, Ernst

    2016-01-01

    Abstract The transmembrane ABC transporters P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are widely recognized for their role in cancer multidrug resistance and absorption and distribution of compounds. Furthermore, they are linked to drug–drug interactions and toxicity. Nevertheless, due to their polyspecificity, a molecular understanding of the ligand‐transporter interaction, which allows designing of both selective and dual inhibitors, is still in its infancy. This study comprises a combined approach of synthesis, in silico prediction, and in vitro testing to identify molecular features triggering transporter selectivity. Synthesis and testing of a series of 15 propafenone analogues with varied rigidity and basicity of substituents provide first trends for selective and dual inhibitors. Results indicate that both the flexibility of the substituent at the nitrogen atom, as well as the basicity of the nitrogen atom, trigger transporter selectivity. Furthermore, inhibitory activity of compounds at P‐gp seems to be much more influenced by logP than those at BCRP. Exploiting these differences further should thus allow designing specific inhibitors for these two polyspecific ABC‐transporters. PMID:26970257

  6. ABC Technology Development Program

    SciTech Connect

    1994-10-14

    The Accelerator-Based Conversion (ABC) facility will be designed to accomplish the following mission: `Provide a weapon`s grade plutonium disposition capability in a safe, economical, and environmentally sound manner on a prudent schedule for [50] tons of weapon`s grade plutonium to be disposed on in [20] years.` This mission is supported by four major objectives: provide a reliable plutonium disposition capability within the next [15] years; provide a level of safety and of safety assurance that meets or exceeds that afforded to the public by modern commercial nuclear power plants; meet or exceed all applicable federal, state, and local regulations or standards for environmental compliance; manage the program in a cost effective manner. The ABC Technology Development Program defines the technology development activities that are required to accomplish this mission. The technology development tasks are related to the following topics: blanket system; vessel systems; reactivity control systems; heat transport system components; energy conversion systems; shutdown heat transport systems components; auxiliary systems; technology demonstrations - large scale experiments.

  7. Electroosmosis in Membranes: Effects of Unstirred Layers and Transport Numbers

    PubMed Central

    Barry, P. H.; Hope, A. B.

    1969-01-01

    When a current is passed through a membrane system, differences in transport numbers between the membrane and the adjacent solutions will, in general, result in depletion and enhancement of concentrations at the membrane-solution interfaces. This will be balanced by diffusion back into the bulk solution, diffusion of solute back across the membrane itself, and osmosis resulting from these local concentration gradients. The two main results of such a phenomenon are (1) that there is a current-induced volume flow, which may be mistaken for electroosmosis, and (2) that there will generally develop transient changes in potential difference (PD) across membranes during and after the passage of current through them. PMID:5786317

  8. Controlled Transport of Functionalized Nanochannel though Lipid Membrane

    NASA Astrophysics Data System (ADS)

    Dutt, Meenakshi; Kuksenok, Olga; Balazs, Anna C.

    2012-02-01

    Via the Dissipative Particle Dynamics approach, we study the directed transport of a transmembrane nanochannel to a desired location within a lipid bilayer. Each nanochannel encompasses an ABA architecture, with a hydrophobic shaft (B) with two hydrophilic ends (A). One of the ends of the nanochannel is functionalized with hydrophilic functional groups, or hairs. The hydrophilic hairs serve a dual role: (a) control transport across the membrane barrier, and (b) enable the channel relocation to a specific membrane site. Our system comprises a lipid membrane with an embedded transmembrane nanochannel with the hairs extending into solution. First, we hold a suitably functionalized pipette above the membrane while the nanochannel freely diffuses within the membrane. For an optimal range of parameters, we demonstrate that the hairs find the pipette and spontaneously anchor onto it. We then show that by moving the pipette for a range of velocities, we can effectively transport the channel to any location within the membrane. This prototype assembly can provide guidelines for designing a number of systems for biomimetic applications.

  9. RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization

    PubMed Central

    Zou, Wei; Yadav, Smita; DeVault, Laura; Jan, Yuh Nung; Sherwood, David R.

    2015-01-01

    Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth. PMID:26394140

  10. Method of making a hydrogen transport membrane, and article

    DOEpatents

    Schwartz, Joseph M.; Corpus, Joseph M.; Lim, Hankwon

    2015-07-21

    The present invention relates to a method of manufacturing a hydrogen transport membrane and the composite article itself. More specifically, the invention relates to producing a membrane substrate, wherein the ceramic substrate is coated with a metal oxide slurry, thereby eliminating the need for an activation step prior to plating the ceramic membrane through an electroless plating process. The invention also relates to modifying the pore size and porosity of the substrate by oxidation or reduction of the particles deposited by the metal oxide slurry.

  11. Comparative genomic analysis of integral membrane transport proteins in ciliates.

    PubMed

    Kumar, Ujjwal; Saier, Milton H

    2015-01-01

    Integral membrane transport proteins homologous to those found in the Transporter Classification Database (TCDB; www.tcdb.org) were identified and bioinformatically characterized by transporter class, family, and substrate specificity in three ciliates, Paramecium tetraurelia (Para), Tetrahymena thermophila (Tetra), and Ichthyophthirius multifiliis (Ich). In these three organisms, 1,326 of 39,600 proteins (3.4%), 1,017 of 24,800 proteins (4.2%), and 504 out of 8,100 proteins (6.2%) integral membrane transport proteins were identified, respectively. Thus, an inverse relationship was observed between the % transporters identified and the number of total proteins per genome reported. This surprising observation provides insight into the evolutionary process, giving rise to genome reduction following whole genome duplication (as in the case of Para) or during pathogenic association with a host organism (Ich). Of these transport proteins in Para and Tetra, about 41% were channels (more than any other type of organism studied), 31% were secondary carriers (fewer than most eukaryotes) and 26% were primary active transporters, mostly ATP-hydrolysis driven (more than most other eukaryotes). In Ich, the number of channels was selectively reduced by 66%, relative to Para and Tetra. Para has four times more inorganic anion transporters than Tetra, and Ich has nonselectively lost most of these. Tetra and Ich preferentially transport sugars and monocarboxylates while Para prefers di- and tricarboxylates. These observations serve to characterize the transport proteins of these related ciliates, providing insight into their nutrition and metabolism. PMID:25099884

  12. Does hindered transport theory apply to desalination membranes?

    PubMed

    Dražević, Emil; Košutić, Krešimir; Kolev, Vesselin; Freger, Viatcheslav

    2014-10-01

    As reverse osmosis (RO) and nanofiltration polyamide membranes become increasingly used for water purification, prediction of pollutant transport is required for membrane development and process engineering. Many popular models use hindered transport theory (HTT), which considers a spherical solute moving through an array of fluid-filled rigid cylindrical pores. Experiments and molecular dynamic simulations, however, reveal that polyamide membranes have a distinctly different structure of a "molecular sponge", a network of randomly connected voids widely distributed in size. In view of this disagreement, this study critically examined the validity of HTT by directly measuring diffusivities of several alcohols within a polyamide film of commercial RO membrane using attenuated total reflection-FTIR. It is found that measured diffusivities deviate from HTT predictions by as much as 2-3 orders of magnitude. This result indicates that HTT does not adequately describe solute transport in desalination membranes. As a more adequate alternative, the concept of random resistor networks is suggested, with resistances described by models of activated transport in "soft" polymers without a sharp size cutoff and with a proper address of solute partitioning. PMID:25137614

  13. Transport in nanoporous carbon membranes: Experiments and analysis

    SciTech Connect

    Acharya, M.; Foley, H.C.

    2000-05-01

    Single-component permeances of six gases were measured on three different supported nanoporous carbon membranes prepared by spray coating and pyrolysis of poly(furfuryl alcohol) on porous stainless-steel disks. Global activation energies were regressed from data collected as a function of temperature. Permeances and global activation energies were correlated to molecular size, assuming that entropic affects dominated the transport. The permeance was best correlated to the minimum projected area of the molecule computed from first principles. The free-energy barriers to transport within the membranes were derived from the temperature dependence of the permeance data, after accounting for porosity differences between the membranes and differences in molecular adsorption. Using transition-state theory and an entropic model derived, the free energy, enthalpy, and entropic barriers to transport within the membrane were examined as a function of molecular size. Computed on the basis of size, the entropic component of this barrier did not account for the large differences in the transition-state free energies. However, when these entropic barrier values were used to compute the enthalpic portion of the barrier free energies, the minimum projected area of each molecule correlated strongly. Furthermore, these enthalpic components of the barriers were fitted nicely by the Everett-Powl mean field potential, using only the pore size as the adjustable parameter. These results shed light on the underlying mechanism by which shape-selective transport takes place in the NPC membranes and small molecules are separated.

  14. Fluorimetric Methods for Analysis of Permeability, Drug Transport Kinetics, and Inhibition of the ABCB1 Membrane Transporter.

    PubMed

    Armada, Ana; Martins, Célia; Spengler, Gabriella; Molnar, Joseph; Amaral, Leonard; Rodrigues, António Sebastião; Viveiros, Miguel

    2016-01-01

    The cell membrane P-glycoprotein (P-gp; MDR1, ABCB1) is an energy-dependent efflux pump that belongs to the ATP-binding cassette (ABC) family of transporters, and has been associated with drug resistance in eukaryotic cells. Multidrug resistance (MDR) is related to an increased expression and function of the ABCB1 (P-gp) efflux pump that often causes chemotherapeutic failure in cancer. Modulators of this efflux pump, such as the calcium channel blocker verapamil (VP) and cyclosporine A (CypA), can reverse the MDR phenotype but in vivo studies have revealed disappointing results due to adverse side effects. Currently available methods are unable to visualize and assess in a real-time basis the effectiveness of ABCB1 inhibitors on the uptake and efflux of ABCB1 substrates. However, predicting and testing ABCB1 modulation activity using living cells during drug development are crucial. The use of ABCB1-transfected mouse T-lymphoma cell line to study the uptake/efflux of fluorescent probes like ethidium bromide (EB), rhodamine 123 (Rh-123), and carbocyanine dye DiOC2, in the presence and absence of potential inhibitors, is currently used in our laboratories to evaluate the ability of a drug to inhibit ABCB1-mediated drug accumulation and efflux. Here we describe and compare three in vitro methods, which evaluate the permeability, transport kinetics of fluorescent substrates, and inhibition of the ABCB1 efflux pump by drugs of chemical synthesis or extracted from natural sources, using model cancer cell lines overexpressing this transporter, namely (1) real-time fluorimetry that assesses the accumulation of ethidium bromide, (2) flow cytometry, and (3) fluorescent microscopy using rhodamine 123 and DiOC2. PMID:26910071

  15. Characterization of Nanostructured Silicon Membranes for Control of Molecular Transport

    NASA Astrophysics Data System (ADS)

    Srijanto, Bernadeta; Retterer, Scott; Fowlkes, Jason; Doktycz, Mitchel

    2011-03-01

    Fabrication of nanoporous membranes for selective transport of molecular species requires precise engineering at the nanoscale. The membrane permeability can be tuned by controlling the physical structure and the surface chemistry of the pores. We use a combination of electron-beam and optical lithography, along with cryogenic deep reactive ion etching, to fabricate silicon membranes that are physically robust and have uniform pore sizes. Pore sizes are further reduced using plasma enhanced chemical vapor deposition and atomic layer deposition of silicon dioxide onto the membrane surfaces. Integrating nanoporous membranes within a microfluidic network provides a platform for tailoring molecular exchange between microchannels, independent of hydrodynamic effects. In enzymatic reactions, for example, tuning the pores size will allow smaller enzymatic substrates to traverse the membrane at controlled rates while larger enzymes remain spatially separated. Our results from membrane cross-sectioning using focused ion beam milling show that pore sizes can be controlled at dimensions below 10nm. Functional characterization was performed by quantitative fluorescence microscopy to observe the selective transport of molecular species of different sizes.

  16. Computer Simulations of Ion Transport in Polymer Electrolyte Membranes.

    PubMed

    Mogurampelly, Santosh; Borodin, Oleg; Ganesan, Venkat

    2016-06-01

    Understanding the mechanisms and optimizing ion transport in polymer membranes have been the subject of active research for more than three decades. We present an overview of the progress and challenges involved with the modeling and simulation aspects of the ion transport properties of polymer membranes. We are concerned mainly with atomistic and coarser level simulation studies and discuss some salient work in the context of pure binary and single ion conducting polymer electrolytes, polymer nanocomposites, block copolymers, and ionic liquid-based hybrid electrolytes. We conclude with an outlook highlighting future directions. PMID:27070764

  17. Continuous Modeling of Calcium Transport Through Biological Membranes

    NASA Astrophysics Data System (ADS)

    Jasielec, J. J.; Filipek, R.; Szyszkiewicz, K.; Sokalski, T.; Lewenstam, A.

    2016-06-01

    In this work an approach to the modeling of the biological membranes where a membrane is treated as a continuous medium is presented. The Nernst-Planck-Poisson model including Poisson equation for electric potential is used to describe transport of ions in the mitochondrial membrane—the interface which joins mitochondrial matrix with cellular cytosis. The transport of calcium ions is considered. Concentration of calcium inside the mitochondrion is not known accurately because different analytical methods give dramatically different results. We explain mathematically these differences assuming the complexing reaction inside mitochondrion and the existence of the calcium set-point (concentration of calcium in cytosis below which calcium stops entering the mitochondrion).

  18. Nonisothermal water transport through hydrophobic membranes in a stirred cell

    SciTech Connect

    Vazquez-Gonzalez, M.I.; Martinez, L.

    1994-10-01

    This paper studies the transport of pure water through microporous hydrophobic membranes in a stirred cell when bathed by two phases at different temperatures. The dependence of the phenomena on the stirring rate and on the average temperature has been investigated. The influence of these operating conditions on the mass transfer rate is discussed while keeping in mind the theories of mass and heat transfer within the membrane and adjoining liquids. The concept of temperature polarization is introduced in the transport equations, and it is shown to be important in the interpretation of our experimental results.

  19. Induction of CYP1A and ABC transporters in European sea bass (Dicentrarchus labrax) upon 2,3,7,8-TCDD waterborne exposure.

    PubMed

    Della Torre, Camilla; Mariottini, Michela; Vannuccini, Maria Luisa; Trisciani, Anna; Marchi, Davide; Corsi, Ilaria

    2014-08-01

    The aim of this study was to characterize the responsiveness of CYP1A and ABC transport proteins in European Sea bass (Dicentrarchus labrax) waterborne exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) (46 pg/L) for 24 h and 7 days. Genes modulation (abcb1, abcc1-2, cyp1a), EROD activity were investigated in liver and 2,3,7,8-TCDD bioconcentration in liver and muscle. TCDD induced significantly cyp1a gene expression and EROD activity at 24 h and 7 d. A significant up-regulation of abcb1 was also observed but only after 7 days. No modulation of abcc1 and abcc2 genes was observed. Waterborne TCDD exposure was able to induce CYP1A and abcb1 encoding for P-glycoprotein in juvenile of European sea bass. PMID:25016329

  20. Prism-patterned Nafion membrane for enhanced water transport in polymer electrolyte membrane fuel cell

    NASA Astrophysics Data System (ADS)

    Kim, Sang Moon; Kang, Yun Sik; Ahn, Chiyeong; Jang, Segeun; Kim, Minhyoung; Sung, Yung-Eun; Yoo, Sung Jong; Choi, Mansoo

    2016-06-01

    Here, we report a simple and effective strategy to enhance the performance of the polymer electrolyte membrane fuel cell by imprinting prism-patterned arrays onto the Nafion membrane, which provides three combined effects directly related to the device performance. First, a locally thinned membrane via imprinted micro prism-structures lead to reduced membrane resistance, which is confirmed by electrochemical impedance spectroscopy. Second, increments of the geometrical surface area of the prism-patterned Nafion membrane compared to a flat membrane result in the increase in the electrochemical active surface area. Third, the vertically asymmetric geometry of prism structures in the cathode catalyst layer lead to enhanced water transport, which is confirmed by oxygen gain calculation. To explain the enhanced water transport, we propose a simple theoretical model on removal of water droplets existing in the asymmetric catalyst layer. These three combined effects achieved via incorporating prism patterned arrays into the Nafion membrane effectively enhance the performance of the polymer electrolyte membrane fuel cell.

  1. Bioinformatic Analyses of Integral Membrane Transport Proteins Encoded Within the Genome of the Planctomycetes species, Rhodopirellula baltica

    PubMed Central

    Paparoditis, Philipp; Vastermark, Ake; Le, Andrew J.; Fuerst, John A.; Saier, Milton H.

    2013-01-01

    Rhodopirellula baltica (R. baltica) is a Planctomycete, known to have intracellular membranes. Because of its unusual cell structure and ecological significance, we have conducted comprehensive analyses of its transmembrane transport proteins. The complete proteome of R. baltica was screened against the Transporter Classification Database (TCDB) to identify recognizable integral membrane transport proteins. 342 proteins were identified with a high degree of confidence, and these fell into several different classes. R. baltica encodes in its genome channels (12%), secondary carriers (33%), and primary active transport proteins (41%) in addition to classes represented in smaller numbers. Relative to most non-marine bacteria, R. baltica possesses a larger number of sodium-dependent symporters but fewer proton-dependent symporters, and it has dimethylsulfoxide (DMSO) and trimethyl-amine-oxide (TMAO) reductases, consistent with its Na+-rich marine environment. R. baltica also possesses a Na+-translocating NADH:quinone dehydrogenase (Na+-NDH), a Na+ efflux decarboxylase, two Na+-exporting ABC pumps, two Na+-translocating F-type ATPases, two Na+:H+ antiporters and two K+:H+ antiporters. Flagellar motility probably depends on the sodium electrochemical gradient. Surprisingly, R. baltica also has a complete set of H+-translocating electron transport complexes similar to those present in β-proteobacteria and eukaryotic mitochondria. The transport proteins identified proved to be typical of the bacterial domain with little or no indication of the presence of eukaryotic-type transporters. However, novel functionally uncharacterized multispanning membrane proteins were identified, some of which are found only in Rhodopirellula species, but others of which are widely distributed in bacteria. The analyses lead to predictions regarding the physiology, ecology and evolution of R. baltica. PMID:23969110

  2. O antigen of FranconibacterpulverisG3872 (O1) is a 4-deoxy-d-arabino-hexose-containing polysaccharide synthesized by the ABC-transporter-dependent pathway.

    PubMed

    Wang, Min; Arbatsky, Nikolay P; Xu, Lingling; Shashkov, Alexander S; Wang, Lei; Knirel, Yuriy A

    2016-07-01

    Franconibacter (Enterobacter, Cronobacter) pulveris bacteria share several typical characteristics with, and hence pose a challenge for the detection of, Cronobacter sakazakii, an emerging opportunistic pathogen, which can cause severe infections in neonates. A structurally variable O-specific polysaccharide (OPS) called O antigen provides the major basis for the typing of Gram-negative bacteria. We investigated the structure and genetics of the O antigen of F. pulveris G3872 (designated O1). An OPS was isolated by mild alkaline degradation of the LPS, whereas the same polysaccharide and its oligosaccharide fragments were obtained by mild acid degradation. Studies by sugar analysis and NMR spectroscopy showed that the OPS contained d-ribose, l-rhamnose (l-Rha) and a rarely occurring monosaccharide 4-deoxy-d-arabino-hexose, and the OPS structure was established. The O-antigen gene cluster of F. pulveris G3872 between JUMPStart and gnd genes includes putative genes for glycosyltransferases, ATP-binding cassette (ABC)-transporter genes wzm and wzt, and genes for the synthesis of l-Rha, but no genes for the synthesis of 4-deoxy-d-arabino-hexose. A mutation test with the wzm gene confirmed that the OPS is synthesized and exported by the ABC-transporter-dependent pathway. A trifunctional transferase was suggested to catalyse formation of two glycosidic linkages and add a methyl group to the non-reducing end of the OPS to terminate the chain elongation. A carbohydrate-binding module that presumably recognizes the terminal methyl-modified monosaccharide was found at the C-terminus of Wzt. Primers specific for F. pulveris G3872 were designed based on the wzm gene, which has potential to be used for identification and detection of the O1 serogroup. PMID:27166227

  3. Identification of the minimal region in lipase ABC transporter recognition domain of Pseudomonas fluorescens for secretion and fluorescence of green fluorescent protein

    PubMed Central

    2012-01-01

    Background TliA is a thermostable lipase secreted by the type 1 secretion system (T1SS) of Pseudomonas fluorescens. The secretion is promoted by its secretion/chaperone domain located near the C-terminus, which is composed mainly of four Repeat-in-Toxin (RTX) repeats. In order to identify the minimal region of TliA responsible for its secretion, five different copies of the secretion/chaperone domain, each involving truncated N-terminal residues and a common C-terminus, were acquired and named as lipase ABC transporter recognition domains (LARDs). Each LARD was fused to epidermal growth factor (EGF) or green fluorescent protein (GFP), and the secretion of EGF-LARD or GFP-LARD fusion proteins was assessed in Escherichia coli with ABC transporter. Results Among the fusion proteins, GFP or EGF with 105-residue LARD3 was most efficiently secreted. In addition, GFP-LARD3 emitted wild type GFP fluorescence. Structurally, LARD3 had the 4 RTX repeats exposed at the N-terminus, while other LARDs had additional residues prior to them or missed some of the RTX repeats. LARD3 was both necessary and sufficient for efficient secretion and maintenance of GFP fluorescence in E. coli, which was also confirmed in P. fluorescens and P. fluorescens ▵tliA, a knock-out mutant of tliA. Conclusion LARD3 was a potent secretion signal in T1SS for its fusion flanking RTX motif, which enhanced secretion and preserved the fluorescence of GFP. LARD3-mediated secretion in E. coli or P. fluorescens will enable the development of enhanced protein manufacturing factory and recombinant microbe secreting protein of interest in situ. PMID:22578275

  4. Lr34 multi-pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species.

    PubMed

    Krattinger, Simon G; Lagudah, Evans S; Wicker, Thomas; Risk, Joanna M; Ashton, Anthony R; Selter, Liselotte L; Matsumoto, Takashi; Keller, Beat

    2011-02-01

    The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat. PMID:21265893

  5. No facilitator required for membrane transport of hydrogen sulfide

    PubMed Central

    Mathai, John C.; Missner, Andreas; Kügler, Philipp; Saparov, Sapar M.; Zeidel, Mark L.; Lee, John K.; Pohl, Peter

    2009-01-01

    Hydrogen sulfide (H2S) has emerged as a new and important member in the group of gaseous signaling molecules. However, the molecular transport mechanism has not yet been identified. Because of structural similarities with H2O, it was hypothesized that aquaporins may facilitate H2S transport across cell membranes. We tested this hypothesis by reconstituting the archeal aquaporin AfAQP from sulfide reducing bacteria Archaeoglobus fulgidus into planar membranes and by monitoring the resulting facilitation of osmotic water flow and H2S flux. To measure H2O and H2S fluxes, respectively, sodium ion dilution and buffer acidification by proton release (H2S ⇆ H+ + HS−) were recorded in the immediate membrane vicinity. Both sodium ion concentration and pH were measured by scanning ion-selective microelectrodes. A lower limit of lipid bilayer permeability to H2S, PM,H2S ≥ 0.5 ± 0.4 cm/s was calculated by numerically solving the complete system of differential reaction diffusion equations and fitting the theoretical pH distribution to experimental pH profiles. Even though reconstitution of AfAQP significantly increased water permeability through planar lipid bilayers, PM,H2S remained unchanged. These results indicate that lipid membranes may well act as a barrier to water transport although they do not oppose a significant resistance to H2S diffusion. The fact that cholesterol and sphingomyelin reconstitution did not turn these membranes into an H2S barrier indicates that H2S transport through epithelial barriers, endothelial barriers, and membrane rafts also occurs by simple diffusion and does not require facilitation by membrane channels. PMID:19805349

  6. Molecular level water and solute transport in reverse osmosis membranes

    NASA Astrophysics Data System (ADS)

    Lueptow, Richard M.; Shen, Meng; Keten, Sinan

    2015-11-01

    The water permeability and rejection characteristics of six solutes, methanol, ethanol, 2-propanol, urea, Na+, and Cl-, were studied for a polymeric reverse osmosis (RO) membrane using non-equilibrium molecular dynamics simulations. Results indicate that water flux increases with an increasing fraction of percolated free volume in the membrane polymer structure. Solute molecules display Brownian motion and hop from pore to pore as they pass through the membrane. The solute rejection depends on both the size of the solute molecule and the chemical interaction of the solute with water and the membrane. When the open spaces in the polymeric structure are such that solutes have to shed at least one water molecule from their solvation shell to pass through the membrane molecular structure, the water-solute pair interaction energy governs solute rejection. Organic solutes more easily shed water molecules than ions to more readily pass through the membrane. Hydrogen-bonding sites for molecules like urea also lead to a higher rejection. These findings underline the importance of the solute's solvation shell and solute-water-membrane chemistry in solute transport and rejection in RO membranes. Funded by the Institute for Sustainability and Energy at Northwestern with computing resources from XSEDE (NSF grant ACI-1053575).

  7. Revisiting the ABCs of multidrug resistance in cancer chemotherapy.

    PubMed

    Tiwari, Amit K; Sodani, Kamlesh; Dai, Chun-Ling; Ashby, Charles R; Chen, Zhe-Sheng

    2011-04-01

    The adenosine tri-phosphate binding cassette (ABC) transporters are one of the largest transmembrane gene families in humans. The ABC transporters are present in a number of tissues, providing protection against xenobiotics and certain endogenous molecules. Unfortunately, their presence produces suboptimal chemotherapeutic outcomes in cancer patient tumor cells. It is well established that they actively efflux antineoplastic agents from cancer cells, producing the multidrug resistance (MDR) phenotype. The inadequate response to chemotherapy and subsequent poor prognosis in cancer patients can be in part the result of the clinical overexpression of ABC transporters. In fact, one of the targeted approaches for overcoming MDR in cancer cells is that directed towards blocking or inhibiting ABC transporters. Indeed, for almost three decades, research has been conducted to overcome MDR through pharmacological inhibition of ABC transporters with limited clinical success. Therefore, contemporary strategies to identify or to synthesize selective "resensitizers" of ABC transporters with limited nonspecific toxicity have been undertaken. Innovative approaches en route to understanding specific biochemical role of ABC transporters in MDR and tumorigenesis will prove essential to direct our knowledge towards more effective targeted therapies. This review briefly discusses the current knowledge regarding the clinical involvement of ABC transporters in MDR to antineoplastic drugs and highlights approaches undertaken so far to overcome ABC transporter-mediated MDR in cancer. PMID:21118094

  8. Molecular mechanisms of reduced glutathione transport: role of the MRP/CFTR/ABCC and OATP/SLC21A families of membrane proteins

    SciTech Connect

    Ballatori, Nazzareno . E-mail: Ned_Ballatori@urmc.rochester.edu; Hammond, Christine L.; Cunningham, Jennifer B.; Krance, Suzanne M.; Marchan, Rosemarie

    2005-05-01

    The initial step in reduced glutathione (GSH) turnover in all mammalian cells is its transport across the plasma membrane into the extracellular space; however, the mechanisms of GSH transport are not clearly defined. GSH export is required for the delivery of its constituent amino acids to other tissues, detoxification of drugs, metals, and other reactive compounds of both endogenous and exogenous origin, protection against oxidant stress, and secretion of hepatic bile. Recent studies indicate that some members of the multidrug resistance-associated protein (MRP/CFTR or ABCC) family of ATP-binding cassette (ABC) proteins, as well as some members of the organic anion transporting polypeptide (OATP or SLC21A) family of transporters contribute to this process. In particular, five of the 12 members of the MRP/CFTR family appear to mediate GSH export from cells namely, MRP1, MRP2, MRP4, MRP5, and CFTR. Additionally, two members of the OATP family, rat Oatp1 and Oatp2, have been identified as GSH transporters. For the Oatp1 transporter, efflux of GSH may provide the driving force for the uptake of extracellular substrates. In humans, OATP-B and OATP8 do not appear to transport GSH; however, other members of this family have yet to be characterized in regards to GSH transport. In yeast, the ABC proteins Ycf1p and Bpt1p transport