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Sample records for abc transporter abca4

  1. Differential Phospholipid Substrates and Directional Transport by ATP-binding Cassette Proteins ABCA1, ABCA7, and ABCA4 and Disease-causing Mutants*♦

    PubMed Central

    Quazi, Faraz; Molday, Robert S.

    2013-01-01

    ABCA1, ABCA7, and ABCA4 are members of the ABCA subfamily of ATP-binding cassette transporters that share extensive sequence and structural similarity. Mutations in ABCA1 cause Tangier disease characterized by defective cholesterol homeostasis and high density lipoprotein (HDL) deficiency. Mutations in ABCA4 are responsible for Stargardt disease, a degenerative disorder associated with severe loss in central vision. Although cell-based studies have implicated ABCA proteins in lipid transport, the substrates and direction of transport have not been firmly established. We have purified and reconstituted ABCA1, ABCA7, and ABCA4 into liposomes for fluorescent-lipid transport studies. ABCA1 actively exported or flipped phosphatidylcholine, phosphatidylserine, and sphingomyelin from the cytoplasmic to the exocytoplasmic leaflet of membranes, whereas ABCA7 preferentially exported phosphatidylserine. In contrast, ABCA4 transported phosphatidylethanolamine in the reverse direction. The same phospholipids stimulated the ATPase activity of these ABCA transporters. The transport and ATPase activities of ABCA1 and ABCA4 were reduced by 25% in the presence of 20% cholesterol. Nine ABCA1 Tangier mutants and the corresponding ABCA4 Stargardt mutants showed significantly reduced phospholipid transport activity and subcellular mislocalization. These studies provide the first direct evidence for ABCA1 and ABCA7 functioning as phospholipid transporters and suggest that this activity is an essential step in the loading of apoA-1 with phospholipids for HDL formation. PMID:24097981

  2. ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer.

    PubMed

    Quazi, Faraz; Lenevich, Stepan; Molday, Robert S

    2012-06-26

    ATP-binding cassette (ABC) transporters comprise a superfamily of proteins, which actively transport a variety of compounds across cell membranes. Mammalian and most eukaryotic ABC transporters function as exporters, flipping or extruding substrates from the cytoplasmic to the extracellular or lumen side of cell membranes. Prokaryotic ABC transporters function either as exporters or importers. Here we show that ABCA4, an ABC transporter found in retinal photoreceptor cells and associated with Stargardt macular degeneration, is a novel importer that actively flips N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of disc membranes, thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptors. ABCA4 also actively transports phosphatidylethanolamine in the same direction. Mutations known to cause Stargardt disease decrease N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine transport activity of ABCA4. These studies provide the first direct evidence for a mammalian ABC transporter that functions as an importer and provide insight into mechanisms underlying substrate transport and the molecular basis of Stargardt disease.

  3. Thermodynamics of ABC transporters.

    PubMed

    Zhang, Xuejun C; Han, Lei; Zhao, Yan

    2016-01-01

    ABC transporters form the largest of all transporter families, and their structural study has made tremendous progress over recent years. However, despite such advances, the precise mechanisms that determine the energy-coupling between ATP hydrolysis and the conformational changes following substrate binding remain to be elucidated. Here, we present our thermodynamic analysis for both ABC importers and exporters, and introduce the two new concepts of differential-binding energy and elastic conformational energy into the discussion. We hope that the structural analysis of ABC transporters will henceforth take thermodynamic aspects of transport mechanisms into account as well.

  4. 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

  5. Structural diversity of ABC transporters

    PubMed Central

    ter Beek, Josy; Guskov, Albert

    2014-01-01

    ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains that hydrolyze ATP are structurally related in all ABC transporters, the membrane-embedded domains, where the substrates are translocated, adopt four different unrelated folds. Here, we review the structural characteristics of ABC transporters and discuss the implications of this structural diversity for mechanistic diversity. PMID:24638992

  6. Reduced macular function in ABCA4 carriers

    PubMed Central

    2015-01-01

    Purpose To study retinal function and morphology in ABCA4 carriers to investigate if ABCA4 carriership is associated with any functional or morphological changes and, if so, to explore whether certain mutations may be associated with particularly severe alterations. Methods Eighteen subjects were recruited by means of being the parents of 10 teenagers/young adults with genetically confirmed ABCA4-associated retinal degenerations. The teenagers/young adults are well-known patients and have been followed in our clinic for many years. The eighteen subjects underwent careful ophthalmological examinations, including fundus photography and autofluorescence imaging, Goldmann perimetry, optical coherence tomography (OCT), full-field electroretinography (ffERG), multifocal electroretinography (mERG), and ABCA4 gene sequencing. The ffERG and mERG results were compared with those of healthy controls. Results All subjects carried at least one ABCA4 mutation. Two subjects were compound heterozygous and therefore were excluded from the group-wise statistical analysis. Thirteen different ABCA4 mutations were found. C.2894 A>G (5/18) and c.768 G>T (4/18) were most common. Fourteen of 16 ABCA4 carriers demonstrated significantly altered mERG parameters (reduced amplitudes and/or delayed implicit times (ITs)) compared to normal values. In addition, the two subjects with compound heterozygous ABCA4 mutations had altered mERG parameters. A statistical comparison to the control group showed significantly reduced amplitudes and delayed ITs; p≤0.003 for all mERG parameters. FfERG parameters were altered in two ABCA4 carriers and one of the subjects with compound heterozygous ABCA4 mutations (reduced amplitude and delayed IT for the 30 Hz flicker ERG). No significant alterations were found for the whole group of ABCA4 carriers compared to the ffERG control group. Fundus photographs showed subtle to extensive pigmentary changes in several ABCA4 carriers. Conclusions In this study, ABCA4

  7. Mapping the functional yeast ABC transporter interactome

    PubMed Central

    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.; Luis, Bryan-Joseph San; Shevelev, Igor; Sturley, Stephen L; Boone, Charles; Greenblatt, Jack F.; Zhang, Zhaolei; Paumi, Christian M.; Babu, Mohan; Park, Hay-Oak; Michaelis, Susan; Stagljar, Igor

    2013-01-01

    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 (MYTH) technology to map the protein interactome of all non-mitochondrial ABC transporters in the model organism Saccharomy cescerevisiae, 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 a surprisingly 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. Improving the stability and function of purified ABCB1 and ABCA4: the influence of membrane lipids.

    PubMed

    Pollock, Naomi L; McDevitt, Christopher A; Collins, Richard; Niesten, Petronella H M; Prince, Stephen; Kerr, Ian D; Ford, Robert C; Callaghan, Richard

    2014-01-01

    ATP Binding Cassette (ABC) transporters play prominent roles in numerous cellular processes and many have been implicated in human diseases. Unfortunately, detailed mechanistic information on the majority of ABC transporters has not yet been elucidated. The slow rate of progress of molecular and high resolution structural studies may be attributed to the difficulty in the investigation of integral membrane proteins. These difficulties include the expression of functional, non-aggregated protein in heterologous systems. Furthermore, the extraction of membrane proteins from source material remains a major bottle-neck in the process since there are relatively few guidelines for selection of an appropriate detergent to achieve optimal extraction. Whilst affinity tag strategies have simplified the purification of membrane proteins; many challenges remain. For example, the chromatographic process and associated steps can rapidly lead to functional inactivation, random aggregation, or even precipitation of the target protein. Furthermore, optimisation of high yield and purity, does not guarantee successful structure determination. Based on this series of potential issues, any investigation into structure-function of membrane proteins requires a systematic evaluation of preparation quality. In particular, the evaluation should focus on function, homogeneity and mono-dispersity. The present investigation provides a detailed assessment of the quality of purified ATP Binding Cassette (ABC) transporters; namely ABCB1 (P-gp) and ABCA4 (ABCR). A number of suggestions are provided to facilitate the production of functional, homogeneous and mono-disperse preparations using the insect cell expression system. Finally, the ABCA4 samples have been used to provide structural insights into this essential photo-receptor cell protein. PMID:24036079

  9. 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

  10. 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

  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.

  12. ABC transporters, atherosclerosis and inflammation.

    PubMed

    Fitzgerald, Michael L; Mujawar, Zahedi; Tamehiro, Norimasa

    2010-08-01

    Atherosclerosis, driven by inflamed lipid-laden lesions, can occlude the coronary arteries and lead to myocardial infarction. This chronic disease is a major and expensive health burden. However, the body is able to mobilize and excrete cholesterol and other lipids, thus preventing atherosclerosis by a process termed reverse cholesterol transport (RCT). Insight into the mechanism of RCT has been gained by the study of two rare syndromes caused by the mutation of ABC transporter loci. In Tangier disease, loss of ABCA1 prevents cells from exporting cholesterol and phospholipid, thus resulting in the build-up of cholesterol in the peripheral tissues and a loss of circulating HDL. Consistent with HDL being an athero-protective particle, Tangier patients are more prone to develop atherosclerosis. Likewise, sitosterolemia is another inherited syndrome associated with premature atherosclerosis. Here mutations in either the ABCG5 or G8 loci, prevents hepatocytes and enterocytes from excreting cholesterol and plant sterols, including sitosterol, into the bile and intestinal lumen. Thus, ABCG5 and G8, which from a heterodimer, constitute a transporter that excretes cholesterol and dietary sterols back into the gut, while ABCA1 functions to export excess cell cholesterol and phospholipid during the biogenesis of HDL. Interestingly, a third protein, ABCG1, that has been shown to have anti-atherosclerotic activity in mice, may also act to transfer cholesterol to mature HDL particles. Here we review the relationship between the lipid transport activities of these proteins and their anti-atherosclerotic effect, particularly how they may reduce inflammatory signaling pathways. Of particular interest are recent reports that indicate both ABCA1 and ABCG1 modulate cell surface cholesterol levels and inhibit its partitioning into lipid rafts. Given lipid rafts may provide platforms for innate immune receptors to respond to inflammatory signals, it follows that loss of ABCA1 and ABCG1

  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. Analysis of the ABCA4 genomic locus in Stargardt disease.

    PubMed

    Zernant, Jana; Xie, Yajing Angela; Ayuso, Carmen; Riveiro-Alvarez, Rosa; Lopez-Martinez, Miguel-Angel; Simonelli, Francesca; Testa, Francesco; Gorin, Michael B; Strom, Samuel P; Bertelsen, Mette; Rosenberg, Thomas; Boone, Philip M; Yuan, Bo; Ayyagari, Radha; Nagy, Peter L; Tsang, Stephen H; Gouras, Peter; Collison, Frederick T; Lupski, James R; Fishman, Gerald A; Allikmets, Rando

    2014-12-20

    Autosomal recessive Stargardt disease (STGD1, MIM 248200) is caused by mutations in the ABCA4 gene. Complete sequencing of ABCA4 in STGD patients identifies compound heterozygous or homozygous disease-associated alleles in 65-70% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing disease-causing ABCA4 variation by a combination of next-generation sequencing (NGS), array-Comparative Genome Hybridization (aCGH) screening, familial segregation and in silico analyses. The entire 140 kb ABCA4 genomic locus was sequenced in 114 STGD patients with one known ABCA4 exonic mutation revealing, on average, 200 intronic variants per sample. Filtering of these data resulted in 141 candidates for new mutations. Two variants were detected in four samples, two in three samples, and 20 variants in two samples, the remaining 117 new variants were detected only once. Multimodal analysis suggested 12 new likely pathogenic intronic ABCA4 variants, some of which were specific to (isolated) ethnic groups. No copy number variation (large deletions and insertions) was detected in any patient suggesting that it is a very rare event in the ABCA4 locus. Many variants were excluded since they were not conserved in non-human primates, were frequent in African populations and, therefore, represented ancestral, and not disease-associated, variants. The sequence variability in the ABCA4 locus is extensive and the non-coding sequences do not harbor frequent mutations in STGD patients of European-American descent. Defining disease-associated alleles in the ABCA4 locus requires exceptionally well characterized large cohorts and extensive analyses by a combination of various approaches. PMID:25082829

  15. 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

  16. 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

  17. MDR-ABC transporters: biomarkers in rheumatoid arthritis.

    PubMed

    Márki-Zay, János; Tauberné Jakab, Katalin; Szerémy, Péter; Krajcsi, Peter

    2013-01-01

    MDR-ABC transporters are widely expressed in cell types relevant to pathogenesis of rheumatoid arthritis. Many reports demonstrate the interaction of small molecule drugs with MDR-ABC transporters. Cell-based assays for disease relevant cell types can be easily gated and could reveal specific drug targets and may increase significance and utilisation of data in clinical practice. Many commonly used DMARDs (e.g. methotrexate, sulfasalazine, leflunomide/teriflunomide, hydroxychloroquine) are ABCG2 substrates. Consequently, the activity of this transporter in patients should be determined to understand the disposition and pharmacokinetics of the therapy. In addition, MDR-ABC transporters transport a variety of endobiotics that play important roles in cell proliferation, cell migration, angiogenesis and inflammation. Therefore, MDR-ABC transporters are important biomarkers in rheumatoid arthritis. PMID:23711386

  18. Structural basis for the mechanism of ABC transporters.

    PubMed

    Beis, Konstantinos

    2015-10-01

    The ATP-binding cassette (ABC) transporters are primary transporters that couple the energy stored in adenosine triphosphate (ATP) to the movement of molecules across the membrane. ABC transporters can be divided into exporters and importers; importers mediate the uptake of essential nutrients into cells and are found predominantly in prokaryotes whereas exporters transport molecules out of cells or into organelles and are found in all organisms. ABC exporters have been linked with multi-drug resistance in both bacterial and eukaryotic cells. ABC transporters are powered by the hydrolysis of ATP and transport their substrate via the alternating access mechanism, whereby the protein alternates between a conformation in which the substrate-binding site is accessible from the outside of the membrane, outward-facing and one in which it is inward-facing. In this mini-review, the structures of different ABC transporter types in different conformations are presented within the context of the alternating access mechanism and how they have shaped our current understanding of the mechanism of ABC transporters.

  19. 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

  20. 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

  1. Protein misfolding and the pathogenesis of ABCA4-associated retinal degenerations

    PubMed Central

    Zhang, Ning; Tsybovsky, Yaroslav; Kolesnikov, Alexander V.; Rozanowska, Malgorzata; Swider, Malgorzata; Schwartz, Sharon B.; Stone, Edwin M.; Palczewska, Grazyna; Maeda, Akiko; Kefalov, Vladimir J.; Jacobson, Samuel G.; Cideciyan, Artur V.; Palczewski, Krzysztof

    2015-01-01

    Mutations in the ABCA4 gene are a common cause of autosomal recessive retinal degeneration. All mouse models to date are based on knockouts of Abca4, even though the disease is often caused by missense mutations such as the complex allele L541P;A1038V (PV). We now show that the PV mutation causes severe human disease whereas the V mutation alone causes mild disease. Mutant ABCA4 proteins expressed heterologously in mammalian cells retained normal cellular localization. However, basal and all-trans-retinal-stimulated ATPase activities were reduced substantially for P and PV but only mildly for V. Electron microscopy revealed marked structural changes and misfolding for the P and PV mutants but few changes for the V mutant, consistent with the disease severity difference in patients. We generated Abca4PV/PV knock-in mice homozygous for the complex PV allele to investigate the effects of this misfolding mutation in vivo. Mutant ABCA4 RNA levels approximated WT ABCA4 RNA levels but, surprisingly, only trace amounts of mutant ABCA4 protein were noted in the retina. RNA sequencing of WT, Abca4−/− and Abca4PV/PV mice revealed mild gene expression alterations in the retina and RPE. Similar to Abca4−/− mice, Abca4PV/PV mice showed substantial A2E and lipofuscin accumulation in their RPE cells but no retinal degeneration up to 12 months of age. Thus, rapid degradation of this large misfolded mutant protein in mouse retina caused little detectable photoreceptor degeneration. These findings suggest likely differences in the unfolded protein response between murine and human photoreceptors and support development of therapies directed at increasing this capability in patients. PMID:25712131

  2. Genetic and Epigenetic Factors at COL2A1 and ABCA4 Influence Clinical Outcome in Congenital Toxoplasmosis

    PubMed Central

    Jamieson, Sarra E.; de Roubaix, Lee-Anne; Cortina-Borja, Mario; Tan, Hooi Kuan; Mui, Ernest J.; Cordell, Heather J.; Kirisits, Michael J.; Miller, E. Nancy; Peacock, Christopher S.; Hargrave, Aubrey C.; Coyne, Jessica J.; Boyer, Kenneth; Bessieres, Marie-Hélène; Buffolano, Wilma; Ferret, Nicole; Franck, Jacqueline; Kieffer, François; Meier, Paul; Nowakowska, Dorota E.; Paul, Malgorzata; Peyron, François; Stray-Pedersen, Babill; Prusa, Andrea-Romana; Thulliez, Philippe; Wallon, Martine; Petersen, Eskild; McLeod, Rima; Gilbert, Ruth E.; Blackwell, Jenefer M.

    2008-01-01

    Background Primary Toxoplasma gondii infection during pregnancy can be transmitted to the fetus. At birth, infected infants may have intracranial calcification, hydrocephalus, and retinochoroiditis, and new ocular lesions can occur at any age after birth. Not all children who acquire infection in utero develop these clinical signs of disease. Whilst severity of disease is influenced by trimester in which infection is acquired by the mother, other factors including genetic predisposition may contribute. Methods and Findings In 457 mother-child pairs from Europe, and 149 child/parent trios from North America, we show that ocular and brain disease in congenital toxoplasmosis associate with polymorphisms in ABCA4 encoding ATP-binding cassette transporter, subfamily A, member 4. Polymorphisms at COL2A1 encoding type II collagen associate only with ocular disease. Both loci showed unusual inheritance patterns for the disease allele when comparing outcomes in heterozygous affected children with outcomes in affected children of heterozygous mothers. Modeling suggested either an effect of mother's genotype, or parent-of-origin effects. Experimental studies showed that both ABCA4 and COL2A1 show isoform-specific epigenetic modifications consistent with imprinting. Conclusions These associations between clinical outcomes of congenital toxoplasmosis and polymorphisms at ABCA4 and COL2A1 provide novel insight into the molecular pathways that can be affected by congenital infection with this parasite. PMID:18523590

  3. Plant cuticular lipid export requires an ABC transporter.

    PubMed

    Pighin, Jamie A; Zheng, Huanquan; Balakshin, Laura J; Goodman, Ian P; Western, Tamara L; Jetter, Reinhard; Kunst, Ljerka; Samuels, A Lacey

    2004-10-22

    A waxy protective cuticle coats all primary aerial plant tissues. Its synthesis requires extensive export of lipids from epidermal cells to the plant surface. Arabidopsis cer5 mutants had reduced stem cuticular wax loads and accumulated sheetlike inclusions in the cytoplasm of wax-secreting cells. These inclusions represented abnormal deposits of cuticular wax and resembled inclusions found in a human disorder caused by a defective peroxisomal adenosine triphosphate binding cassette (ABC) transporter. We found that the CER5 gene encodes an ABC transporter localized in the plasma membrane of epidermal cells and conclude that it is required for wax export to the cuticle.

  4. ABC transporters involved in export of cell surface glycoconjugates.

    PubMed

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-09-01

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

  5. ABC Transporters Involved in Export of Cell Surface Glycoconjugates

    PubMed Central

    Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

    2010-01-01

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

  6. Placental ABC transporters, cellular toxicity and stress in pregnancy.

    PubMed

    Aye, Irving L M H; Keelan, Jeffrey A

    2013-04-25

    The human placenta, in addition to its roles as a nutrient transfer and endocrine organ, functions as a selective barrier to protect the fetus against the harmful effects of exogenous and endogenous toxins. Members of the ATP-binding cassette (ABC) family of transport proteins limit the entry of xenobiotics into the fetal circulation via vectorial efflux from the placenta to the maternal circulation. Several members of the ABC family, including proteins from the ABCA, ABCB, ABCC and ABCG subfamilies, have been shown to be functional in the placenta with clinically significant roles in xenobiotic efflux. However, recent findings suggest that these transporters also protect placental tissue by preventing the cellular accumulation of cytotoxic compounds such as lipids, sterols and their derivatives. Such protective functions are likely to be particularly important in pregnancies complicated by inflammatory or oxidative stress, where the generation of toxic metabolites is enhanced. For example, ABC transporters have been shown to protect against the harmful effects of hypoxia and oxidative stress through increased expression and efflux of oxysterols and glutathione conjugated xenobiotics. However, this protective capacity may be diminished in response to the same stressors. Several studies in primary human trophoblast cells and animal models have demonstrated decreased expression and activity of placental ABC transporters with inflammatory, oxidative or metabolic stress. Several clinical studies in pregnancies complicated by inflammatory conditions such as preeclampsia and gestational diabetes support these findings, although further studies are required to determine the clinical relevance of the relationships between placental ABC transporter expression and activity, and placental function in stressed pregnancies. Such studies are necessary to fully understand the consequences of pregnancy disorders on placental function and viability in order to optimise pregnancy

  7. 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

  8. 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.

  9. 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

  10. 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

  11. Complex inheritance of ABCA4 disease: four mutations in a family with multiple macular phenotypes.

    PubMed

    Lee, Winston; Xie, Yajing; Zernant, Jana; Yuan, Bo; Bearelly, Srilaxmi; Tsang, Stephen H; Lupski, James R; Allikmets, Rando

    2016-01-01

    Over 800 mutations in the ABCA4 gene cause autosomal recessive Stargardt disease. Due to extensive genetic heterogeneity, observed variant-associated phenotypes can manifest tremendous variability of expression. Furthermore, the high carrier frequency of pathogenic ABCA4 alleles in the general population (~1:20) often results in pseudo-dominant inheritance patterns further complicating the diagnosis and characterization of affected individuals. This study describes a genotype/phenotype analysis of an unusual family with multiple macular disease phenotypes spanning across two generations and segregating four distinct ABCA4 mutant alleles. Complete sequencing of ABCA4 discovered two known missense mutations, p.C54Y and p.G1961E. Array comparative genomic hybridization revealed a large novel deletion combined with a small insertion, c.6148-698_c.6670del/insTGTGCACCTCCCTAG, and complete sequencing of the entire ABCA4 genomic locus uncovered a new deep intronic variant, c.302+68C>T. Patients with the p.G1961E mutation had the mildest, confined maculopathy phenotype with peripheral flecks while those with all other mutant allele combinations exhibited a more advanced stage of generalized retinal and choriocapillaris atrophy. This family epitomizes the clinical and genetic complexity of ABCA4-associated diseases. It contained variants from all classes of mutations, in the coding region, deep intronic, both single nucleotide variants and copy number variants that accounted for varying phenotypes segregating in an apparent dominant fashion. Unequivocally defining disease-associated alleles in the ABCA4 locus requires a multifaceted approach that includes advanced mutation detection methods and a thorough analysis of clinical phenotypes.

  12. Class C ABC transporters and Saccharomyces cerevisiae vacuole fusion

    PubMed Central

    Sasser, Terry L; Fratti, Rutilio A

    2014-01-01

    Membrane fusion is carried out by core machinery that is conserved throughout eukaryotes. This is comprised of Rab GTPases and their effectors, and SNARE proteins, which together are sufficient to drive the fusion of reconstituted proteoliposomes. However, an outer layer of factors that are specific to individual trafficking pathways in vivo regulates the spatial and temporal occurrence of fusion. The homotypic fusion of Saccharomyces cerevisiae vacuolar lysosomes utilizes a growing set of factors to regulate the fusion machinery that include members of the ATP binding cassette (ABC) transporter family. Yeast vacuoles have five class C ABC transporters that are known to transport a variety of toxins into the vacuole lumen as part of detoxifying the cell. We have found that ABCC transporters can also regulate vacuole fusion through novel mechanisms. For instance Ybt1 serves as negative regulator of fusion through its effects on vacuolar Ca2+ homeostasis. Additional studies showed that Ycf1 acts as a positive regulator by affecting the efficient recruitment of the SNARE Vam7. Finally, we discuss the potential interface between the translocation of lipids across the membrane bilayer, also known as lipid flipping, and the efficiency of fusion. PMID:25610719

  13. Mammalian P4-ATPases and ABC transporters and their role in phospholipid transport.

    PubMed

    Coleman, Jonathan A; Quazi, Faraz; Molday, Robert S

    2013-03-01

    Transport of phospholipids across cell membranes plays a key role in a wide variety of biological processes. These include membrane biosynthesis, generation and maintenance of membrane asymmetry, cell and organelle shape determination, phagocytosis, vesicle trafficking, blood coagulation, lipid homeostasis, regulation of membrane protein function, apoptosis, etc. P(4)-ATPases and ATP binding cassette (ABC) transporters are the two principal classes of membrane proteins that actively transport phospholipids across cellular membranes. P(4)-ATPases utilize the energy from ATP hydrolysis to flip aminophospholipids from the exocytoplasmic (extracellular/lumen) to the cytoplasmic leaflet of cell membranes generating membrane lipid asymmetry and lipid imbalance which can induce membrane curvature. Many ABC transporters play crucial roles in lipid homeostasis by actively transporting phospholipids from the cytoplasmic to the exocytoplasmic leaflet of cell membranes or exporting phospholipids to protein acceptors or micelles. Recent studies indicate that some ABC proteins can also transport phospholipids in the opposite direction. The importance of P(4)-ATPases and ABC transporters is evident from the findings that mutations in many of these transporters are responsible for severe human genetic diseases linked to defective phospholipid transport. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

  14. 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.

  15. PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters in Vivo

    PubMed Central

    Mairinger, Severin; Erker, Thomas; Müller, Markus; Langer, Oliver

    2013-01-01

    Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer’s and Parkinson’s disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications. PMID:21434859

  16. Modulation of Expression and Activity of ABC Transporters by the Phytoestrogen Genistein. Impact on Drug Disposition.

    PubMed

    Rigalli, Juan Pablo; Ciriaci, Nadia; Mottino, Aldo Domingo; Catania, Viviana Alicia; Ruiz, María Laura

    2016-01-01

    ATP binding cassette (ABC) transporters are involved in drug absorption, distribution and elimination. They also mediate multidrug resistance in cancer cells. Isoflavones, such as genistein (GNT), belong to a class of naturally-occurring compounds found at high concentrations in commonly consumed soya based-foods and dietary supplements. GNT and its metabolites interact with ABC transporters as substrates, inhibitors and/or modulators of their expression. This review compiles information about regulation of ABC transporters by GNT with special emphasis on the three major groups of ABC transporters involved in excretion of endo- and xenobiotics as follows: Pglycoprotein (MDR1, ABCB1), a group of multidrug resistance associated proteins (MRPs, ABCC subfamily) and ABCG2 (BCRP), an ABC half-transporter. The impact of these regulations on potential GNT-drug interactions is further considered. PMID:27048380

  17. Structural and Genetic Assessment of the ABCA4-Associated Optical Gap Phenotype

    PubMed Central

    Nõupuu, Kalev; Lee, Winston; Zernant, Jana; Tsang, Stephen H.; Allikmets, Rando

    2014-01-01

    Purpose. To investigate the developmental stages and genetic etiology of the optical gap phenotype in recessive Stargardt disease (STGD1). Methods. Single and longitudinal data points from 15 patients, including four sibling pairs, exhibiting an optical gap phenotype on spectral-domain optical coherence tomography (SD-OCT) with confirmed disease-causing ABCA4 alleles were retrospectively analyzed. Fundus images with corresponding SD-OCT scans were collected with a confocal scanning laser ophthalmoscope. Structural phenotypes were assigned to three developmental stages according to SD-OCT. The ABCA4 gene was screened in all patients. Results. At least two disease-causing ABCA4 variants where identified in each patient; all except one (91%) were compound heterozygous for the p.G1961E mutation. All patients exhibited structural findings on SD-OCT that grouped into three progressive developmental stages over several years. Stage 1 was characterized by mild disruptions of the ellipsoid zone (EZ) band over the fovea. Stage 2 was a progressive expansion of the EZ band loss resulting in an empty lesion devoid of photoreceptors. Stage 3 observed a structural collapse of the inner retinal layers into the optical gap space leading to involvement and atrophy of the RPE thereafter. Conclusions. The optical gap phenotype in STGD1 can be structurally divided into three progressive stages spanning several years. This particular phenotype also appears to be highly associated with the p.G1961E mutation of ABCA4. Taken together, it appears that a focal loss of photoreceptors sequentially precedes RPE dysfunction in the early development of ABCA4-associated optical gap lesions. PMID:25301883

  18. 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

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

    PubMed Central

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

    2013-01-01

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

  20. 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

  1. Transcription factors that mediate epithelial–mesenchymal transition lead to multidrug resistance by upregulating ABC transporters

    PubMed Central

    Saxena, M; Stephens, M A; Pathak, H; Rangarajan, A

    2011-01-01

    Development of multidrug resistance (MDR) is a major deterrent in the effective treatment of metastatic cancers by chemotherapy. Even though MDR and cancer invasiveness have been correlated, the molecular basis of this link remains obscure. We show here that treatment with chemotherapeutic drugs increases the expression of several ATP binding cassette transporters (ABC transporters) associated with MDR, as well as epithelial–mesenchymal transition (EMT) markers, selectively in invasive breast cancer cells, but not in immortalized or non-invasive cells. Interestingly, the mere induction of an EMT in immortalized and non-invasive cell lines increased their expression of ABC transporters, migration, invasion, and drug resistance. Conversely, reversal of EMT in invasive cells by downregulating EMT-inducing transcription factors reduced their expression of ABC transporters, invasion, and rendered them more chemosensitive. Mechanistically, we demonstrate that the promoters of ABC transporters carry several binding sites for EMT-inducing transcription factors, and overexpression of Twist, Snail, and FOXC2 increases the promoter activity of ABC transporters. Furthermore, chromatin immunoprecipitation studies revealed that Twist binds directly to the E-box elements of ABC transporters. Thus, our study identifies EMT inducers as novel regulators of ABC transporters, thereby providing molecular insights into the long-standing association between invasiveness and MDR. Targeting EMT transcription factors could hence serve as novel strategies to curb both metastasis and the associated drug resistance. PMID:21734725

  2. 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

  3. Nitrite Transport Activity of the ABC-Type Cyanate Transporter of the Cyanobacterium Synechococcus elongatus▿

    PubMed Central

    Maeda, Shin-ichi; Omata, Tatsuo

    2009-01-01

    In addition to the ATP-binding cassette (ABC)-type nitrate/nitrite-bispecific transporter, which has a high affinity for both substrates (Km, ∼1 μM), Synechococcus elongatus has an active nitrite transport system with an apparent Km (NO2−) value of 20 μM. We found that this activity depends on the cynABD genes, which encode a putative cyanate (NCO−) ABC-type transporter. Accordingly, nitrite transport by CynABD was competitively inhibited by NCO− with a Ki value of 0.025 μM. The transporter was induced under conditions of nitrogen deficiency, and the induced cells showed a Vmax value of 11 to 13 μmol/mg of chlorophyll per h for cyanate or nitrite, which could supply ∼30% of the amount of nitrogen required for optimum growth. Its relative specificity for the substrates and regulation at transcriptional and posttranslational levels suggested that the physiological role of the bispecific cyanate/nitrite transporter in S. elongatus is to allow nitrogen-deficient cells to assimilate low concentrations of cyanate in the medium. Its contribution to nitrite assimilation was significant in a mutant lacking the ABC-type nitrate/nitrite transporter, suggesting a possible role for CynABD in nitrite assimilation by cyanobacterial species that lack another high-affinity mechanism(s) for nitrite transport. PMID:19286804

  4. 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

  5. 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....

  6. 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

  7. The role of ABC transporters in drug resistance, metabolism and toxicity.

    PubMed

    Glavinas, Hristos; Krajcsi, Péter; Cserepes, Judit; Sarkadi, Balázs

    2004-01-01

    ATP Binding Cassette (ABC) transporters form a special family of membrane proteins, characterized by homologous ATP-binding, and large, multispanning transmembrane domains. Several members of this family are primary active transporters, which significantly modulate the absorption, metabolism, cellular effectivity and toxicity of pharmacological agents. This review provides a general overview of the human ABC transporters, their expression, localization and basic mechanism of action. Then we shortly deal with the human ABC transporters as targets of therapeutic interventions in medicine, including cancer drug resistance, lipid and other metabolic disorders, and even gene therapy applications. We place a special emphasis on the three major groups of ABC transporters involved in cancer multidrug resistance (MDR). These are the classical P-glycoprotein (MDR1, ABCB1), the multidrug resistance associated proteins (MRPs, in the ABCC subfamily), and the ABCG2 protein, an ABC half-transporter. All these proteins catalyze an ATP-dependent active transport of chemically unrelated compounds, including anticancer drugs. MDR1 (P-glycoprotein) and ABCG2 preferentially extrude large hydrophobic, positively charged molecules, while the members of the MRP family can extrude both hydrophobic uncharged molecules and water-soluble anionic compounds. Based on the physiological expression and role of these transporters, we provide examples for their role in Absorption-Distribution-Metabolism-Excretion (ADME) and toxicology, and describe several basic assays which can be applied for screening drug interactions with ABC transporters in the course of drug research and development.

  8. Crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus.

    PubMed

    Manjula, M; Pampa, K J; Kumar, S M; Mukherjee, S; Kunishima, N; Rangappa, K S; Lokanath, N K

    2015-03-27

    The ATP binding cassette (ABC) transporters, represent one of the largest superfamilies of primary transporters, which are very essential for various biological functions. The crystal structure of ATP-binding subunit of an ABC transporter from Geobacillus kaustophilus has been determined at 1.77 Å resolution. The crystal structure revealed that the protomer has two thick arms, (arm I and II), which resemble 'L' shape. The ATP-binding pocket is located close to the end of arm I. ATP molecule is docked into the active site of the protein. The dimeric crystal structure of ATP-binding subunit of ABC transporter from G. kaustophilus has been compared with the previously reported crystal structure of ATP-binding subunit of ABC transporter from Salmonella typhimurium.

  9. 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

  10. GintABC1 encodes a putative ABC transporter of the MRP subfamily induced by Cu, Cd, and oxidative stress in Glomus intraradices.

    PubMed

    González-Guerrero, Manuel; Benabdellah, Karim; Valderas, Ascensión; Azcón-Aguilar, Concepción; Ferrol, Nuria

    2010-02-01

    A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintABC1) was isolated from the extraradical mycelia of the arbuscular mycorrhizal fungus Glomus intraradices. Bioinformatic analysis of the sequence indicated that GintABC1 encodes a 1513 amino acid polypeptide, containing two six-transmembrane clusters (TMD) intercalated with sequences characteristics of the nucleotide binding domains (NBD) and an extra N-terminus extension (TMD0). GintABC1 presents a predicted TMD0-(TMD-NBD)(2) topology, typical of the multidrug resistance-associated protein subfamily of ABC transporters. Gene expression analyses revealed no difference in the expression levels of GintABC1 in the extra- vs the intraradical mycelia. GintABC1 was up-regulated by Cd and Cu, but not by Zn, suggesting that this transporter might be involved in Cu and Cd detoxification. Paraquat, an oxidative agent, also induced the transcription of GintABC1. These data suggest that redox changes may be involved in the transcriptional regulation of GintABC1 by Cd and Cu.

  11. Control of Plasma Membrane Permeability by ABC Transporters

    PubMed Central

    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

    2014-01-01

    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

  12. Presentation of Complex Homozygous Allele in ABCA4 Gene in a Patient with Retinitis Pigmentosa.

    PubMed

    Audere, Māreta; Rutka, Katrīna; Šepetiene, Svetlana; Lāce, Baiba

    2015-01-01

    Retinitis pigmentosa is a degenerative retinal disease characterized by progressive photoreceptor damage, which causes loss of peripheral and night vision and the development of tunnel vision and may result in loss of central vision. This study describes a patient with retinitis pigmentosa caused by a mutation in the ABCA4 gene with complex allele c.1622T>C, p.L541P; c.3113C>T, p.A1038V in homozygous state. PMID:26229699

  13. Clinical and genetic analyses reveal novel pathogenic ABCA4 mutations in Stargardt disease families

    PubMed Central

    Lin, Bing; Cai, Xue-Bi; Zheng, Zhi-Li; Huang, Xiu-Feng; Liu, Xiao-Ling; Qu, Jia; Jin, Zi-Bing

    2016-01-01

    Stargardt disease (STGD1) is a juvenile macular degeneration predominantly inherited in an autosomal recessive pattern, characterized by decreased central vision in the first 2 decades of life. The condition has a genetic basis due to mutation in the ABCA4 gene, and arises from the deposition of lipofuscin-like substance in the retinal pigmented epithelium (RPE) with secondary photoreceptor cell death. In this study, we describe the clinical and genetic features of Stargardt patients from four unrelated Chinese cohorts. The targeted exome sequencing (TES) was carried out in four clinically confirmed patients and their family members using a gene panel comprising 164 known causative inherited retinal dystrophy (IRD) genes. Genetic analysis revealed eight ABCA4 mutations in all of the four pedigrees, including six mutations in coding exons and two mutations in adjacent intronic areas. All the affected individuals showed typical manifestations consistent with the disease phenotype. We disclose two novel ABCA4 mutations in Chinese patients with STGD disease, which will expand the existing spectrum of disease-causing variants and will further aid in the future mutation screening and genetic counseling, as well as in the understanding of phenotypic and genotypic correlations. PMID:27739528

  14. Multidrug-Resistance Transporter AbcA Secretes Staphylococcus aureus Cytolytic Toxins.

    PubMed

    Yoshikai, Hirono; Kizaki, Hayato; Saito, Yuki; Omae, Yosuke; Sekimizu, Kazuhisa; Kaito, Chikara

    2016-01-15

    Phenol-soluble modulins (PSMs) are Staphylococcus aureus cytolytic toxins that lyse erythrocytes and neutrophils and have important functions in the S. aureus infectious process. The molecular mechanisms of PSM secretion, however, are not well understood. Here we report that knockout of the multidrug-resistance ABC transporter AbcA, which contributes to S. aureus resistance against antibiotics and chemicals, diminished the secreted amount of PSM, leading to the accumulation of PSM in the intracellular fraction. The amount of PSM in the culture supernatants of the abcA knockout mutants was restored by introduction of the wild-type abcA gene, whereas it was not completely restored by introduction of mutant abcA genes encoding AbcA mutant proteins carrying amino acid substitutions in the adenosine triphosphate binding motifs. The abcA knockout mutant exhibited attenuated virulence in a mouse systemic infection model. These findings suggest that the multidrug resistance transporter AbcA secretes PSMs and contributes to S. aureus virulence.

  15. Multidrug-Resistance Transporter AbcA Secretes Staphylococcus aureus Cytolytic Toxins.

    PubMed

    Yoshikai, Hirono; Kizaki, Hayato; Saito, Yuki; Omae, Yosuke; Sekimizu, Kazuhisa; Kaito, Chikara

    2016-01-15

    Phenol-soluble modulins (PSMs) are Staphylococcus aureus cytolytic toxins that lyse erythrocytes and neutrophils and have important functions in the S. aureus infectious process. The molecular mechanisms of PSM secretion, however, are not well understood. Here we report that knockout of the multidrug-resistance ABC transporter AbcA, which contributes to S. aureus resistance against antibiotics and chemicals, diminished the secreted amount of PSM, leading to the accumulation of PSM in the intracellular fraction. The amount of PSM in the culture supernatants of the abcA knockout mutants was restored by introduction of the wild-type abcA gene, whereas it was not completely restored by introduction of mutant abcA genes encoding AbcA mutant proteins carrying amino acid substitutions in the adenosine triphosphate binding motifs. The abcA knockout mutant exhibited attenuated virulence in a mouse systemic infection model. These findings suggest that the multidrug resistance transporter AbcA secretes PSMs and contributes to S. aureus virulence. PMID:26160745

  16. 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

  17. 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

  18. 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

  19. On the energy-dependence of Hoechst 33342 transport by the ABC transporter LmrA.

    PubMed

    Venter, Henrietta; Velamakanni, Saroj; Balakrishnan, Lekshmy; van Veen, Hendrik W

    2008-02-15

    LmrA is an ATP-binding cassette (ABC) multidrug transporter from Lactococcus lactis, and is a structural homologue of the human multidrug resistance P-glycoprotein (ABCB1), the overexpression of which is associated with multidrug resistance in tumours. We recently observed that a truncated version of LmrA lacking the nucleotide-binding domain mediates a proton motive force-dependent ethidium transport reaction by catalyzing proton-ethidium symport. This finding raised the question whether proton motive force-dependent transport can also be observed for other drugs, and whether this reaction is also relevant for full-length LmrA. Furthermore, the observations on LmrA-MD raised the question whether ATP-dependent transport by LmrA in intact cells could be due to the activity of independent ABC transporters that might become upregulated in the lactococcal cells due to the overexpression of LmrA; the recently identified ABC multidrug transporter LmrCD was put forward as a possible candidate. Here, we investigated the energy coupling to the transport of the amphiphilic dye Hoechst 33342 in proteoliposomes containing purified LmrA. For this purpose, LmrA was obtained from lactococcal cells lacking the genomic lmrA and lmrCD genes, in which LmrA was expressed from a plasmid. To separate ATP-dependence from proton motive force-dependence, we also used mutant LmrA proteins, which were affected in their ability to hydrolyse ATP. Our studies in proteoliposomes demonstrate that LmrA can catalyze Hoechst 33342 transport independent of auxiliary proteins, in an ATP-dependent fashion and a transmembrane chemical proton gradient (interior acidic)-dependent fashion.

  20. Multidrug resistance-associated ABC transporters - too much of one thing, good for nothing.

    PubMed

    Prochazkova, Jirina; Lanova, Martina; Pachernik, Jiri

    2012-08-01

    Abstract Overexpression of ATP-binding cassette (ABC) transporters in cancer cells results in multidrug resistance (MDR) which leads to unsuccessful chemotherapy. The most important MDR-associated members of ABC superfamily are ABC B1/P-glycoprotein/MDR1, ABC C1/multidrug resistance associated protein 1 (MRP1), and ABC G2/BCRP. This study is not only focused on function, substrates, and localization of these popular proteins but also on other ABC C family members such as ABC C2-6/MRP2-6 and ABC C7/CFTR. Current research is mainly oriented on the cancer-promoting role of these proteins, but important lessons could also be learned from the physiological roles of these proteins or from polymorphisms affecting their function. Thorough knowledge of structure and detailed mechanism of efflux can aid in the discovery of new chemotherapy targets in the future. Although the best way on how to deal with MDR would be to prevent its development, we describe some new promising strategies on how to conquer both inherited and induced MDRs.

  1. 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

  2. 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

  3. 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

  4. Inhibition or knockdown of ABC transporters enhances susceptibility of adult and juvenile schistosomes to Praziquantel.

    PubMed

    Kasinathan, Ravi S; Sharma, Lalit Kumar; Cunningham, Charles; Webb, Thomas R; Greenberg, Robert M

    2014-10-01

    Parasitic flatworms of the genus Schistosoma cause schistosomiasis, a neglected tropical disease that affects hundreds of millions. Treatment of schistosomiasis depends almost entirely on the drug praziquantel (PZQ). Though essential to treating and controlling schistosomiasis, a major limitation of PZQ is that it is not active against immature mammalian-stage schistosomes. Furthermore, there are reports of field isolates with heritable reductions in PZQ susceptibility, and researchers have selected for PZQ-resistant schistosomes in the laboratory. P-glycoprotein (Pgp; ABCB1) and other ATP binding cassette (ABC) transporters remove a wide variety of toxins and xenobiotics from cells, and have been implicated in multidrug resistance (MDR). Changes in ABC transporter structure or expression levels are also associated with reduced drug susceptibility in parasitic helminths, including schistosomes. Here, we show that the activity of PZQ against schistosome adults and juveniles ex vivo is potentiated by co-administration of either the highly potent Pgp inhibitor tariquidar or combinations of inhibitors targeting multiple ABC multidrug transporters. Adult worms exposed to sublethal PZQ concentrations remain active, but co-administration of ABC transporter inhibitors results in complete loss of motility and disruption of the tegument. Notably, juvenile schistosomes (3-4 weeks post infection), normally refractory to 2 µM PZQ, become paralyzed when transporter inhibitors are added in combination with the PZQ. Experiments using the fluorescent PZQ derivative (R)-PZQ-BODIPY are consistent with the transporter inhibitors increasing effective intraworm concentrations of PZQ. Adult worms in which expression of ABC transporters has been suppressed by RNA interference show increased responsiveness to PZQ and increased retention of (R)-PZQ-BODIPY consistent with an important role for these proteins in setting levels of PZQ susceptibility. These results indicate that parasite ABC

  5. Single liposome analysis of peptide translocation by the ABC transporter TAPL

    PubMed Central

    Zollmann, Tina; Moiset, Gemma; Tumulka, Franz; Tampé, Robert; Poolman, Bert; Abele, Rupert

    2015-01-01

    ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce. We reconstituted the human lysosomal polypeptide ABC transporter TAPL, expressed in Pichia pastoris, into lipid vesicles (liposomes) and performed explicit transport measurements. We analyzed solute transport at the single liposome level by monitoring the coincident fluorescence of solutes and proteoliposomes in the focal volume of a confocal microscope. We determined a turnover number of eight peptides per minute, which is two orders of magnitude higher than previously estimated from macroscopic measurements. Moreover, we show that TAPL translocates peptides against a large concentration gradient. Maximal filling is not limited by an electrochemical gradient but by trans-inhibition. Countertransport and reversibility studies demonstrate that peptide translocation is a strictly unidirectional process. Altogether, these data are included in a refined model of solute transport by ABC exporters. PMID:25646430

  6. 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...

  7. 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

  8. 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.

  9. Watching conformational dynamics of ABC transporters with single-molecule tools.

    PubMed

    Husada, Florence; Gouridis, Giorgos; Vietrov, Ruslan; Schuurman-Wolters, Gea K; Ploetz, Evelyn; de Boer, Marijn; Poolman, Bert; Cordes, Thorben

    2015-10-01

    ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

  10. An ABC pleiotropic drug resistance transporter of Fusarium graminearum with a role in crown and root diseases of wheat.

    PubMed

    Gardiner, Donald M; Stephens, Amber E; Munn, Alan L; Manners, John M

    2013-11-01

    FgABC1 (FGSG_04580) is predicted to encode a pleiotropic drug resistance class ABC transporter in Fusarium graminearum, a globally important pathogen of wheat. Deletion mutants of FgABC1 showed reduced virulence towards wheat in crown and root infection assays but were unaltered in infectivity on barley. Expression of FgABC1 during head blight and crown rot disease increases during the necrotrophic phases of infection suggestive of a role for FgABC1 in late infection stages in different tissue types. Deletion of FgABC1 also led to increased sensitivity of the fungus to the antifungal compound benalaxyl in culture, but the response to known cereal defence compounds, gramine, 2-benzoxazalinone and tryptamine was unaltered. FgABC1 appears to have a role in protecting the fungus from antifungal compounds and is likely to help combat as yet unidentified wheat defence compounds during disease development.

  11. 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

  12. Generating Symmetry in the Asymmetric ATP-binding Cassette (ABC) Transporter Pdr5 from Saccharomyces cerevisiae*

    PubMed Central

    Gupta, Rakeshkumar P.; Kueppers, Petra; Hanekop, Nils; Schmitt, Lutz

    2014-01-01

    Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely “regenerated” Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters. PMID:24733388

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

    PubMed

    Aryal, Bibek; Laurent, Christophe; Geisler, Markus

    2015-10-01

    The ABC (ATP-binding cassette) transporter family in higher plants is highly expanded compared with those of mammalians. Moreover, some members of the plant ABC subfamily B (ABCB) display very high substrate specificity compared with their mammalian counterparts that are often associated with multi-drug resistance 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 plant Arabidopsis reveals a surprisingly high degree of similarity. Both physically interact with orthologues of the FK506-binding proteins that chaperon both transporters to the plasma membrane in an action that seems to involve heat shock protein (Hsp)90. Further, both transporters are phosphorylated at regulatory domains that connect both nt-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:26517911

  14. How much do we know about drug handling by SLC and ABC drug transporters in children?

    PubMed

    Nigam, S K; Bhatnagar, V

    2013-07-01

    Although solute carrier (SLC) and ATP-binding cassette (ABC) transporters are critical to the absorption, distribution, and elimination of many small-molecule drugs in children, how these transporters regulate pediatric drug handling remains unclear. For proper dosing and to diminish toxicity, we need a better understanding of how organ development and functional maturation, as well as developmental changes in systemic physiology, impact transporter-mediated drug handling at pediatric developmental stages from the preterm infant through adolescence.

  15. 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...

  16. 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

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

    PubMed

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

    2014-07-15

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

  18. 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.

  19. An ABC transporter in the mitochondrial inner membrane is required for normal growth of yeast.

    PubMed Central

    Leighton, J; Schatz, G

    1995-01-01

    In an attempt to identify a mitochondrial ATP binding cassette (ABC) transporter, we have used the polymerase chain reaction to amplify 10 DNA fragments homologous to members of the ABC family from the yeast Saccharomyces cerevisiae. We disrupted five of the corresponding genes and found that one of the resulting null mutants barely grew on rich medium and failed to grow on minimal medium. This gene, termed ATM1, encodes a putative 'half-transporter' of 694 amino acids. Atm1p is synthesized with an N-terminal mitochondrial matrix-targeting signal and is located in the mitochondrial inner membrane, with its C-terminal ATPase domain exposed to the matrix. Cells lacking a functional ATM1 gene have an unstable mitochondrial genome and have white mitochondria that completely lack cytochromes. Atm1p is the first mitochondrial member of the ABC family to be identified and the only eukaryotic ABC transporter that has been shown to be necessary for normal cellular growth. Images PMID:7828591

  20. Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain barrier

    PubMed Central

    Miller, David S.

    2010-01-01

    ATP-binding cassette (ABC) transporters are important, selective elements of the blood-brain barrier. They line the luminal plasma membrane of the brain capillary endothelium, facing the vascular space, both protecting the CNS from entry of neurotoxicants and limiting access of therapeutic drugs to the brain parenchyma. Recent studies highlight the multiple signaling pathways through which the expression and activity of P-glycoprotein and other ABC transporters are modulated in response to xenobiotics, stress and disease. They show that increased transporter expression occurs in response to signals that activate specific transcription factors including, PXR, CAR, NF-κB and AP-1, and reduced transporter activity occurs rapidly and reversibly in response to signaling through Src kinase, protein kinase C and estrogen receptors. A detailed understanding of such regulation can provide the basis for improved neuroprotection and enhanced therapeutic drug delivery to the brain. PMID:20417575

  1. Putative ABC transporter responsible for acetic acid resistance in Acetobacter aceti.

    PubMed

    Nakano, Shigeru; Fukaya, Masahiro; Horinouchi, Sueharu

    2006-01-01

    Two-dimensional gel electrophoretic analysis of the membrane fraction of Acetobacter aceti revealed the presence of several proteins that were produced in response to acetic acid. A 60-kDa protein, named AatA, which was mostly induced by acetic acid, was prepared; aatA was cloned on the basis of its NH2-terminal amino acid sequence. AatA, consisting of 591 amino acids and containing ATP-binding cassette (ABC) sequences and ABC signature sequences, belonged to the ABC transporter superfamily. The aatA mutation with an insertion of the neomycin resistance gene within the aatA coding region showed reduced resistance to acetic acid, formic acid, propionic acid, and lactic acid, whereas the aatA mutation exerted no effects on resistance to various drugs, growth at low pH (adjusted with HCl), assimilation of acetic acid, or resistance to citric acid. Introduction of plasmid pABC101 containing aatA under the control of the Escherichia coli lac promoter into the aatA mutant restored the defect in acetic acid resistance. In addition, pABC101 conferred acetic acid resistance on E. coli. These findings showed that AatA was a putative ABC transporter conferring acetic acid resistance on the host cell. Southern blot analysis and subsequent nucleotide sequencing predicted the presence of aatA orthologues in a variety of acetic acid bacteria belonging to the genera Acetobacter and Gluconacetobacter. The fermentation with A. aceti containing aatA on a multicopy plasmid resulted in an increase in the final yield of acetic acid.

  2. The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation

    PubMed Central

    Sasser, Terry L.; Padolina, Mark; Fratti, Rutilio A.

    2013-01-01

    Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca2+ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50 % relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. As vacuole fusion can be inhibited by high Ca2+ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function. PMID:22970809

  3. An overview of ABC and SLC drug transporter gene regulation.

    PubMed

    Chen, Qiu-Xia; Hu, Hai-Hong; Zhou, Quan; Yu, Ai-Ming; Zeng, Su

    2013-02-01

    Membrane transporters play a significant role in drug absorption, distribution and excretion, and they consequently affect the pharmacokinetics, efficacy and safety of a drug. Under certain circumstances, such as pathological processes or exposure to certain substances, the expression of drug transporters is modified in cells. Change in transporter expression and function may affect cellular drug disposition resulting in different drug responses. This raises a number of questions such as which drugs are likely to modulate the expression of drug transporters, what factors support this process, and which transporters are influenced in a particular situation. In this paper, we summarize recent findings to find an answer to these questions. Particularly, we present an overview of the transcription factors involved in the regulation of a given drug transporter, the signaling transduction pathways that contribute to drug transporter gene expression, and xenobiotics and endobiotics that initiate the processes.

  4. Structural basis for substrate specificity of an amino acid ABC transporter.

    PubMed

    Yu, Jie; Ge, Jingpeng; Heuveling, Johanna; Schneider, Erwin; Yang, Maojun

    2015-04-21

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate a variety of substrates, ranging from ions to macromolecules, either out of or into the cytosol (hence defined as importers or exporters, respectively). It has been demonstrated that ABC exporters and importers function through a common mechanism involving conformational switches between inward-facing and outward-facing states; however, the mechanism underlying their functions, particularly substrate recognition, remains elusive. Here we report the structures of an amino acid ABC importer Art(QN)2 from Thermoanaerobacter tengcongensis composed of homodimers each of the transmembrane domain ArtQ and the nucleotide-binding domain ArtN, either in its apo form or in complex with substrates (Arg, His) and/or ATPs. The structures reveal that the straddling of the TMDs around the twofold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD has a negatively charged pocket that together create a negatively charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.

  5. Estimation of Candida albicans ABC Transporter Behavior in Real-Time via Fluorescence.

    PubMed

    Szczepaniak, Joanna; Łukaszewicz, Marcin; Krasowska, Anna

    2015-01-01

    We present a fluorometric method for determining ABC transporter activity in the pathogenic fungus C. albicans during different growth phases and in response to glucose. The carbocyanine dye diS-C3(3) was previously used to monitor plasma membrane potentials and test the influence of surface-active compounds in membrane polarization. We used diS-C3(3) to show changes in fluorescence kinetics that reflect changes in the activity of ABC transporters in C. albicans growth. Cdr1-GFP fluorescence, revealed that Cdr1p relocates to the inside of the cell after the early-log growth phase. Addition of glucose to the cell suspension resulted in Cdr1p transporter expression in the CDR2-knockout strain. We confirmed the diS-C3(3) results by standard RT-PCR and Western blotting.

  6. 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.

  7. Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ.

    PubMed

    Gouridis, Giorgos; Schuurman-Wolters, Gea K; Ploetz, Evelyn; Husada, Florence; Vietrov, Ruslan; de Boer, Marijn; Cordes, Thorben; Poolman, Bert

    2015-01-01

    The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

  8. 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

  9. 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

  10. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  11. 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

  12. Clinico-Pathologic Function of Cerebral ABC Transporters – Implications for the Pathogenesis of Alzheimer’s Disease

    PubMed Central

    Pahnke, Jens; Wolkenhauer, Olaf; Krohn, Markus; Walker, Lary C.

    2009-01-01

    In recent years it has become evident that ABC transporters fulfill important barrier functions in normal organs and during disease processes. Most importantly, resistance to drugs in cancer cells led to intense oncological and pharmacological investigations in which researchers were able to highlight important pharmacological interactions of chemotherapeuticals with ABC transporter function. Recently, the development of neurodegenerative diseases and the maintenance of neuronal stem cells have been linked to the activity of ABC transporters. Here, we summarize findings from cell culture experiments, animal models and studies of patients with Alzheimer’s disease. Furthermore, we discuss pharmacological interactions and computational methods for risk assessment. PMID:18690837

  13. 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

  14. 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.

  15. 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

  16. A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase

    PubMed Central

    Morisaki, J. Hiroshi; Smith, Peter A.; Date, Shailesh V.; Kajihara, Kimberly K.; Truong, Chau Linda; Modrusan, Zora; Yan, Donghong; Kang, Jing; Xu, Min; Shah, Ishita M.; Mintzer, Robert; Kofoed, Eric M.; Cheung, Tommy K.; Arnott, David; Koehler, Michael F. T.; Heise, Christopher E.; Brown, Eric J.

    2016-01-01

    ABSTRACT The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro. These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB. Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo. PMID:27601569

  17. Substrate binding by a bacterial ABC transporter involved in polysaccharide export

    SciTech Connect

    Cuthbertson, Leslie; Kimber, Matthew S.; Whitfield, Chris

    2008-04-02

    ATP-binding-cassette (ABC) transporters are responsible for the export of a wide variety of cell-surface glycoconjugates in both Gram-positive and Gram-negative bacteria. These include the O-antigenic polysaccharide (O-PS) portion of lipopolysaccharide, a crucial virulence determinant in Gram-negative pathogens. O-PSs are synthesized by one of two fundamentally different pathways. Escherichia coli O serotypes O8 and O9a provide the prototype systems for studying O-PS export via ABC transporters. The transporter is composed of the transmembrane component Wzm and the nucleotide-binding component Wzt. Although the N-terminal domain of Wzt is a conventional ABC protein, the C-terminal domain of Wzt (C-Wzt) is a unique structural element that determines the specificity of the transporter for either the O8 or O9a O-PS. We show here that the two domains of Wzt can function when expressed as separate polypeptides; both are essential for export. In vitro, C-Wzt binds its cognate O-PS by recognizing a residue located at the nonreducing end of the polymer. The crystal structure of C-WztO9a is reported here and reveals a {beta} sandwich with an immunoglobulin-like topology that contains the O-PS-binding pocket. Substrate interactions with nucleotide-binding domains have been demonstrated in an ABC exporter previously. However, to our knowledge substrate binding by a discrete, cytoplasmic accessory domain in an extended nucleotide-binding domain polypeptide has not previously been demonstrated. Elucidation of the substrate-recognition system involved in O-PS export provides insight into the mechanism that coordinates polymer biosynthesis, termination, and export.

  18. Rescue of the Stargardt phenotype in Abca4 knockout mice through inhibition of vitamin A dimerization.

    PubMed

    Charbel Issa, Peter; Barnard, Alun R; Herrmann, Philipp; Washington, Ilyas; MacLaren, Robert E

    2015-07-01

    Stargardt disease, an ATP-binding cassette, subfamily A, member 4 (ABCA4)-related retinopathy, is a genetic condition characterized by the accelerated accumulation of lipofuscin in the retinal pigment epithelium, degeneration of the neuroretina, and loss of vision. No approved treatment exists. Here, using a murine model of Stargardt disease, we show that the propensity of vitamin A to dimerize is responsible for triggering the formation of the majority of lipofuscin and transcriptional dysregulation of genes associated with inflammation. Data further demonstrate that replacing vitamin A with vitamin A deuterated at the carbon 20 position (C20-D3-vitamin A) impedes the dimerization rate of vitamin A--by approximately fivefold for the vitamin A dimer A2E--and subsequent lipofuscinogenesis and normalizes the aberrant transcription of complement genes without impairing retinal function. Phenotypic rescue by C20-D3-vitamin A was also observed noninvasively by quantitative autofluorescence, an imaging technique used clinically, in as little as 3 months after the initiation of treatment, whereas upon interruption of treatment, the age-related increase in autofluorescence resumed. Data suggest that C20-D3-vitamin A is a clinically amiable tool to inhibit vitamin A dimerization, which can be used to determine whether slowing the dimerization of vitamin A can prevent vision loss caused by Stargardt disease and other retinopathies associated with the accumulation of lipofuscin in the retina.

  19. 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

  20. 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

  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. 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

  3. 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.

  4. 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

  5. Accelerated Accumulation of Lipofuscin Pigments in the RPE of a Mouse Model for ABCA4-Mediated Retinal Dystrophies following Vitamin A Supplementation

    PubMed Central

    Radu, Roxana A.; Yuan, Quan; Hu, Jane; Peng, Jennifer H.; Lloyd, Marcia; Nusinowitz, Steven; Bok, Dean; Travis, Gabriel H.

    2010-01-01

    Purpose Dietary supplementation with vitamin A is sometimes prescribed as a treatment for retinitis pigmentosa, a group of inherited retinal degenerations that cause progressive blindness. Loss-of-function mutations in the ABCA4 gene are responsible for a subset of recessive retinitis pigmentosa. Other mutant alleles of ABCA4 cause the related diseases, recessive cone-rod dystrophy, and recessive Stargardt macular degeneration. Mice with a knockout mutation in the abca4 gene massively accumulate toxic lipofuscin pigments in the retinal pigment epithelium. Treatment of these mice with fenretinide, an inhibitor of vitamin A delivery to the eye, blocks formation of these toxic pigments. Here the authors tested the hypothesis that dietary supplementation with vitamin A may accelerate lipofuscin pigment formation in abca4−/− mice. Methods Wild-type and abca4−/− mice were fed normal or vitamin A–supplemented diets. Tissues from these mice were analyzed biochemically for retinoids and lipofuscin pigments. Eyes from these mice were analyzed morphologically for lipofuscin in the retinal pigment epithelium and for degeneration of photoreceptors. Visual function in these mice was analyzed by electroretinography. Results Mice that received vitamin A supplementation had dramatically higher levels of retinyl esters in the liver and retinal pigment epithelium. Lipofuscin pigments were significantly increased by biochemical and morphologic analysis in wild-type and abca4−/− mice fed the vitamin A–supplemented diet. Photoreceptor degeneration was observed in 11-month-old albino, but not pigmented, abca4−/− mice on both diets. Conclusions Vitamin A supplementation should be avoided in patients with ABCA4 mutations or other retinal or macular dystrophies associated with lipofuscin accumulation in the retinal pigment epithelium. PMID:18515570

  6. 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.

  7. 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

  8. 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

  9. Structural analysis of bacterial ABC transporter inhibition by an antibody fragment.

    PubMed

    Ahuja, Shivani; Rougé, Lionel; Swem, Danielle L; Sudhamsu, Jawahar; Wu, Ping; Russell, Stephen J; Alexander, Mary Kate; Tam, Christine; Nishiyama, Mireille; Starovasnik, Melissa A; Koth, Christopher M

    2015-04-01

    Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment.

  10. 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

  11. 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

  12. Differential contributions of five ABC transporters to mutidrug resistance, antioxidion and virulence of Beauveria bassiana, an entomopathogenic fungus.

    PubMed

    Song, Ting-Ting; Zhao, Jing; Ying, Sheng-Hua; Feng, Ming-Guang

    2013-01-01

    Multidrug resistance (MDR) confers agrochemical compatibility to fungal cells-based mycoinsecticdes but mechanisms involved in MDR remain poorly understood for entomopathogenic fungi, which have been widely applied as biocontrol agents against arthropod pests. Here we characterized the functions of five ATP-binding cassette (ABC) transporters, which were classified to the subfamilies ABC-B (Mdr1), ABC-C (Mrp1) and ABC-G (Pdr1, Pdr2 and Pdr5) and selected from 54 full-size ABC proteins of Beauveria bassiana based on their main domain architecture, membrane topology and transcriptional responses to three antifungal inducers. Disruption of each transporter gene resulted in significant reduction in resistance to four to six of eight fungicides or antifungal drugs tested due to their differences in structure and function. Compared with wild-type and complemented (control) strains, disruption mutants of all the five transporter genes became significantly less tolerant to the oxidants menadione and H₂O₂ based on 22-41% and 10-31% reductions of their effective concentrations required for the suppression of 50% colony growth at 25°C. Under a standardized spray, the killing actions of ΔPdr5 and ΔMrp1 mutants against Spodoptera litura second-instar larvae were delayed by 59% and 33% respectively. However, no significant virulence change was observed in three other delta mutants. Taken together, the examined five ABC transporters contribute differentially to not only the fungal MDR but antioxidant capability, a phenotype rarely associated with ABC efflux pumps in previous reports; at least some of them are required for the full virulence of B. bassiana, thereby affecting the fungal biocontrol potential. Our results indicate that ABC pump-dependent MDR mechanisms exist in entomopathogenic fungi as do in yeasts and human and plant pathogenic fungi.

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

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

    PubMed

    Staud, Frantisek; Cerveny, Lukas; Ceckova, Martina

    2012-11-01

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

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

    PubMed Central

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

    2014-01-01

    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. PMID:25377891

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

    PubMed

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

    2002-11-01

    Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein ( MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers. PMID:12430019

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

    PubMed

    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

    2014-09-01

    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.

  2. 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.

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

    DOE PAGES

    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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Predicting Progression of ABCA4-Associated Retinal Degenerations Based on Longitudinal Measurements of the Leading Disease Front

    PubMed Central

    Cideciyan, Artur V.; Swider, Malgorzata; Schwartz, Sharon B.; Stone, Edwin M.; Jacobson, Samuel G.

    2015-01-01

    Purpose To evaluate the progression of the earliest stage of disease in ABCA4-associated retinal degenerations (RDs). Methods Near-infrared excited reduced-illuminance autofluorescence imaging was acquired across the retina up to 80 degrees eccentricity in 44 patients with two ABCA4 alleles. The eccentricity of the leading disease front (LDF) corresponding to the earliest stage of disease was measured along the four meridians. A mathematical model describing the expansion of the LDF was developed based on 6 years of longitudinal follow-up. Results The extent of LDF along the superior, inferior, and temporal meridians showed a wide spectrum from 3.5 to 70 degrees. In patients with longitudinal data, the average centrifugal expansion rate was 2 degrees per year. The nasal extent of LDF between the fovea and ONH ranged from 4.3 to 16.5 degrees and expanded at 0.35 degrees per year. The extent of LDF beyond ONH ranged from 19 to 75 degrees and expanded on average at 2 degrees per year. A mathematical model fit well to the longitudinal data describing the expansion of the LDF. Conclusions The eccentricity of the LDF in ABCA4-RD shows a continuum from parafovea to far periphery along all four meridians consistent with a wide spectrum of severity observed clinically. The model of progression may provide a quantitative prediction of the LDF expansion based on the age and eccentricity of the LDF at a baseline visit, and thus contribute significantly to the enrollment of candidates appropriate for clinical trials planning specific interventions, efficacy outcomes, and durations. PMID:26377081

  11. 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

  12. 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

  13. ABC Transporter Required for Intercellular Transfer of Developmental Signals in a Heterocystous Cyanobacterium

    PubMed Central

    Videau, Patrick; Rivers, Orion S.; Higa, Kelly C.

    2015-01-01

    ABSTRACT In the filamentous cyanobacterium Anabaena, patS and hetN encode peptide-derived signals with many of the properties of morphogens. These signals regulate the formation of a periodic pattern of heterocysts by lateral inhibition of differentiation. Here we show that intercellular transfer of the patS- and hetN-dependent developmental signals from heterocysts to vegetative cells requires HetC, a predicted ATP-binding cassette transporter (ABC transporter). Relative to the wild type, in a hetC mutant differentiation resulted in a reduced number of heterocysts that were incapable of nitrogen fixation, but deletion of patS or hetN restored heterocyst number and function in a hetC background. These epistasis results suggest that HetC is necessary for conferring self-immunity to the inhibitors on differentiating cells. Nine hours after induction of differentiation, HetC was required for neither induction of transcription of patS nor intercellular transfer of the patS-encoded signal to neighboring cells. Conversely, in strains lacking HetC, the patS- and hetN-encoded signals were not transferred from heterocyst cells to adjacent vegetative cells. The results support a model in which the patS-dependent signal is initially transferred between vegetative cells in a HetC-independent fashion, but some time before morphological differentiation of heterocysts is complete, transfer of both signals transitions to a HetC-dependent process. IMPORTANCE How chemical cues that regulate pattern formation in multicellular organisms move from one cell to another is a central question in developmental biology. In this study, we show that an ABC transporter, HetC, is necessary for transport of two developmental signals between different types of cells in a filamentous cyanobacterium. ABC transporters are found in organisms as diverse as bacteria and humans and, as the name implies, are often involved in the transport of molecules across a cellular membrane. The activity of HetC was

  14. Structure, biosynthesis, and function of bacterial capsular polysaccharides synthesized by ABC transporter-dependent pathways.

    PubMed

    Willis, Lisa M; Whitfield, Chris

    2013-08-30

    Bacterial capsules are formed primarily from long-chain polysaccharides with repeat-unit structures. A given bacterial species can produce a range of capsular polysaccharides (CPSs) with different structures and these help distinguish isolates by serotyping, as is the case with Escherichia coli K antigens. Capsules are important virulence factors for many pathogens and this review focuses on CPSs synthesized via ATP-binding cassette (ABC) transporter-dependent processes in Gram-negative bacteria. Bacteria utilizing this pathway are often associated with urinary tract infections, septicemia, and meningitis, and E. coli and Neisseria meningitidis provide well-studied examples. CPSs from ABC transporter-dependent pathways are synthesized at the cytoplasmic face of the inner membrane through the concerted action of glycosyltransferases before being exported across the inner membrane and translocated to the cell surface. A hallmark of these CPSs is a conserved reducing terminal glycolipid composed of phosphatidylglycerol and a poly-3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) linker. Recent discovery of the structure of this conserved lipid terminus provides new insights into the early steps in CPS biosynthesis.

  15. [Role of the ABC transporters A1 and G1, key reverse cholesterol transport proteins, in atherosclerosis].

    PubMed

    Demina, E P; Miroshnikova, V V; Schwarzman, A L

    2016-01-01

    Atherosclerosis is one of the most common causes of death worldwide. Epidemiology studies firmly established an inverse relationship between atherogenesis and distorted lipid metabolism, in particular, higher levels of total cholesterol, an accumulation of CH-laden macrophages (foam cells), and lower plasma levels of antiatherogenic high density lipoprotein (HDL). It is believed that the reverse cholesterol transport, a process that removes excess cholesterol from peripheral tissues/cells including macrophages to circulating HDL, is one of the main mechanisms responsible for anti-atherogenic properties of HDL. The key proteins of reverse cholesterol transport-ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1)-mediate the cholesterol efflux from macrophages and prevent their transformation into foam cells. This review focuses on the role of ABC transporters A1 and G1 in the pathogenesis of atherosclerosis.

  16. Overexpression, Membrane Preparation, and Purification of a Typical Multidrug ABC Transporter BmrA.

    PubMed

    Wiseman, Benjamin; Jault, Jean-Michel

    2016-01-01

    The production and purification is normally the first step in any biophysical or biochemical study of a new target protein. For membrane proteins, due to their generally low expression levels and hydrophobic properties this is often a major hurdle. Some multidrug transporters are members of one of the largest families of membrane proteins, the ABC ("ATP-binding cassette"), and are responsible for the uptake and export of a wide variety of molecules. This can lead to resistance when those molecules are antibiotics or chemotherapy drugs. To better understand their role in multidrug resistance pure and active protein is required. Here we outline a protocol to produce a highly pure and functionally active multidrug transporter BmrA that is suitable for use in biophysical and biochemical studies. We show that BmrA can be heterologously overexpressed in huge amount in E. coli and extracted from the membrane in a functionally active form. PMID:27485334

  17. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

  20. 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

  1. 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

  2. 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

  3. Identification of Novel Mutations in ABCA4 Gene: Clinical and Genetic Analysis of Indian Patients with Stargardt Disease

    PubMed Central

    Battu, Rajani; Verma, Anshuman; Hariharan, Ramesh; Krishna, Shuba; Kiran, Ravi; Jacob, Jemima; Ganapathy, Aparna; Ramprasad, Vedam L.; Kumaramanickavel, Govindasamy; Jeyabalan, Nallathambi; Ghosh, Arkasubhra

    2015-01-01

    Stargardt disease (STGD) is the leading cause of juvenile macular degeneration associated with progressive central vision loss, photophobia, and colour vision abnormalities. In this study, we have described the clinical and genetic features of Stargardt patients from an Indian cohort. The next generation sequencing was carried out in five clinically confirmed unrelated patients and their family members using a gene panel comprising 184 retinal specific genes. Sequencing results were analyzed by read mapping and variant calling in genes of interest, followed by their verification and interpretation. Genetic analysis revealed ABCA4 mutations in all of the five unrelated patients. Among these, four patients were found with compound heterozygous mutations and another one had homozygous mutation. All the affected individuals showed signs and symptoms consistent with the disease phenotype. We report two novel ABCA4 mutations in Indian patients with STGD disease, which expands the existing spectrum of disease-causing variants and the understanding of phenotypic and genotypic correlations. Screening for causative mutations in patients with STGD using panel of targeted gene sequencing by NGS would be a cost effective tool, might be helpful in confirming the precise diagnosis, and contributes towards the genetic counselling of asymptomatic carriers and isolated patients. PMID:25922843

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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

  9. Ontogeny of ABC and SLC transporters in the microvessels of developing rat brain.

    PubMed

    Soares, Ricardo V; Do, Tuan M; Mabondzo, Aloïse; Pons, Gérard; Chhun, Stéphanie

    2016-04-01

    The blood-brain barrier (BBB) is responsible for the control of solutes' concentration in the brain. Tight junctions and multiple ATP-binding cassette (ABC) and SoLute Carrier (SLC) efflux transporters protect brain cells from xenobiotics, therefore reducing brain exposure to intentionally administered drugs. In epilepsy, polymorphisms and overexpression of efflux transporters genes could be associated with pharmacoresistance. The ontogeny of these efflux transporters should also be addressed because their expression during development may be related to different brain exposure to antiepileptic drugs in the immature brain. We detected statistically significant higher expression of Abcb1b and Slc16a1 genes, and lower expression of Abcb1a and Abcg2 genes between the post-natal day 14 (P14) and the adult rat microvessels. P-gP efflux activity was also shown to be lower in P14 rats when compared with the adults. The P-gP proteins coded by rodent genes Abcb1a and Abcb1b are known to have different substrate affinities. The role of the Abcg2 gene is less clear in pharmacoresistance in epilepsy, nonetheless the coded protein Bcrp is frequently associated with drug resistance. Finally, we observed a higher expression of the Mct1 transporter gene in the P14 rat brain microvessels. Accordingly to our results, we suppose that age may be another factor influencing brain exposure to antiepileptics as a consequence of different expression patterns of efflux transporters between the adult and immature BBB.

  10. 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

  11. 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

  12. 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...

  13. Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

    PubMed

    Biswas, E E

    2001-07-27

    Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP > UTP. These

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

    PubMed

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

    2015-06-15

    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

  15. 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

  16. Macular Function in Macular Degenerations: Repeatability of Microperimetry as a Potential Outcome Measure for ABCA4-Associated Retinopathy Trials

    PubMed Central

    Swider, Malgorzata; Aleman, Tomas S.; Feuer, Willam J.; Schwartz, Sharon B.; Russell, Robert C.; Steinberg, Janet D.; Stone, Edwin M.; Jacobson, Samuel G.

    2012-01-01

    Purpose. To measure macular visual function in patients with unstable fixation, to define the photoreceptor source of this function, and to estimate its test-retest repeatability as a prerequisite to clinical trials. Methods. Patients (n = 38) with ABCA4-associated retinal degeneration (RD) or with retinitis pigmentosa (RP) were studied with retina-tracking microperimetry along the foveo-papillary profile between the fovea and the optic nerve head, and point-by-point test-retest repeatability was estimated. A subset with foveal fixation was also studied with dark-adapted projection perimetry using monochromatic blue and red stimuli along the horizontal meridian. Results. Macular function in ABCA4-RD patients transitioned from lower sensitivity at the parafovea to higher sensitivity in the perifovea. RP patients had the inverse pattern. Red-on-red microperimetric sensitivities successfully avoided ceiling effects and were highly correlated with absolute sensitivities. Point-by-point test-retest limits (95% confidence intervals) were ±4.2 dB; repeatability was not related to mean sensitivity, eccentricity from the fovea, age, fixation location, or instability. Repeatability was also not related to the local slope of sensitivity and was unchanged in the parapapillary retina. Conclusions. Microperimetry allows reliable testing of macular function in RD patients without foveal fixation in longitudinal studies evaluating natural disease progression or efficacy of therapeutic trials. A single estimate of test-retest repeatability can be used to determine significant changes in visual function at individual retinal loci within diseased regions that are homogeneous and those that are heterogeneous and also in transition zones at high risk for disease progression. PMID:22247458

  17. 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

  18. 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

  19. 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.

  20. Arbuscular mycorrhiza affects nickel translocation and expression of ABC transporter and metallothionein genes in Festuca arundinacea.

    PubMed

    Shabani, Leila; Sabzalian, Mohammad R; Mostafavi pour, Sodabeh

    2016-01-01

    Mycorrhizal fungi are key microorganisms for enhancing phytoremediation of soils contaminated with heavy metals. In this study, the effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae (=Glomus mosseae) on physiological and molecular mechanisms involved in the nickel (Ni) tolerance of tall fescue (Festuca arundinacea = Schedonorus arundinaceus) were investigated. Nickel addition had a pronounced negative effect on tall fescue growth and photosynthetic pigment contents, as well as on AMF colonization. Phosphorus content increased markedly in mycorrhizal plants (M) compared to non-inoculated (NM) ones. However, no significant difference was observed in root carbohydrate content between AMF-inoculated and non-inoculated plants. For both M and NM plants, Ni concentrations in shoots and roots increased according to the addition of the metal into soil, but inoculation with F. mosseae led to significantly lower Ni translocation from roots to the aboveground parts compared to non-inoculated plants. ABC transporter and metallothionein transcripts accumulated to considerably higher levels in tall fescue plants colonized by F. mosseae than in the corresponding non-mycorrhizal plants. These results highlight the importance of mycorrhizal colonization in alleviating Ni-induced stress by reducing Ni transport from roots to shoots of tall fescue plants.

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

    PubMed

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

    2013-12-03

    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.

  2. The role of ABC and SLC transporters in the pharmacokinetics of dietary and herbal phytochemicals and their interactions with xenobiotics.

    PubMed

    Li, Yan; Lu, Jun; Paxton, James W

    2012-06-01

    There is accumulating evidence that many compounds, known as phytochemicals (PCs), which are derived from dietary plants and herbs, may have a role in combating a number of chronic diseases. Despite many in vitro studies elucidating the mechanism(s) of action of various PCs, there are still reservations with regard to their health benefits in vivo, particularly as there is a paucity of research on their oral bioavailability, their pharmacokinetics, and the concentrations achieved at their site(s) of action. Recently various transporters, including the ATP-binding cassette (ABC) and the solute carrier (SLC) transporters, have been cloned and functional analyses have suggested that they play significant roles in the absorption and disposition of most drugs and PCs. While some SLC transporters facilitate absorption of PCs into the systemic circulation, various efflux pumps, including the ABC transporters, actively transport the PC back into the gastro-intestinal (GI) lumen, thus preventing further penetration into the body. Some ABC transporters also act in concert with Phase 1 and 2 metabolizing enzymes as a defensive barrier in the intestines and liver. If the PC overcomes the defence mechanisms of the gut and the liver, it will enter the systemic circulation and be distributed to the other organs of the body and possible site(s) of action. PCs can usually pass with ease through the pores of the capillaries of organs such as the heart and lungs, but with difficulty into pharmacological sanctuaries, such as the brain, testis, or foetus. Such sanctuaries contain a number of efflux transporters in their protective membrane, which restrict the penetration of xenobiotics, including PCs. The ABC and SLC transporters are also abundantly expressed in the liver and kidney and regulate the excretion of many compounds, including PCs and their metabolites. It is also becoming apparent that there is a complex interplay between various PCs and their ability to modulate the

  3. The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

    PubMed Central

    Rivers, Damien M. R.

    2015-01-01

    ABSTRACT Rhamnose catabolism in Rhizobium leguminosarum was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded by rhaSTPQ and on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis of rhaK showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles rhaK72 and rhaK73 were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined by rhaSTPQ. In this work, we show that both N- and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the rhaK72 or rhaK73 allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly. IMPORTANCE ABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters

  4. 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

  5. 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

  6. Impaired mitochondrial energy production and ABC transporter function-A crucial interconnection in dementing proteopathies of the brain.

    PubMed

    Pahnke, Jens; Fröhlich, Christina; Krohn, Markus; Schumacher, Toni; Paarmann, Kristin

    2013-10-01

    Ageing is the main risk factor for the development of dementing neurodegenerative diseases (NDs) and it is accompanied by the accumulation of variations in mitochondrial DNA. The resulting tissue-specific alterations in ATP production and availability cause deteriorations of cerebral clearance mechanisms that are important for the removal of toxic peptides and its aggregates. ABC transporters were shown to be the most important exporter superfamily for toxic peptides, e.g. β-amyloid and α-synuclein. Their activity is highly dependent on the availability of ATP and forms a directed energy-exporter network, linking decreased mitochondrial function with highly impaired ABC transporter activity and disease progression. In this paper, we describe a network based on interactions between ageing, energy metabolism, regeneration, accumulation of toxic peptides and the development of proteopathies of the brain with a focus on Alzheimer's disease (AD). Additionally, we provide new experimental evidence for interactions within this network in regenerative processes in AD.

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

    PubMed Central

    2008-01-01

    Background The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. Results We demonstrate that the two maltose ATP binding cassette (ABC) transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively) are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to have been horizontally

  8. 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

  9. A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence.

    PubMed

    Brown, J S; Gilliland, S M; Holden, D W

    2001-05-01

    Restricted iron availability is a major obstacle to growth and survival of pathogenic bacteria during infection. In contrast to Gram-negative pathogens, little is known about how Gram-positive pathogens obtain this essential metal. We have identified two Streptococcus pneumoniae genetic loci, pit1 and pit2, encoding homologues of ABC iron transporters that are required for iron uptake by this organism. S. pneumoniae strains containing disrupted copies of either pit1 or pit2 had decreased sensitivity to the iron-dependent antibiotic streptonigrin, and a strain containing disrupted copies of both pit1 and pit2 was unable to use haemoglobin as an iron source and had a reduced rate of iron uptake. The pit2- strain was moderately and the pit1-/pit2- strain strongly attenuated in virulence in mouse models of pulmonary and systemic infection, showing that the pit loci play a critical role during in vivo growth of S. pneumoniae. The pit2 locus is contained within a 27 kb region of chromosomal DNA that has several features of Gram-negative bacterial pathogenicity islands. This probable pathogenicity island (PPI-1) is the first to be described for S. pneumoniae, and its acquisition is likely to have played a significant role in the evolution of this important human pathogen.

  10. Tissue distribution and phenobarbital induction of target SLC- and ABC- transporters in cattle.

    PubMed

    Zancanella, V; Giantin, M; Lopparelli, R M; Nebbia, C; Dacasto, M

    2013-08-01

    In veterinary pharmaco-toxicological sciences, few data about uptake and efflux drug transporters (DTs) expression and regulation phenomena have been published. In this study, the tissue distribution and transcriptional modulation of solute carrier (SLC) and ATP-binding cassette (ABC) DTs were investigated in cattle orally administered with phenobarbital (PB) by using a quantitative real-time RT-PCR approach. The criterion for target gene selection was the PB-responsiveness in human and rodent model species. All target DTs were expressed in the liver. Only two of the seven PB-responsive target DTs (SLCO1B3 and SLC10A1) were not constitutively expressed in cattle extra-hepatic tissues. The greatest number of DTs (SLCO2B1, ABCB1, ABCC2, ABCG2) were expressed in intestine and testis, followed by, adrenal gland (SLCO2B1, ABCB1, ABCG2), lung (ABCB1, ABCG2), kidney, and skeletal muscle (ABCG2). PB administration never altered DTs mRNA levels, except for an increase in hepatic ABCC2 mRNA and a down-regulation of renal ABCG2. Altogether, these results confirm only to some extent data obtained in humans and laboratory species; clearly, they should be considered a preliminary step for further molecular investigations about species-differences in DT gene expression and regulation as well as in DT expression and function.

  11. 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

  12. 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

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

    PubMed

    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.

  14. 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

  15. A Two-Component System Regulates the Expression of an ABC Transporter for Xylo-Oligosaccharides in Geobacillus stearothermophilus▿

    PubMed Central

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

    2007-01-01

    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 μM 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 (ΔCp) 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

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

    PubMed

    Huang, Chun-Lin; Ho, Kuo-Chieh

    2007-10-01

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

  17. 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

  18. A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain.

    PubMed

    Song, Won-Yong; Yamaki, Tomohiro; Yamaji, Naoki; Ko, Donghwi; Jung, Ki-Hong; Fujii-Kashino, Miho; An, Gynheung; Martinoia, Enrico; Lee, Youngsook; Ma, Jian Feng

    2014-11-01

    Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice.

  19. A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain

    PubMed Central

    Song, Won-Yong; Yamaki, Tomohiro; Yamaji, Naoki; Ko, Donghwi; Jung, Ki-Hong; Fujii-Kashino, Miho; An, Gynheung; Martinoia, Enrico; Lee, Youngsook; Ma, Jian Feng

    2014-01-01

    Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice. PMID:25331872

  20. 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

  1. 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

  2. AztD, a Periplasmic Zinc Metallochaperone to an ATP-binding Cassette (ABC) Transporter System in Paracoccus denitrificans.

    PubMed

    Handali, Melody; Roychowdhury, Hridindu; Neupane, Durga P; Yukl, Erik T

    2015-12-11

    Bacterial ATP-binding cassette (ABC) transporters of transition metals are essential for acquisition of necessary elements from the environment. A large number of Gram-negative bacteria, including human pathogens, have a fourth conserved gene of unknown function adjacent to the canonical permease, ATPase, and solute-binding protein (SBP) genes of the AztABC zinc transporter system. To assess the function of this putative accessory factor (AztD) from Paracoccus denitrificans, we have analyzed its transcriptional regulation, metal binding properties, and interaction with the SBP (AztC). Transcription of the aztD gene is significantly up-regulated under conditions of zinc starvation. Recombinantly expressed AztD purifies with slightly substoichiometric zinc from the periplasm of Escherichia coli and is capable of binding up to three zinc ions with high affinity. Size exclusion chromatography and a simple intrinsic fluorescence assay were used to determine that AztD as isolated is able to transfer bound zinc nearly quantitatively to apo-AztC. Transfer occurs through a direct, associative mechanism that prevents loss of metal to the solvent. These results indicate that AztD is a zinc chaperone to AztC and likely functions to maintain zinc homeostasis through interaction with the AztABC system. This work extends our understanding of periplasmic zinc trafficking and the function of chaperones in this process.

  3. Transepithelial transport of fusariotoxin nivalenol: mediation of secretion by ABC transporters.

    PubMed

    Tep, Jonathan; Videmann, Bernadette; Mazallon, Michèle; Balleydier, Sabine; Cavret, Séverine; Lecoeur, Sylvaine

    2007-05-15

    Mycotoxin nivalenol (NIV) is a natural contaminant of various cereal crops, animal feed and processed grains throughout the world. Human and animal contamination occurs mainly orally, and the toxin must traverse the intestinal epithelial barrier before inducing potential health effects. In this study, we investigated the mechanisms involved in NIV transepithelial transfer. The human intestinal Caco-2 cell line showed a basal-to-apical polarized transport of NIV. Using metabolic inhibitors and temperature-dependent experiments, we demonstrated that basolateral-apical (BL-AP) transfer of NIV involved an energy-dependent transport whereas apical-basolateral (AP-BL) transfer was governed by passive diffusion. NIV efflux was significantly decreased in the presence of the P-glycoprotein (P-gp) inhibitor valspodar, the multi-drug resistance-associated proteins (MRPs) inhibitor MK571, but was not modified by the breast cancer resistance protein (BCRP) inhibitor Ko143. Intracellular NIV accumulation was investigated using epithelial cell lines transfected with either human P-glycoprotein or MRP2. This accumulation was significantly decreased in LLCPK1/MDR1 and MDCKII/MRP2 cells, compared to wild-type cells, and this effect was reversed by valspodar and MK571, respectively. These in vitro results suggested that NIV was a substrate for both P-glycoprotein and MRP2. This interaction may play a key role in weak intestinal absorption of NIV and the mainly predominant excretion of NIV in faeces in animal studies.

  4. 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

  5. Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis.

    PubMed

    Ghoshal, Angana; Mukhopadhyay, Sumi; Demine, Rodion; Forgber, Michael; Jarmalavicius, Saulius; Saha, Bibhuti; Sundar, Shyam; Walden, Peter; Mandal, Chhabinath; Mandal, Chitra

    2009-08-01

    We report the discovery and characterization of a glycosylated bacterial ABC-type phosphate transporter isolated from the peripheral blood mononuclear cell (PBMC) fraction of patients with visceral leishmaniasis (VL). Three disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) of 19, 56 and 65 kDa, respectively, had been identified and their purity, apparent mass and pI established by SDS-PAGE and isoelectric focusing. Western blot analyses showed that the 9-O-acetylated sialic acid is linked via alpha2-->6 linkage to a subterminal N-acetylgalactosamine. For the 56 kDa protein, N- as well as O-glycosylations were demonstrated by specific glycosidase treatment and found to account for more than 9 kDa of the protein mass. The presence of sialic acids was further confirmed through thin layer chromatography, fluorimetric HPLC and electrospray ionization-mass spectrometry. The protein was identified by mass spectrometry and de novo sequencing of five tryptic fragments as a periplasmic ABC-type phosphate transporter of Pseudomonas aeruginosa. The amino acid sequences of the assigned peptides had 83-100% identity with the NCBI entry for a Pseudomonas transporter protein. Based on the recently reported X-ray structure of a human phosphate-binding protein, we predicted a 3D structural model for the 56 kDa protein using homology and threading methods. The most probable N- and O-glycosylation sites were identified by combinations of sequence motif-searching bioinformatics tools, solvent accessibility calculations, structural environment analyses and mass spectrometric data. This is the first reported glycosylation as well as sialylation of the periplasmic component of an ABC-type phosphate transporter protein and of one of few identified bacterial glycoproteins.

  6. 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

  7. 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.

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

    PubMed Central

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

    2015-01-01

    The mycotoxin deoxynivalenol (DON) acts as a disease virulence factor for Fusarium fungi, and tolerance of DON enhances wheat resistance to Fusarium head blight (FHB) disease. Two variants of an ATP-binding cassette (ABC) family C transporter gene were cloned from DON-treated wheat mRNA, namely TaABCC3.1 and TaABCC3.2. These represent two of three putative genes identified on chromosomes 3A, 3B, and 3D of the wheat genome sequence. Variant TaABCC3.1 represents the DON-responsive transcript previously associated with DON resistance in wheat. PCR-based mapping and in silico sequence analyses located TaABCC3.1 to the short arm of wheat chromosome 3B (not within the FHB resistance quantitative trait locus Fhb1). In silico analyses of microarray data indicated that TaABCC3 genes are expressed in reproductive tissue and roots, and in response to the DON producer Fusarium graminearum. Gene expression studies showed that TaABCC3.1 is activated as part of the early host response to DON and in response to the FHB defence hormone jasmonic acid. Virus-induced gene silencing (VIGS) confirmed that TaABCC3 genes contributed to DON tolerance. VIGS was performed using two independent viral construct applications: one specifically targeted TaABCC3.1 for silencing, while the other targeted this gene and the chromosome 3A homeologue. In both instances, VIGS resulted in more toxin-induced discoloration of spikelets, compared with the DON effects in non-silenced spikelets at 14 d after toxin treatment (≥2.2-fold increase, P<0.05). Silencing by both VIGS constructs enhanced head ripening, and especially so in DON-treated heads. VIGS of TaABCC3 genes also reduced the grain number by more than 28% (P<0.05), both with and without DON treatment, and the effects were greater for the construct that targeted the two homeologues. Hence, DON-responsive TaABCC3 genes warrant further study to determine their potential as disease resistance breeding targets and their function in grain formation

  9. 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

  10. 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

  11. Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms

    PubMed Central

    Young, Rosanna E.B.; Twelkmeyer, Brigitte; Vitiazeva, Varvara; Power, Peter M.; Schweda, Elke K.H.; Hood, Derek W.

    2013-01-01

    Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host–bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component. PMID:24035104

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

    PubMed

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

    2015-03-30

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

  13. ABC and SLC transporter expression and proton oligopeptide transporter (POT) mediated permeation across the human blood--brain barrier cell line, hCMEC/D3 [corrected].

    PubMed

    Carl, Stephen M; Lindley, David J; Das, Debanjan; Couraud, Pierre O; Weksler, Babette B; Romero, Ignacio; Mowery, Stephanie A; Knipp, Gregory T

    2010-08-01

    Initial studies indicate that the newly developed hCMEC/D3 cell line may prove to be a useful model for studying the physiology of the human blood-brain barrier (BBB) endothelium. The purpose of this study was to assess the mRNA expression of several ABC and SLC transporters, with an emphasis on the proton-coupled oligopeptide transporter superfamily (POT) transporters in this immortalized BBB cell model. The transport kinetics of POT-substrates was also evaluated. The hCMEC/D3 cell line was maintained in a modified EGM-2 medium in collagenated culture flasks and passaged every 3-4 days at approximately 85%-95% confluence. Messenger RNA (mRNA) expression of a variety of ABC and SLC transporters was evaluated using qRT-PCR arrays, while additional qRT-PCR primers were designed to assess the expression of POT members. The transport kinetics of mannitol and urea were utilized to quantitatively estimate the intercellular pore radius, while POT substrate transport was also determined to assess the suitability of the cell model from a drug screening perspective. Optimization of the cell line was attempted by culturing with on laminin and fibronectin enhanced collagen and in the presence of excess Ca(2+). hCMEC/D3 cells express both hPHT1 and hPHT2, while little to no expression of either hPepT1 or hPepT2 was observed. The relative expression of other ABC and SLC transporters is discussed. While POT substrate transport does suggest suitability for BBB drug permeation screening, the relative intercellular pore radius was estimated at 19 A, significantly larger than that approximated in vivo. Culturing with extracellular matrix proteins did not alter mannitol permeability. These studies characterized this relevant human hCMEC/D3 BBB cell line with respect to both the relative mRNA expression of various ABC and SLC transporters and its potential utility as an in vitro screening tool for brain permeation. Additional studies are required to adequately determine the potential

  14. 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

    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

  15. 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

  16. Functional characterization of the nucleotide binding domain of the Cryptosporidium parvum CpABC4 transporter: an iron-sulfur cluster transporter homolog.

    PubMed

    Benitez, Alvaro J; Arrowood, Michael J; Mead, Jan R

    2009-06-01

    In a previous study, we showed that the Cryptosporidium parvum ATP half-transporter CpABC4 (cgd1_1350) transcript was up-regulated in response to drug treatment with paromomycin and cyclosporine A in an in vitro infection model. CpABC4 may be directly or indirectly involved in the metabolic interactions between host and parasite in response to drug treatment and/or be involved in the intrinsic resistance to chemotherapy. In order to characterize the catalytic site of this transporter, an extended region of the nucleotide-binding domain of CpABC4 (H6-1350NBD) was expressed and purified as an N-terminal hexahistidine-tagged protein in E. coli. The presence of a single tryptophan residue enabled the intrinsic fluorescence to be monitored in response to binding of different compounds. A dose-dependent quenching of the domain's intrinsic fluorescence was observed with its natural substrate, ATP and the fluorescent analogue TNP-ATP. A similar effect was observed with progesterone as well as the flavonoids quercetin and silibinin, previously shown to inhibit parasite development in a cell-based assay. The purified domain also exhibited ATPase activity in the nanomolar range, which further confirmed correct folding and activity of the recombinant domain. The H6-1350NBD serves as a tool to test and design stereospecific inhibitors of the catalytic site, as well as other compounds that bind elsewhere in the domain that may indirectly interact with the catalytic site of the NBD of the CpABC4 transporter.

  17. 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

  18. Function of the Caenorhabditis elegans ABC Transporter PGP-2 in the Biogenesis of a Lysosome-related Fat Storage Organelle

    PubMed Central

    Schroeder, Lena K.; Kremer, Susan; Kramer, Maxwell J.; Currie, Erin; Kwan, Elizabeth; Watts, Jennifer L.; Lawrenson, Andrea L.

    2007-01-01

    Caenorhabditis elegans gut granules are intestine specific lysosome-related organelles with birefringent and autofluorescent contents. We identified pgp-2, which encodes an ABC transporter, in screens for genes required for the proper formation of gut granules. pgp-2(−) embryos mislocalize birefringent material into the intestinal lumen and are lacking in acidified intestinal V-ATPase–containing compartments. Adults without pgp-2(+) function similarly lack organelles with gut granule characteristics. These cellular phenotypes indicate that pgp-2(−) animals are defective in gut granule biogenesis. Double mutant analysis suggests that pgp-2(+) functions in parallel with the AP-3 adaptor complex during gut granule formation. We find that pgp-2 is expressed in the intestine where it functions in gut granule biogenesis and that PGP-2 localizes to the gut granule membrane. These results support a direct role of an ABC transporter in regulating lysosome biogenesis. Previously, pgp-2(+) activity has been shown to be necessary for the accumulation of Nile Red–stained fat in C. elegans. We show that gut granules are sites of fat storage in C. elegans embryos and adults. Notably, levels of triacylglycerides are relatively normal in animals defective in the formation of gut granules. Our results provide an explanation for the loss of Nile Red–stained fat in pgp-2(−) animals as well as insight into the specialized function of this lysosome-related organelle. PMID:17202409

  19. 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

  20. 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

  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. TetAB(46), a predicted heterodimeric ABC transporter conferring tetracycline resistance in Streptococcus australis isolated from the oral cavity

    PubMed Central

    Warburton, Philip J.; Ciric, Lena; Lerner, Avigdor; Seville, Lorna A.; Roberts, Adam P.; Mullany, Peter; Allan, Elaine

    2013-01-01

    Objectives To identify the genes responsible for tetracycline resistance in a strain of Streptococcus australis isolated from pooled saliva from healthy volunteers in France. S. australis is a viridans Streptococcus, originally isolated from the oral cavity of children in Australia, and subsequently reported in the lungs of cystic fibrosis patients and as a cause of invasive disease in an elderly patient. Methods Agar containing 2 mg/L tetracycline was used for the isolation of tetracycline-resistant organisms. A genomic library in Escherichia coli was used to isolate the tetracycline resistance determinant. In-frame deletions and chromosomal repair were used to confirm function. Antibiotic susceptibility was determined by agar dilution and disc diffusion assay. Results The tetracycline resistance determinant from S. australis FRStet12 was isolated from a genomic library in E. coli and DNA sequencing showed two open reading frames predicted to encode proteins with similarity to multidrug resistance-type ABC transporters. Both genes were required for tetracycline resistance (to both the naturally occurring and semi-synthetic tetracyclines) and they were designated tetAB(46). Conclusions This is the first report of a predicted ABC transporter conferring tetracycline resistance in a member of the oral microbiota. PMID:22941900

  3. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  4. Poloxamines display a multiple inhibitory activity of ATP-binding cassette (ABC) transporters in cancer cell lines.

    PubMed

    Cuestas, María L; Sosnik, Alejandro; Mathet, Verónica L

    2011-08-01

    Primary hepatocellular carcinoma is the third most common fatal cancer worldwide with more than 500,000 annual deaths. Approximately 40% of the patients with HCC showed tumoral overexpression of transmembrane proteins belonging to the ATP-binding cassette protein superfamily (ABC) which pump drugs out of cells. The overexpression of these efflux transporters confers on the cells a multiple drug resistance phenotype, which is considered a crucial cause of treatment refractoriness in patients with cancer. The aim of this study was to investigate the inhibitory effect of different concentrations of pH- and temperature-responsive X-shaped poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines, Tetronic, PEO-PPO) showing a wide range of molecular weights and EO/PO ratios on the functional activity of three different ABC proteins, namely P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein MRP1, in two human hepatocarcinoma cell lines, HepG2 and Huh7. First, the cytotoxicity of the different copolymers (at different concentrations) on both liver carcinoma cell lines was thoroughly evaluated by means of apoptosis analysis using annexin V and propidium iodide (PI). Thus, viable cells (AV-/PI-), early apoptotic cells (AV+/PI-) and late apoptotic cells (V-FITC+/PI+) were identified. Results pointed out copolymers of intermediate to high hydrophobicity and intermediate molecular weight (e.g., T904) as the most cytotoxic. Then, DiOC2, rhodamine 123 and vinblastine were used as differential substrates of these pumps. HeLa, an epithelial cell line of human cervical cancer that does not express P-gp, was used exclusively as a control and enabled the discerning between P-gp and MRP1 inhibition. Moderate to highly hydrophobic poloxamines T304, T904 and T1301 showed inhibitory activity against P-gp and BCRP but not against MRP1 in both hepatic cell lines. A remarkable dependence of this effect on the

  5. 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

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

    PubMed

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

    2016-09-01

    ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. PMID:27602881

  7. 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

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

    PubMed

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

    2016-04-20

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

  9. 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

  10. 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

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

    PubMed

    Xu, Yongbin; Guo, Jianyun; 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

  12. 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

  13. 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

  14. 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.

  15. 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...

  16. Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis.

    PubMed Central

    Choudhuri, Baisakhee Saha; Bhakta, Sanjib; Barik, Rajib; Basu, Joyoti; Kundu, Manikuntala; Chakrabarti, Parul

    2002-01-01

    The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis. However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2',7'-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters. PMID:12057006

  17. 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

  18. Essential letters in the fungal alphabet: ABC and MFS transporters and their roles in survival and pathogenicity.

    PubMed

    Perlin, Michael H; Andrews, Jared; Toh, Su San

    2014-01-01

    Fungi depend heavily on their ability to exploit resources that may become available to them in their myriad of possible lifestyles. Whether this requires simple uptake of sugars as saprobes or competition for host-derived carbohydrates or peptides, fungi must rely on transporters that effectively allow the fungus to accumulate such nutrients from their environments. In other cases, fungi secrete compounds that facilitate their interactions with potential hosts and/or neutralize their competition. Finally, fungi that find themselves on the receiving end of insults, from hosts, competitors, or the overall environment are better served if they can get rid of such toxins or xenobiotics. In this chapter, we update studies on the most ubiquitous transporters, the ABC and MFS superfamilies. In addition, we discuss the importance of subsets of these proteins with particular relevance to plant pathogenic fungi. The availability of ever-increasing numbers of sequenced fungal genomes, combined with high-throughput methods for transcriptome analysis, provides insights previously inaccessible prior to the -omics era. As examples of such broader perspectives, we point to revelations about exploitive use of sugar transporters by plant pathogens, expansion of trichothecene efflux pumps in fungi that do not produce these mycotoxins, and the discovery of a fungal-specific oligopeptide transporter class that, so far, is overrepresented in the plant pathogenic fungi.

  19. The ABC transporter AtABCB14 is a malate importer and modulates stomatal response to CO2.

    PubMed

    Lee, Miyoung; Choi, Yongwook; Burla, Bo; Kim, Yu-Young; Jeon, Byeongwook; Maeshima, Masayoshi; Yoo, Joo-Yeon; Martinoia, Enrico; Lee, Youngsook

    2008-10-01

    Carbon dioxide uptake and water vapour release in plants occur through stomata, which are formed by guard cells. These cells respond to light intensity, CO2 and water availability, and plant hormones. The predicted increase in the atmospheric concentration of CO2 is expected to have a profound effect on our ecosystem. However, many aspects of CO2-dependent stomatal movements are still not understood. Here we show that the ABC transporter AtABCB14 modulates stomatal closure on transition to elevated CO2. Stomatal closure induced by high CO2 levels was accelerated in plants lacking AtABCB14. Apoplastic malate has been suggested to be one of the factors mediating the stomatal response to CO2 (Refs 4,5) and indeed, exogenously applied malate induced a similar AtABCB14-dependent response as high CO2 levels. In isolated epidermal strips that contained only guard cells, malate-dependent stomatal closure was faster in plants lacking the AtABCB14 and slower in AtABCB14-overexpressing plants, than in wild-type plants, indicating that AtABCB14 catalyses the transport of malate from the apoplast into guard cells. Indeed, when AtABCB14 was heterologously expressed in Escherichia coli and HeLa cells, increases in malate transport activity were observed. We therefore suggest that AtABCB14 modulates stomatal movement by transporting malate from the apoplast into guard cells, thereby increasing their osmotic pressure.

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

    PubMed

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

    2007-06-01

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

  1. Separating the Roles of Acropetal and Basipetal Auxin Transport on Gravitropism with Mutations in Two Arabidopsis Multidrug Resistance-Like ABC Transporter Genes[W][OA

    PubMed Central

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

    2007-01-01

    Two Arabidopsis thaliana ABC transporter genes linked to auxin transport by various previous results were studied in a reverse-genetic fashion. Mutations in Multidrug Resistance-Like1 (MDR1) reduced acropetal auxin transport in roots by 80% without affecting basipetal transport. Conversely, mutations in MDR4 blocked 50% of basipetal transport without affecting acropetal transport. Developmental and auxin distribution phenotypes associated with these altered auxin flows were studied with a high-resolution morphometric system and confocal microscopy, respectively. Vertically grown mdr1 roots produced positive and negative curvatures threefold greater than the wild type, possibly due to abnormal auxin distribution observed in the elongation zone. However, upon 90° reorientation, mdr1 gravitropism was inseparable from the wild type. Thus, acropetal auxin transport maintains straight growth but contributes surprisingly little to gravitropism. Conversely, vertically maintained mdr4 roots grew as straight as the wild type, but their gravitropism was enhanced. Upon reorientation, curvature in this mutant developed faster, was distributed more basally, and produced a greater total angle than the wild type. An amplified auxin asymmetry may explain the mdr4 hypertropism. Double mutant analysis indicated that the two auxin transport streams are more independent than interdependent. The hypothesis that flavanols regulate MDR-dependent auxin transport was supported by the epistatic relationship of mdr4 to the tt4 phenylpropanoid pathway mutation. PMID:17557805

  2. 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

  3. 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.

  4. Nucleotide-binding sites of the heterodimeric LmrCD ABC-multidrug transporter of Lactococcus lactis are asymmetric.

    PubMed

    Lubelski, Jacek; van Merkerk, Ronald; Konings, Wil N; Driessen, Arnold J M

    2006-01-17

    LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[alpha-32P]ATP, which, at 4 degrees C, occurs predominantly at LmrC, while aluminiumfluoride (AlF(x))-induced trapping of the hydrolyzed nucleotide at 30 degrees C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs. PMID:16401093

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

    PubMed

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

    1997-04-01

    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

  6. Molecular Cloning of a 32-Kilodalton Lipoprotein Component of a Novel Iron-Regulated Staphylococcus epidermidis ABC Transporter

    PubMed Central

    Cockayne, Alan; Hill, Philip J.; Powell, Nick B. L.; Bishop, Keith; Sims, Cate; Williams, Paul

    1998-01-01

    Our previous studies identified two iron-regulated cytoplasmic membrane proteins of 32 and 36 kDa expressed by both Staphylococcus epidermidis and Staphylococcus aureus. In this study we show by Triton X-114 phase partitioning and tritiated palmitic acid labelling that these proteins are lipoproteins which are anchored into the cytoplasmic membrane by their lipid-modified N termini. In common with those of some other gram-positive bacteria, these highly immunogenic lipoproteins were released from the bacterial cell into the culture supernatants, with release being promoted by growth of the bacteria under iron-restricted conditions. Immunoelectron microscopy with a monospecific rabbit antiserum to the 32-kDa S. epidermidis lipoprotein showed that the majority of the antigen was distributed throughout the staphylococcal cell wall. Only minor quantities were detected in the cytoplasmic membrane, and exposure of the lipoprotein on the bacterial surface was minimal. A monoclonal antibody raised to the 32-kDa lipoprotein of S. aureus was used in immunoblotting studies to investigate the conservation of this antigen among a variety of staphylococci. The monoclonal antibody reacted with polypeptides of 32 kDa in S. epidermidis and S. aureus and of 40 kDa in Staphylococcus hominis. No reactivity was detected with Staphylococcus lugdunensis, Staphylococcus cohni, or Staphylococcus haemolyticus. The gene encoding the 32-kDa lipoprotein from S. epidermidis has been isolated from a Lambda Zap II genomic DNA library and found to be a component of an iron-regulated operon encoding a novel ABC-type transporter. The operon contains three genes, designated sitA, -B, and -C, encoding an ATPase, a cytoplasmic membrane protein, and the 32-kDa lipoprotein, respectively. SitC shows significant homology both with a number of bacterial adhesins, including FimA of Streptococcus parasanguis and ScaA of Streptococcus gordonii, and with lipoproteins of a recently described family of ABC

  7. 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

  8. Changes in mRNA expression of ABC and SLC transporters in liver and intestines of the adjuvant-induced arthritis rat.

    PubMed

    Uno, Satoshi; Uraki, Misato; Ito, Ayami; Shinozaki, Yuki; Yamada, Ayano; Kawase, Atsushi; Iwaki, Masahiro

    2009-01-01

    In this study, a real-time reverse transcription-polymerase chain reaction was used to determine the effects of adjuvant-induced arthritis (AA) on the amounts of mRNA of 12 types of rat ATP-binding cassette (ABC) and solute carrier (SLC) transporters in the liver and small intestine, 7 (D7) and 21 days (D21) after the injection of adjuvant. There were no significant differences in mRNA levels of ABC and SLC transporters between the livers of AA and control rats on D7, except in the case of Mdr1a. However, levels of Mdr1a, Mrp2 and Oatp SLC transporters were significantly lower in AA than in the control livers on D21. In contrast, the mRNA levels of several ABC and SLC transporters, especially Mrp2, Bcrp, LAT2 and Oatp1a5, were significantly lower in the small intestines of AA rats compared with the controls on D7, though there were no significant differences by D21. The time-dependent alterations in mRNA levels of the pregnane X receptor, but not the constitutive androstane receptor, in the liver and intestine were similar to the changes in mRNA levels of most transporters examined. The present study showed that AA was associated with reduced mRNA expression of several ABC and SLC transporters in the liver and small intestine, but that the time courses of the effects of AA on mRNA expression differed between the liver and small intestine. These results raise the possibility of a functional change of the transporters of liver and intestine in AA rats.

  9. YehZYXW of Escherichia coli Is a Low-Affinity, Non-Osmoregulatory Betaine-Specific ABC Transporter.

    PubMed

    Lang, Shenhui; Cressatti, Marisa; Mendoza, Kris E; Coumoundouros, Chelsea N; Plater, Samantha M; Culham, Doreen E; Kimber, Matthew S; Wood, Janet M

    2015-09-22

    Transporter-mediated osmolyte accumulation stimulates the growth of Escherichia coli in high-osmolality environments. YehZYXW was predicted to be an osmoregulatory transporter because (1) osmotic and stationary phase induction of yehZYXW is mediated by RpoS, (2) the Yeh proteins are homologous to the components of known osmoregulatory ABC transporters (e.g., ProU of E. coli), and (3) YehZ models based on the structures of periplasmic betaine-binding proteins suggested that YehZ retains key betaine-binding residues. The betaines choline-O-sulfate, glycine betaine, and dimethylsulfoniopropionate bound YehZ and ProX with millimolar and micromolar affinities, respectively, as determined by equilibrium dialysis and isothermal titration calorimetry. The crystal structure of the YehZ apoprotein, determined at 1.5 Å resolution (PDB ID: 4WEP ), confirmed its similarity to other betaine-binding proteins. Small and nonpolar residues in the hinge region of YehZ (e.g., Gly223) pack more closely than the corresponding residues in ProX, stabilizing the apoprotein. Betaines bound YehZ-Gly223Ser an order of magnitude more tightly than YehZ, suggesting that weak substrate binding in YehZ is at least partially due to apo state stabilization. Neither ProX nor YehZ bound proline. Assays based on osmoprotection or proline auxotrophy failed to detect YehZYXW-mediated uptake of proline, betaines, or other osmolytes. However, transport assays revealed low-affinity glycine betaine uptake, mediated by YehZYXW, that was inhibited at high salinity. Thus, YehZYXW is a betaine transporter that shares substrate specificity, but not an osmoregulatory function, with homologues like E. coli ProU. Other work suggests that yehZYXW may be an antivirulence locus whose expression promotes persistent, asymptomatic bacterial infection.

  10. The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae.

    PubMed

    Amnuaykanjanasin, Alongkorn; Daub, Margaret E

    2009-02-01

    The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.

  11. Ensemble Rule-Based Classification of Substrates of the Human ABC-Transporter ABCB1 Using Simple Physicochemical Descriptors.

    PubMed

    Demel, Michael A; Kraemer, Oliver; Ettmayer, Peter; Haaksma, Eric; Ecker, Gerhard F

    2010-03-15

    Within the last decades, the detailed knowledge on the impact of membrane bound drug efflux transporters of the ATP binding cassette (ABC) protein family on the pharmacological profile of drugs has enormously increased. Especially, ABCB1 (P-glycoprotein, P-gp, MDR1) has attracted particular interest in medicinal chemistry, since it determines the clinical efficacy, side effects and toxicity risks of drug candidates. Based on this, the development of in silico models that provide rapid and cost-effective screening tools for the classification of substrates and nonsubstrates of ABCB1 is an urgent need in contemporary ADMET profiling. A characteristic hallmark feature of this transporter is its polyspecific ligand recognition pattern. In this study we describe a method for classifying ABCB1 ligands in terms of simple, conjunctive rules (RuleFit) based on interpretable ADMET features. The retrieved results showed that models based on large, very diverse data sets gave better classification performance than models based on smaller, more homogenous training sets. The best model achieved gave a correct classification rate of 0.90 for an external validation set. Furthermore, from the interpretation of the best performing model it could be concluded that in comparison to nonsubstrates ABCB1 substrates generally show a higher number of hydrogen-bond acceptors, are more flexible and exhibit higher logP values.

  12. 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

  13. 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

  14. 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

  15. Restoration of Lysosomal pH in RPE Cells from Cultured Human and ABCA4−/− Mice: Pharmacologic Approaches and Functional Recovery

    PubMed Central

    Liu, Ji; Lu, Wennan; Reigada, David; Nguyen, Jonathan; Laties, Alan M.; Mitchell, Claire H.

    2008-01-01

    Purpose Degradation of engulfed material is primarily mediated by lysosomal enzymes that function optimally within a narrow range of acidic pH values. RPE cells are responsible for daily degradation of photoreceptor outer segments and are thus particularly susceptible to perturbations in lysosomal pH. The authors hypothesized that elevated lysosomal pH levels could slow enzyme activity and encourage accumulation of partially digested material. Consequently, treatment to lower perturbed lysosomal pH levels may enhance degradative activity. Methods A high-throughput screening assay was developed to quantify the lysosomal pH of fresh mouse and cultured ARPE-19 cells. The effect of lysosomal pH on outer segment clearance was determined. Results Lysosomal pH is elevated in RPE cells from ABCA4 knockout mice and in cultured human ARPE-19 cells exposed to N-retinylidene-N-retinylethanolamine (A2E), tamoxifen, or chloroquine. The lysosomal pH of fresh RPE cells from ABCA4−/− mice and of chemically compromised RPE cells was reacidified by elevating intracellular cAMP directly. Compromised lysosomal pH was also restored by stimulating A2A adenosine or β-adrenergic receptors, consistent with Gs-protein coupling of these receptors. Restoring lysosomal pH with these treatments enhanced photoreceptor outer segment clearance, demonstrating functional relevance consistent with an enhancement of degradative enzyme activity. Conclusions Elevation of lysosomal pH in RPE cells interferes with the degradation of outer segments and may contribute to the pathologies associated with A2E. Pharmacologic elevation of cAMP can restore an acid pH and improve degradative function. PMID:18235027

  16. 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

  17. 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

  18. The ABCs of membrane transporters in health and disease (SLC series): introduction.

    PubMed

    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.

  19. 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

  20. Specific interactions between the Candida albicans ABC transporter Cdr1p ectodomain and a D-octapeptide derivative inhibitor.

    PubMed

    Niimi, Kyoko; Harding, David R K; Holmes, Ann R; Lamping, Erwin; Niimi, Masakazu; Tyndall, Joel D A; Cannon, Richard D; Monk, Brian C

    2012-08-01

    Overexpression of the Candida albicans ATP-binding cassette transporter CaCdr1p causes clinically significant resistance to azole drugs including fluconazole (FLC). Screening of a ~1.89 × 10(6) member D-octapeptide combinatorial library that concentrates library members at the yeast cell surface identified RC21v3, a 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative of the D-octapeptide D-NH(2) -FFKWQRRR-CONH(2) , as a potent and stereospecific inhibitor of CaCdr1p. RC21v3 chemosensitized Saccharomyces cerevisiae strains overexpressing CaCdr1p but not other fungal ABC transporters, the C. albicans MFS transporter CaMdr1p or the azole target enzyme CaErg11p, to FLC. RC21v3 also chemosensitized clinical C. albicans isolates overexpressing CaCDR1 to FLC, even when CaCDR2 was overexpressed. Specific targeting of CaCdr1p by RC21v3 was confirmed by spontaneous RC21v3 chemosensitization-resistant suppressor mutants of S. cerevisiae expressing CaCdr1p. The suppressor mutations introduced a positive charge beside, or within, extracellular loops 1, 3, 4 and 6 of CaCdr1p or an aromatic residue near the extracytoplasmic end of transmembrane segment 5. The mutations did not affect CaCdr1p localization or CaCdr1p ATPase activity but some increased susceptibility to the CaCdr1p substrates FLC, rhodamine 6G, rhodamine 123 and cycloheximide. The suppressor mutations showed that the drug-like CaCdr1p inhibitors FK506, enniatin, milbemycin α11 and milbemycin β9 have modes of action similar to RC21v3. PMID:22788839

  1. 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

  2. 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.

  3. 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

  4. A step-by-step method for the reconstitution of an ABC transporter into nanodisc lipid particles.

    PubMed

    Bao, Huan; Duong, Franck; Chan, Catherine S

    2012-01-01

    The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted. PMID:22951950

  5. 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

  6. Crystal structure of the phosphate-binding protein (PBP-1) of an ABC-type phosphate transporter from Clostridium perfringens.

    PubMed

    Gonzalez, Daniel; Richez, Magali; Bergonzi, Celine; Chabriere, Eric; Elias, Mikael

    2014-01-01

    Phosphate limitation is an important environmental stress that affects the metabolism of various organisms and, in particular, can trigger the virulence of numerous bacterial pathogens. Clostridium perfringens, a human pathogen, is one of the most common causes of enteritis necroticans, gas gangrene and food poisoning. Here, we focused on the high affinity phosphate-binding protein (PBP-1) of an ABC-type transporter, responsible for cellular phosphate uptake. We report the crystal structure (1.65 Å resolution) of the protein in complex with phosphate. Interestingly, PBP-1 does not form the short, low-barrier hydrogen bond with phosphate that is typical of previously characterized phosphate-binding proteins, but rather a canonical hydrogen bond. In its unique binding configuration, PBP-1 forms an unusually high number of hydrogen bonds (14) with the phosphate anion. Discrimination experiments reveal that PBP-1 is the least selective PBP characterised so far and is able to discriminate phosphate from its close competing anion, arsenate, by ~150-fold.

  7. 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

  8. A Novel Gene Amplification Causes Upregulation of the PatAB ABC Transporter and Fluoroquinolone Resistance in Streptococcus pneumoniae

    PubMed Central

    Baylay, Alison J.; Ivens, Alasdair

    2015-01-01

    Overexpression of the ABC transporter genes patA and patB confers efflux-mediated fluoroquinolone resistance in Streptococcus pneumoniae and is also linked to pneumococcal stress responses. Although upregulation of patAB has been observed in many laboratory mutants and clinical isolates, the regulatory mechanisms controlling expression of these genes are unknown. In this study, we aimed to identify the cause of high-level constitutive overexpression of patAB in M184, a multidrug-resistant mutant of S. pneumoniae R6. Using a whole-genome transformation and sequencing approach, we identified a novel duplication of a 9.2-kb region of the M184 genome which included the patAB genes. This duplication did not affect growth and was semistable with a low segregation rate. The expression levels of patAB in M184 were much higher than those that could be fully explained by doubling of the gene dosage alone, and inactivation of the first copy of patA had no effect on multidrug resistance. Using a green fluorescent protein reporter system, increased patAB expression was ascribed to transcriptional read-through from a tRNA gene upstream of the second copy of patAB. This is the first report of a large genomic duplication causing antibiotic resistance in S. pneumoniae and also of a genomic duplication causing antibiotic resistance by a promoter switching mechanism. PMID:25779578

  9. The function of the ATP-binding cassette (ABC) transporter ABCB1 is not susceptible to actin disruption.

    PubMed

    Meszaros, Peter; Hummel, Ina; Klappe, Karin; Draghiciu, Oana; Hoekstra, Dick; Kok, Jan W

    2013-02-01

    Previously we have shown that the activity of the multidrug transporter ABCC1 (multidrug resistance protein 1), and its localization in lipid rafts, depends on cortical actin (Hummel I, Klappe K, Ercan C, Kok JW. Mol. Pharm. 2011 79, 229-40). Here we show that the efflux activity of the ATP-binding cassette (ABC) family member ABCB1 (P-glycoprotein), did not depend on actin, neither in ABCB1 over expressing murine National Institutes of Health (NIH) 3T3 MDR1 G185 cells nor in human SK-N-FI cells, which endogenously express ABCB1. Disruption of the actin cytoskeleton, upon treatment of the cells with latrunculin B or cytochalasin D, caused severe changes in cell and membrane morphology, and concomitant changes in the subcellular distribution of ABCB1, as revealed by confocal laser scanning and electron microscopy. Nevertheless, irrespective of actin perturbation, the cell surface pool of ABCB1 remained unaltered. In NIH 3T3 MDR1 G185 cells, ABCB1 is partly localized in detergent-free lipid rafts, which partitioned in two different density gradient regions, both enriched in cholesterol and sphingolipids. Interestingly, disruption of the actin cytoskeleton did not change the density gradient distribution of ABCB1. Our data demonstrate that the functioning of ABCB1 as an efflux pump does not depend on actin, which is due to its distribution in both cell surface-localized non-raft membrane areas and lipid raft domains, which do not depend on actin stabilization.

  10. Crystal structure of the phosphate-binding protein (PBP-1) of an ABC-type phosphate transporter from Clostridium perfringens.

    PubMed

    Gonzalez, Daniel; Richez, Magali; Bergonzi, Celine; Chabriere, Eric; Elias, Mikael

    2014-01-01

    Phosphate limitation is an important environmental stress that affects the metabolism of various organisms and, in particular, can trigger the virulence of numerous bacterial pathogens. Clostridium perfringens, a human pathogen, is one of the most common causes of enteritis necroticans, gas gangrene and food poisoning. Here, we focused on the high affinity phosphate-binding protein (PBP-1) of an ABC-type transporter, responsible for cellular phosphate uptake. We report the crystal structure (1.65 Å resolution) of the protein in complex with phosphate. Interestingly, PBP-1 does not form the short, low-barrier hydrogen bond with phosphate that is typical of previously characterized phosphate-binding proteins, but rather a canonical hydrogen bond. In its unique binding configuration, PBP-1 forms an unusually high number of hydrogen bonds (14) with the phosphate anion. Discrimination experiments reveal that PBP-1 is the least selective PBP characterised so far and is able to discriminate phosphate from its close competing anion, arsenate, by ~150-fold. PMID:25338617

  11. 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

  12. 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

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

    PubMed

    Cole, Susan P C

    2014-11-01

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

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

    PubMed Central

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

    2015-01-01

    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

  15. In vitro establishment of ivermectin-resistant Rhipicephalus microplus cell line and the contribution of ABC transporters on the resistance mechanism.

    PubMed

    Pohl, Paula C; Carvalho, Danielle D; Daffre, Sirlei; Vaz, Itabajara da Silva; Masuda, Aoi

    2014-08-29

    The cattle tick Rhipicephalus microplus is one of the most economically damaging livestock ectoparasites, and its widespread resistance to acaricides is a considerable challenge to its control. In this scenario, the establishment of resistant cell lines is a useful approach to understand the mechanisms involved in the development of acaricide resistance, to identify drug resistance markers, and to develop new acaricides. This study describes the establishment of an ivermectin (IVM)-resistant R. microplus embryonic cell line, BME26-IVM. The resistant cells were obtained after the exposure of IVM-sensitive BME26 cells to increasing doses of IVM in a step-wise manner, starting from an initial non-toxic concentration of 0.5 μg/mL IVM, and reaching 6 μg/mL IVM after a 46-week period. BME26-IVM cell line was 4.5 times more resistant to IVM than the parental BME26 cell line (lethal concentration 50 (LC50) 15.1 ± 1.6 μg/mL and 3.35 ± 0.09 μg/mL, respectively). As an effort to determine the molecular mechanisms governing resistance, the contribution of ATP-binding cassette (ABC) transporter was investigated. Increased expression levels of ABC transporter genes were found in IVM-treated cells, and resistance to IVM was significantly reduced by co-incubation with 5 μM cyclosporine A (CsA), an ABC transporter inhibitor, suggesting the involvement of these proteins in IVM-resistance. These results are similar to those already described in IVM-resistant tick populations, and suggest that similar resistance mechanisms are involved in vitro and in vivo. They reinforce the hypothesis that ABC transporters are involved in IVM resistance and support the use of BME26-IVM as an in vitro approach to study acaricide resistance mechanisms. PMID:24956999

  16. 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

  17. 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.

  18. 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...

  19. 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

  20. 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.

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

    PubMed Central

    2016-01-01

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

  2. 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).

  3. 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

  4. 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

    -CDs can significantly reduce the cellular cholesterol content of cells forming atherosclerotic lesions and can subsequently modulate the expression of ABC transporters involved in RCT. The use of methylated β-CDs would represent a valuable and efficient tool to interfere with atherosclerosis pathogenesis in patients, nonetheless their mode of action still needs further investigations to be fully understood and finely controlled at the cellular level. PMID:27252658

  5. 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

  6. 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

  7. Toxicological effects of particulate matter (PM2.5) on rats: Bioaccumulation, antioxidant alterations, lipid damage, and ABC transporter activity.

    PubMed

    Ribeiro, Joaquim de Paula; Kalb, Ana Cristina; Campos, Paula Peixoto; Cruz, Alex Rubén Huaman De La; Martinez, Pablo Elias; Gioda, Adriana; Souza, Marta Marques de; Gioda, Carolina Rosa

    2016-11-01

    Previous studies have demonstrated the harmful effects of atmospheric pollutants on cardiac systems because of the presence of particulate matter (PM), a complex mixture of numerous substances including trace metals. In this study, the toxicity of PM2.5 from two regions, rural (PM2.5 level of 8.5 ± 4.0 μg m(-3)) and industrial (PM2.5 level of 14.4 ± 4.1 μg m(-3)) in Brazil, was investigated through in vivo experiments in rats. Metal accumulation and biochemical responses were evaluated after rats were exposed to three different concentrations of PM2.5 in saline extract (10× dilution, 5× dilution, and concentrated). The experimental data showed the bioaccumulation of diverse trace metals in the hearts of groups exposed to PM2.5 from both regions. Furthermore, mobilization of the antioxidant defenses and an increase in lipid peroxidation of the cardiac tissue was observed in response to the industrial and rural area PM2.5. Glutathione-S-transferase activity was increased in groups exposed to the 5× and concentrated rural PM2.5. Additionally, ATP-binding cassette (ABC) transporter activity in the cardiac tissue exposed to PM2.5 was reduced in response to the 5× dilution of the rural and industrial region PM2.5. Histological analysis showed a decrease in the percentage of cardiac cells in the heart at all tested concentrations. The results indicate that exposure to different concentrations of PM2.5 from both sources causes biochemical and histological changes in the heart with consequent damage to biological structures; these factors can favor the development of cardiac diseases.

  8. Toxicological effects of particulate matter (PM2.5) on rats: Bioaccumulation, antioxidant alterations, lipid damage, and ABC transporter activity.

    PubMed

    Ribeiro, Joaquim de Paula; Kalb, Ana Cristina; Campos, Paula Peixoto; Cruz, Alex Rubén Huaman De La; Martinez, Pablo Elias; Gioda, Adriana; Souza, Marta Marques de; Gioda, Carolina Rosa

    2016-11-01

    Previous studies have demonstrated the harmful effects of atmospheric pollutants on cardiac systems because of the presence of particulate matter (PM), a complex mixture of numerous substances including trace metals. In this study, the toxicity of PM2.5 from two regions, rural (PM2.5 level of 8.5 ± 4.0 μg m(-3)) and industrial (PM2.5 level of 14.4 ± 4.1 μg m(-3)) in Brazil, was investigated through in vivo experiments in rats. Metal accumulation and biochemical responses were evaluated after rats were exposed to three different concentrations of PM2.5 in saline extract (10× dilution, 5× dilution, and concentrated). The experimental data showed the bioaccumulation of diverse trace metals in the hearts of groups exposed to PM2.5 from both regions. Furthermore, mobilization of the antioxidant defenses and an increase in lipid peroxidation of the cardiac tissue was observed in response to the industrial and rural area PM2.5. Glutathione-S-transferase activity was increased in groups exposed to the 5× and concentrated rural PM2.5. Additionally, ATP-binding cassette (ABC) transporter activity in the cardiac tissue exposed to PM2.5 was reduced in response to the 5× dilution of the rural and industrial region PM2.5. Histological analysis showed a decrease in the percentage of cardiac cells in the heart at all tested concentrations. The results indicate that exposure to different concentrations of PM2.5 from both sources causes biochemical and histological changes in the heart with consequent damage to biological structures; these factors can favor the development of cardiac diseases. PMID:27567156

  9. 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

  10. Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm, Bombyx mori.

    PubMed

    Wang, Lingyan; Kiuchi, Takashi; Fujii, Tsuguru; Daimon, Takaaki; Li, Muwang; Banno, Yutaka; Kikuta, Shingo; Kikawada, Takahiro; Katsuma, Susumu; Shimada, Toru

    2013-07-01

    ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.

  11. 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.

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

    PubMed Central

    Braun, Yvonne; Dubiley, Svetlana; Lafon, Corinne; Köhler, Thilo; Page, Malcolm G. P.; Mourez, Michael; Severinov, Konstantin

    2015-01-01

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

  13. 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.

  14. The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use.

    PubMed

    Klein, Markus; Perfus-Barbeoch, Laetitia; Frelet, Annie; Gaedeke, Nicola; Reinhardt, Didier; Mueller-Roeber, Bernd; Martinoia, Enrico; Forestier, Cyrille

    2003-01-01

    Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance. Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels. Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1). Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark. Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements. Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark. In contrast, opening of stomata of mrp5-1 plants was reduced in the light. In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress. In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness. All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct. Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light. Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level. Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use. Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency. Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics. These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning.

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

    PubMed

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

    2015-09-01

    Despite the ubiquitous role of ATP-binding cassette (ABC) importers in nutrient uptake, only the Escherichia 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.

  16. 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

  17. 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

  18. 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

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

    PubMed Central

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

    2015-01-01

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

  20. 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.

  1. 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

  2. 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

  3. The Efflux Pump Inhibitor Reserpine Selects Multidrug-Resistant Streptococcus pneumoniae Strains That Overexpress the ABC Transporters PatA and PatB▿ †

    PubMed Central

    Garvey, Mark I.; Piddock, Laura J. V.

    2008-01-01

    One way to combat multidrug-resistant microorganisms is the use of efflux pump inhibitors (EPIs). Spontaneous mutants resistant to the EPI reserpine selected from Streptococcus pneumoniae NCTC 7465 and R6 at a frequency suggestive of a single mutational event were also multidrug resistant. No mutations in pmrA (which encodes the efflux protein PmrA) were detected, and the expression of pmrA was unaltered in all mutants. In the reserpine-resistant multidrug-resistant mutants, the overexpression of both patA and patB, which encode ABC transporters, was associated with accumulation of low concentrations of antibiotics and dyes. The addition of sodium orthovanadate, an inhibitor of ABC efflux pumps, or the insertional inactivation of either gene restored wild-type antibiotic susceptibility and wild-type levels of accumulation. Only when patA was insertionally inactivated were both multidrug resistance and reserpine resistance lost. Strains in which patA was insertionally inactivated grew significantly more slowly than the wild type. These data indicate that the overexpression of both patA and patB confers multidrug resistance in S. pneumoniae but that only patA is involved in reserpine resistance. The selection of reserpine-resistant multidrug-resistant pneumococci has implications for analogous systems in other bacteria or in cancer. PMID:18362193

  4. New type of osmoregulated solute transporter identified in halophilic members of the bacteria domain: TRAP transporter TeaABC mediates uptake of ectoine and hydroxyectoine in Halomonas elongata DSM 2581(T).

    PubMed

    Grammann, Katrin; Volke, Angela; Kunte, Hans Jörg

    2002-06-01

    The halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. We constructed a deletion mutant of H. elongata, KB1, defective in ectoine synthesis and tolerating elevated salt concentrations only in the presence of external compatible solutes. The dependency of KB1 on solute uptake for growth in high-salt medium was exploited to select insertion mutants unable to accumulate external solutes via osmoregulated transporters. One insertion mutant out of 7,200 failed to accumulate the osmoprotectants ectoine and hydroxyectoine. Genetic analysis of the insertion site proved that the mutation affected an open reading frame (ORF) of 1,281 bp (teaC). The nucleotide sequence upstream of teaC was determined, and two further ORFs of 603 bp (teaB) and 1,023 bp (teaA) were identified. Deletion of teaA and teaB proved that all three genes are mandatory for ectoine uptake. Sequence comparison showed significant identity of TeaA, TeaB, and TeaC to the transport proteins of the recently identified tripartite ATP-independent periplasmic transporter family (TRAP-T). The affinity of the cells for ectoines was determined (K(s) = 21.7 microM), suggesting that the transporter TeaABC exhibits high affinity for ectoines. An elevation of the external osmolarity resulted in a strong increase in ectoine uptake via TeaABC, demonstrating that this transporter is osmoregulated. Deletion of teaC and teaBC in the wild-type strain led to mutants which excreted significant amounts of ectoine into the medium when cultivated at high salt concentrations. Therefore, the physiological role of TeaABC may be primarily to recover ectoine leaking through the cytoplasmic membrane.

  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. The ABC Transporter Encoded at the Pneumococcal Fructooligosaccharide Utilization Locus Determines the Ability To Utilize Long- and Short-Chain Fructooligosaccharides

    PubMed Central

    Linke, Caroline M.; Woodiga, Shireen A.; Meyers, Dustin J.; Buckwalter, Carolyn M.; Salhi, Hussam E.

    2013-01-01

    Streptococcus pneumoniae is an important human pathogen that requires carbohydrates for growth. The significance of carbohydrate acquisition is highlighted by the genome encoding more than 27 predicted carbohydrate transporters. It has long been known that about 60% of pneumococci could utilize the fructooligosaccharide inulin as a carbohydrate source, but the mechanism of utilization was unknown. Here we demonstrate that a predicted sucrose utilization locus is actually a fructooligosaccharide utilization locus and imparts the ability of pneumococci to utilize inulin. Genes in strain TIGR4 predicted to encode an ABC transporter (SP_1796-8) and a β-fructosidase (SP_1795) are required for utilization of several fructooligosaccharides longer than kestose, which consists of two β(2-1)-linked fructose molecules with a terminal α(1-2)-linked glucose molecule. Similar to other characterized pneumococcal carbohydrate utilization transporter family 1 transporters, growth is dependent on the gene encoding the ATPase MsmK. While the majority of pneumococcal strains encode SP_1796-8 at this genomic location, 19% encode an alternative transporter. Although strains encoding either transporter can utilize short-chain fructooligosaccharides for growth, only strains encoding SP_1796-8 can utilize inulin. Exchange of genes encoding the SP_1796-8 transporter for those encoding the alternative transporter resulted in a TIGR4 strain that could utilize short-chain fructooligosaccharide but not inulin. These data demonstrate that the transporter encoded at this locus determines the ability of the bacteria to utilize long-chain fructooligosaccharides and explains the variation in inulin utilization between pneumococcal strains. PMID:23264576

  7. 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...

  8. 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

  9. TrmB, a sugar sensing regulator of ABC transporter genes in Pyrococcus furiosus exhibits dual promoter specificity and is controlled by different inducers.

    PubMed

    Lee, Sung-Jae; Moulakakis, Christina; Koning, Sonja M; Hausner, Winfried; Thomm, Michael; Boos, Winfried

    2005-09-01

    TrmB is the transcriptional repressor for the gene cluster of the trehalose/maltose ABC transporter of the hyperthermophilic archaea Thermococcus litoralis and Pyrococcus furiosus (malE or TM operon), with maltose and trehalose acting as inducers. We found that TrmB (the protein is identical in both organisms) also regulated the transcription of genes encoding a separate maltodextrin ABC transporter in P. furiosus (mdxE or MD operon) with maltotriose, longer maltodextrins and sucrose acting as inducers, but not with maltose or trehalose. In vitro transcription of the malE and the mdxE operons was inhibited by TrmB binding to the different operator sequences. Inhibition of the TM operon was released by maltose and trehalose whereas inhibition of the MD operon was released by maltotriose and larger maltodextrins as well as by sucrose. Scanning mutagenesis of the TM operator revealed the role of the palindromic TACTNNNAGTA sequence for TrmB recognition. TrmB exhibits a broad spectrum of sugar-binding specificity, binding maltose, sucrose, maltotriose and trehalose in decreasing order of affinity, half-maximal binding occurring at 20, 60, 250 and 500 microM substrate concentration respectively. Of all substrates, only maltose shows sigmoidal binding characteristics with a Hill coefficient of 2. As measured by molecular sieve chromatography and cross-linking TrmB behaved as dimer in dilute buffer solution at room temperature. We conclude that TrmB acts as a bifunctional transcriptional regulator acting on two different promoters and being differentially controlled by binding to different sugars. We believe this to represent a novel strategy of prokaryotic transcription regulation.

  10. Nilotinib enhances the efficacy of conventional chemotherapeutic drugs in CD34⁺CD38⁻ stem cells and ABC transporter overexpressing leukemia cells.

    PubMed

    Wang, Fang; Wang, Xiao-Kun; Shi, Cheng-Jun; Zhang, Hui; Hu, Ya-Peng; Chen, Yi-Fan; Fu, Li-Wu

    2014-03-19

    Incomplete chemotherapeutic eradication of leukemic CD34⁺CD38⁻ stem cells is likely to result in disease relapse. The purpose of this study was to evaluate the effect of nilotinib on eradicating leukemia stem cells and enhancing the efficacy of chemotherapeutic agents. Our results showed that ABCB1 and ABCG2 were preferentially expressed in leukemic CD34⁺CD38⁻ cells. Nilotinib significantly enhanced the cytotoxicity of doxorubicin and mitoxantrone in CD34⁺CD38⁻ cells and led to increased apoptosis. Moreover, nilotinib strongly reversed multidrug resistance and increased the intracellular accumulation of rhodamine 123 in primary leukemic blasts overexpressing ABCB1 and/or ABCG2. Studies with ABC transporter-overexpressing carcinoma cell models confirmed that nilotinib effectively reversed ABCB1- and ABCG2-mediated drug resistance, while showed no significant reversal effect on ABCC1- and ABCC4-mediated drug resistance. Results from cytotoxicity assays showed that CD34⁺CD38⁻ cells exhibited moderate resistance (2.41-fold) to nilotinib, compared with parental K562 cells. Furthermore, nilotinib was less effective in blocking the phosphorylation of Bcr-Abl and CrkL (a substrate of Bcr-Abl kinase) in CD34⁺CD38⁻ cells. Taken together, these data suggest that nilotinib particularly targets CD34⁺CD38⁻ stem cells and MDR leukemia cells, and effectively enhances the efficacy of chemotherapeutic drugs by blocking the efflux function of ABC transporters.

  11. 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.

  12. Effects of in vitro exposure to ivermectin and levamisole on the expression patterns of ABC transporters in Haemonchus contortus larvae.

    PubMed

    Raza, Ali; Kopp, Steven R; Bagnall, Neil H; Jabbar, Abdul; Kotze, Andrew C

    2016-08-01

    This study investigated the interaction of ATP binding cassette (ABC) transport proteins with ivermectin (IVM) and levamisole (LEV) in larvae of susceptible and resistant isolates of Haemonchus contortus in vitro by measuring transcription patterns following exposure to these anthelmintics. Furthermore, we studied the consequences of drug exposure by measuring the sensitivity of L3 to subsequent exposure to higher drug concentrations using larval migration assays. The most highly transcribed transporter genes in both susceptible and resistant L3 were pgp-9.3, abcf-1, mrp-5, abcf-2, pgp-3, and pgp-10. The resistant isolate showed significantly higher transcription of pgp-1, pgp-9.1 and pgp-9.2 compared to the susceptible isolate. Five P-gp genes and the haf-6 gene showed significantly higher transcription (up to 12.6-fold) after 3 h exposure to IVM in the resistant isolate. Similarly, five P-gp genes, haf-6 and abcf-1 were transcribed at significantly higher levels (up to 10.3-fold) following 3 h exposure to LEV in this isolate. On the other hand, there were no significant changes in transcriptional patterns of all transporter genes in the susceptible isolate following 3 and 6 h exposure to IVM or LEV. In contrast to these isolate-specific transcription changes, both isolates showed an increase in R-123 efflux following exposure to the drugs, suggesting that the drugs stimulated activity of existing transporter proteins in both isolates. Exposure of resistant larvae to IVM or LEV resulted, in some instances, in an increase in the proportion of the population able to migrate at the highest IVM concentrations in subsequent migration assays. The significant increase in transcription of some ABC transporter genes following 3 h exposure to both IVM and LEV in the resistant isolate only, suggests that an ability to rapidly upregulate protective pathways in response to drugs may be a component of the resistance displayed by this isolate. PMID:27164439

  13. OusB, a Broad-Specificity ABC-Type Transporter from Erwinia chrysanthemi, Mediates Uptake of Glycine Betaine and Choline with a High Affinity

    PubMed Central

    Choquet, Gwénaëlle; Jehan, Nathalie; Pissavin, Christine; Blanco, Carlos; Jebbar, Mohamed

    2005-01-01

    The ability of Erwinia chrysanthemi to cope with environments of elevated osmolality is due in part to the transport and accumulation of osmoprotectants. In this study we have identified a high-affinity glycine betaine and choline transport system in E. chrysanthemi. By using a pool of Tn5-B21 ousA mutants, we isolated a mutant that could grow in the presence of a toxic analogue of glycine betaine (benzyl-glycine betaine) at high osmolalities. This mutant was impaired in its ability to transport all effective osmoprotectants in E. chrysanthemi. The DNA sequence of the regions flanking the transposon insertion site revealed three chromosomal genes (ousVWX) that encode components of an ABC-type transporter (OusB): OusV (ATPase), OusW (permease), and OusX (periplasmic binding protein). The OusB components showed a significant degree of sequence identity to components of ProU from Salmonella enterica serovar Typhimurium and Escherichia coli. OusB was found to restore the uptake of glycine betaine and choline through functional complementation of an E. coli mutant defective in both ProU and ProP osmoprotectant uptake systems. Competition experiments demonstrated that choline, dimethylsulfoniacetate, dimethylsulfoniopropionate, and ectoine were effective competitors for OusB-mediated betaine transport but that carnitine, pipecolate, and proline were not effective. In addition, the analysis of single and double mutants showed that OusA and OusB were the only osmoprotectant transporters operating in E. chrysanthemi. PMID:16000740

  14. 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

  15. Preliminary time-of-flight neutron diffraction studies of Escherichia coli ABC transport receptor phosphate-binding protein at the Protein Crystallography Station

    PubMed Central

    Sippel, K. H.; Bacik, J.; Quiocho, F. A.; Fisher, S. Z.

    2014-01-01

    Inorganic phosphate is an essential molecule for all known life. Organisms have developed many mechanisms to ensure an adequate supply, even in low-phosphate conditions. In prokaryotes phosphate transport is instigated by the phosphate-binding protein (PBP), the initial receptor for the ATP-binding cassette (ABC) phosphate transporter. In the crystal structure of the PBP–phosphate complex, the phosphate is completely desolvated and sequestered in a deep cleft and is bound by 13 hydrogen bonds: 12 to protein NH and OH donor groups and one to a carboxylate acceptor group. The carboxylate plays a key recognition role by accepting a phosphate hydrogen. PBP phosphate affinity is relatively consistent across a broad pH range, indicating the capacity to bind monobasic (H2PO4 −) and dibasic (HPO4 2−) phosphate; however, the mechanism by which it might accommodate the second hydrogen of monobasic phosphate is unclear. To answer this question, neutron diffraction studies were initiated. Large single crystals with a volume of 8 mm3 were grown and subjected to hydrogen/deuterium exchange. A 2.5 Å resolution data set was collected on the Protein Crystallography Station at the Los Alamos Neutron Science Center. Initial refinement of the neutron data shows significant nuclear density, and refinement is ongoing. This is the first report of a neutron study from this superfamily. PMID:24915101

  16. TrmB, a sugar-specific transcriptional regulator of the trehalose/maltose ABC transporter from the hyperthermophilic archaeon Thermococcus litoralis.

    PubMed

    Lee, Sung-Jae; Engelmann, Afra; Horlacher, Reinhold; Qu, Qiuhao; Vierke, Gudrun; Hebbeln, Carina; Thomm, Michael; Boos, Winfried

    2003-01-10

    We report the characterization of TrmB, a protein of 38,800 apparent molecular weight, that is involved in the maltose-specific regulation of a gene cluster in Thermococcus litoralis, malE malF malG orf trmB malK, encoding a binding protein-dependent ABC transporter for trehalose and maltose. TrmB binds maltose and trehalose half-maximally at 20 microm and 0.5 mm sugar concentration, respectively. Binding of maltose but not of trehalose showed indications of sigmoidality and quenched the intrinsic tryptophan fluorescence by 15%, indicating a conformational change on maltose binding. TrmB causes a shift in electrophoretic mobility of DNA fragments harboring the promoter and upstream regulatory motif identified by footprinting. Band shifting by TrmB can be prevented by maltose. In vitro transcription assays with purified components from Pyrococcus furiosus have been established to show pmalE promoter-dependent transcription at 80 degrees C. TrmB specifically inhibits transcription, and this inhibition is counteracted by maltose and trehalose. These data characterize TrmB as a maltose-specific repressor for the trehalose/maltose transport operon of Thermococcus litoralis.

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

    PubMed Central

    Bartsevich, V V; Pakrasi, H B

    1995-01-01

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

  18. 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

  19. Overexpression of patA and patB, Which Encode ABC Transporters, Is Associated with Fluoroquinolone Resistance in Clinical Isolates of Streptococcus pneumoniae▿

    PubMed Central

    Garvey, Mark I.; Baylay, Alison J.; Wong, Ryan L.; Piddock, Laura J. V.

    2011-01-01

    Fifty-seven clinical isolates of Streptococcus pneumoniae were divided into four groups based on their susceptibilities to the fluoroquinolones ciprofloxacin and norfloxacin and the dyes ethidium bromide and acriflavine. Comparative reverse transcription-PCR was used to determine the level of expression of the genes patA and patB, which encode putative ABC transporters. Overexpression was observed in 14 of the 15 isolates that were resistant to both fluoroquinolones and dyes and in only 3 of 24 of those resistant to fluoroquinolones only. Isolates overexpressing patA and patB accumulated significantly less of the fluorescent dye Hoechst 33342 than wild-type isolates, suggesting that PatA and PatB are involved in efflux. Inactivation of patA and patB by in vitro mariner mutagenesis conferred hypersusceptibility to ethidium bromide and acriflavine in all isolates tested and lowered the MICs of ciprofloxacin in the patAB-overproducing and/or fluoroquinolone-resistant isolates. These data represent the first observation of overexpression of patA and patB in clinical isolates and show that PatA and PatB play a clinically relevant role in fluoroquinolone resistance. PMID:20937787

  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.

  1. 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

  2. Attenuated mutant strain of Salmonella Typhimurium lacking the ZnuABC transporter contrasts tumor growth promoting anti-cancer immune response

    PubMed Central

    Chirullo, Barbara; Ammendola, Serena; Leonardi, Leonardo; Falcini, Roberto; Petrucci, Paola; Pistoia, Claudia; Vendetti, Silvia; Battistoni, Andrea; Pasquali, Paolo

    2015-01-01

    Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens. We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors. PMID:26158862

  3. Involvement of a Novel ABC Transporter and Monoalkyl Phthalate Ester Hydrolase in Phthalate Ester Catabolism by Rhodococcus jostii RHA1▿

    PubMed Central

    Hara, Hirofumi; Stewart, Gordon R.; Mohn, William W.

    2010-01-01

    Phthalate esters (PEs) are important environmental pollutants. While the biodegradation of the parent compound, phthalate (PTH), is well characterized, the biodegradation of PEs is not well understood. In particular, prior to this study, genes involved in the uptake and hydrolysis of these compounds were not conclusively identified. We found that Rhodococcus jostii RHA1 could grow on a variety of monoalkyl PEs, including methyl, butyl, hexyl, and 2-ethylhexyl PTHs. Strain RHA1 could not grow on most dialkyl PEs, but suspensions of cells grown on PTH transformed dimethyl, diethyl, dipropyl, dibutyl, dihexyl and di-(2-ethylhexyl) PTHs. The major products of these dialkyl PEs were PTH and the corresponding monoalkyl PEs, and minor products resulted from the shortening of the alkyl side chains. RHA1 exhibited an inducible, ATP-dependent uptake system for PTH with a Km of 22 μM. The deletion and complementation of the patB gene demonstrated that the ATP-binding cassette (ABC) transporter encoded by patDABC is required for the uptake of PTH and monoalkyl PEs by RHA1. The hydrolase encoded by patE of RHA1 was expressed in Escherichia coli. PatE specifically hydrolyzed monoalkyl PEs to PTH but did not transform dialkyl PEs or other aromatic esters. This investigation of RHA1 elucidates key processes that are consistent with the environmental fate of PEs. PMID:20038686

  4. Differential expression of peroxidase and ABC transporter as the key regulatory components for degradation of azo dyes by Penicillium oxalicum SAR-3.

    PubMed

    Saroj, Samta; Kumar, Karunesh; Prasad, Manoj; Singh, R P

    2014-12-01

    Fungal species are potential dye decomposers since these secrete spectra of extracellular enzymes involved in catabolism. However, cellular mechanisms underlying azo dye catalysis and detoxification are incompletely understood and obscure. A potential strain designated as Penicillium oxalicum SAR-3 demonstrated broad-spectrum catabolic ability of different azo dyes. A forward suppression subtractive hybridization (SSH) cDNA library of P. oxalicum SAR-3 constructed in presence and absence of azo dye Acid Red 183 resulted in identification of 183 unique expressed sequence tags (ESTs) which were functionally classified into 12 functional categories. A number of novel genes that affect specifically organic azo dye degradation were discovered. Although the ABC transporters and peroxidases emerged as prominent hot spot for azo dye detoxification, we also identified a number of proteins that are more proximally related to stress-responsive gene expression. Majority of the ESTs (29.5%) were grouped as hypothetical/unknown indicating the presence of putatively novel genes. Analysis of few ESTs through quantitative real-time reverse transcription polymerase chain reaction revealed their possible role in AR183 degradation. The ESTs identified in the SSH library provide a novel insight on the transcripts that are expressed in P. oxalicum strain SAR-3 in response to AR183.

  5. 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

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

    PubMed

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

    2012-03-01

    Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains. PMID:22203607

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

    PubMed

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

    2014-10-01

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

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

    PubMed Central

    Cole, Susan P. C.

    2014-01-01

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

  9. Ectoine-induced proteins in Sinorhizobium meliloti include an Ectoine ABC-type transporter involved in osmoprotection and ectoine catabolism.

    PubMed

    Jebbar, Mohamed; Sohn-Bösser, Linda; Bremer, Erhard; Bernard, Théophile; Blanco, Carlos

    2005-02-01

    To understand the mechanisms of ectoine-induced osmoprotection in Sinorhizobium meliloti, a proteomic examination of S. meliloti cells grown in minimal medium supplemented with ectoine was undertaken. This revealed the induction of 10 proteins. The protein products of eight genes were identified by using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Five of these genes, with four other genes whose products were not detected on two-dimensional gels, belong to the same gene cluster, which is localized on the pSymB megaplasmid. Four of the nine genes encode the characteristic components of an ATP-binding cassette transporter that was named ehu, for ectoine/hydroxyectoine uptake. This transporter was encoded by four genes (ehuA, ehuB, ehuC, and ehuD) that formed an operon with another gene cluster that contains five genes, named eutABCDE for ectoine utilization. On the basis of sequence homologies, eutABCDE encode enzymes with putative and hypothetical functions in ectoine catabolism. Analysis of the properties of ehuA and eutA mutants suggests that S. meliloti possesses at least one additional ectoine catabolic pathway as well as a lower-affinity transport system for ectoine and hydroxyectoine. The expression of ehuB, as determined by measurements of UidA activity, was shown to be induced by ectoine and hydroxyectoine but not by glycine betaine or by high osmolality.

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

    PubMed

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

    2016-01-01

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

  11. High-throughput screening of dipeptide utilization mediated by the ABC transporter DppBCDF and its substrate-binding proteins DppA1-A5 in Pseudomonas aeruginosa.

    PubMed

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

    2014-01-01

    In this study, we show that the dppBCDF operon of Pseudomonas aeruginosa PA14 encodes an ABC transporter responsible for the utilization of di/tripeptides. The substrate specificity of ABC transporters is determined by its associated substrate-binding proteins (SBPs). Whereas in E. coli only one protein, DppA, determines the specificity of the transporter, five orthologous SBPs, DppA1-A5 are present in P. aeruginosa. Multiple SBPs might broaden the substrate specificity by increasing the transporter capacity. We utilized the Biolog phenotype MicroArray technology to investigate utilization of di/tripeptides in mutants lacking either the transport machinery or all of the five SBPs. This high-throughput method enabled us to screen hundreds of dipeptides with various side-chains, and subsequently, to determine the substrate profile of the dipeptide permease. The substrate spectrum of the SBPs was elucidated by complementation of a penta mutant, deficient of all five SBPs, with plasmids carrying individual SBPs. It became apparent that some dipeptides were utilized with different affinity for each SBP. We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides. DppA5 was not able to complement the penta mutant under our screening conditions. Phaseolotoxin, a toxic tripeptide inhibiting the enzyme ornithine carbamoyltransferase, is also transported into P. aeruginosa via the DppBCDF permease. The SBP DppA1, and with much greater extend DppA3, are responsible for delivering the toxin to the permease. Our results provide a first overview of the substrate pattern of the ABC dipeptide transport machinery in P. aeruginosa. PMID:25338022

  12. 75 FR 49549 - ABC & D Recycling, Inc.-Lease and Operation Exemption-a Line of Railroad in Ware, MA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-13

    ... Surface Transportation Board ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, MA ABC & D Recycling, Inc. (ABC & D), a noncarrier, has filed a verified notice of exemption... operation of this trackage in FD 35356, ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line...

  13. Identification of ABC transporter genes conferring combined pleuromutilin-lincosamide-streptogramin A resistance in bovine methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci.

    PubMed

    Wendlandt, Sarah; Kadlec, Kristina; Feßler, Andrea T; Schwarz, Stefan

    2015-06-12

    The aim of this study was to investigate the genetic basis of combined pleuromutilin-lincosamide-streptogramin A resistance in 26 unrelated methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) from dairy cows suffering from mastitis. The 26 pleuromutilin-resistant staphylococcal isolates were screened for the presence of the genes vga(A), vga(B), vga(C), vga(E), vga(E) variant, sal(A), vmlR, cfr, lsa(A), lsa(B), lsa(C), and lsa(E) by PCR. None of the 26 isolates carried the genes vga(B), vga(C), vga(E), vga(E) variant, vmlR, cfr, lsa(A), lsa(B), or lsa(C). Two Staphylococcus haemolyticus and single Staphylococcus xylosus, Staphylococcus lentus, and Staphylococcus hominis were vga(A)-positive. Twelve S. aureus, two Staphylococcus warneri, as well as single S. lentus and S. xylosus carried the lsa(E) gene. Moreover, single S. aureus, S. haemolyticus, S. xylosus, and Staphylococcus epidermidis were positive for both genes, vga(A) and lsa(E). The sal(A) gene was found in a single Staphylococcus sciuri. All ABC transporter genes were located in the chromosomal DNA, except for a plasmid-borne vga(A) gene in the S. epidermidis isolate. The genetic environment of the lsa(E)-positive isolates was analyzed using previously described PCR assays. Except for the S. warneri and S. xylosus, all lsa(E)-positive isolates harbored a part of the previously described enterococcal multiresistance gene cluster. This is the first report of the novel lsa(E) gene in the aforementioned bovine CoNS species. This is also the first identification of the sal(A) gene in a S. sciuri from a case of bovine mastitis. Moreover, the sal(A) gene was shown to also confer pleuromutilin resistance.

  14. 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

  15. Haemonchus contortus P-Glycoproteins Interact with Host Eosinophil Granules: A Novel Insight into the Role of ABC Transporters in Host-Parasite Interaction

    PubMed Central

    Issouf, Mohamed; Guégnard, Fabrice; Koch, Christine; Le Vern, Yves; Blanchard-Letort, Alexandra; Che, Hua; Beech, Robin N.; Kerboeuf, Dominique; Neveu, Cedric

    2014-01-01

    Eosinophils are one of the major mammalian effector cells encountered by helminths during infection. In the present study, we investigated the effects of eosinophil granule exposure on the sheep parasitic nematode Haemonchus contortus as a model. H. contortus eggs exposed to eosinophil granule products showed increased rhodamine 123 efflux and this effect was not due to loss of egg integrity. Rh123 is known to be a specific P-glycoprotein (Pgp) substrate and led to the hypothesis that in addition to their critical role in xenobiotic resistance, helminth ABC transporters such as Pgp may also be involved in the detoxification of host cytotoxic products. We showed by quantitative RT-PCR that, among nine different H. contortus Pgp genes, Hco-pgp-3, Hco-pgp-9.2, Hco-pgp-11 and, Hco-pgp-16 were specifically up-regulated in parasitic life stages suggesting a potential involvement of these Pgps in the detoxification of eosinophil granule products. Using exsheathed L3 larvae that mimic the first life stage in contact with the host, we demonstrated that eosinophil granules induced a dose dependent overexpression of Hco-pgp-3 and the closely related Hco-pgp-16. Taken together, our results provide the first evidence that a subset of helminth Pgps interact with, and could be involved in the detoxification of, host products. This opens the way for further studies aiming to explore the role of helminth Pgps in the host-parasite interaction, including evasion of the host immune response. PMID:24498376

  16. 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

  17. Physicochemical factors controlling the activity and energy coupling of an ionic strength-gated ATP-binding cassette (ABC) transporter.

    PubMed

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

    2013-10-11

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

  18. Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis

    PubMed Central

    Otsuka, Taketo; Kirkham, Charmaine; Johnson, Antoinette; Jones, Megan M.

    2014-01-01

    Moraxella catarrhalis is a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeable Haemophilus influenzae, M. catarrhalis has become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, of M. catarrhalis. Among 30 clinical isolates tested, the sbp2 gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not the sbp2 mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance of M. catarrhalis from the lung compared to that in the control group at both 25-μg and 50-μg doses (P < 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen against M. catarrhalis. PMID:24914218

  19. Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa leads to increased ampicillin and carbenicillin resistance and decreased virulence.

    PubMed

    Yang, Liang; Chen, Lin; Shen, Lixin; Surette, Michael; Duan, Kangmin

    2011-02-01

    Resistance-Nodulation-Cell Division (RND) pumps play important roles in bacterial resistance to antibiotics. Pseudomonas aeruginosa is an important human pathogen which exhibits high level resistance to antibiotics. There are total of 12 RND pumps present in the P. aeruginosa PAOl genome. The recently characterized MuxABC-OpmB system has been shown to play a role in resistance to novobiocin, aztreonam, macrolides, and tetracycline in a multiple knockout mutation. In this study, we examined the expression levels of all the 12 RND pump gene clusters and tested the involvement of MuxABC-OpmB in pathogenicity. The results indicated that in addition to the four known constitutively expressed RND pumps, mexAB-oprM, mexGHI-opmD, mexVW, and mexXY, relatively high levels of expression were observed with mexJK and muxABC-opmB in the conditions tested. Inactivation of muxA in the muxABC-opmB operon resulted in elevated resistance to ampicillin and carbenicillin. The mutant also showed attenuated virulence in both Brassica rapa pekinensis and Drosophila melanogaster infection models. The decreased virulence at least in part was due to decreased twitching motility in the mutant. These results indicate that the RND pump MuxABC-OpmB is associated with ampicillin and carbenicillin susceptibility and also involved in pathogenesis in P. aeruginosa.

  20. Basal and benzo[a]pyrene-induced expression profile of phase I and II enzymes and ABC transporter mRNA in the early life stage of Chinese rare minnows (Gobiocypris rarus).

    PubMed

    Yuan, Lilai; Lv, Biping; Zha, Jinmiao; Wang, Weimin; Wang, Zijian

    2014-08-01

    ATP-binding cassette (ABC) transporters together with phase I and II detoxification enzymes have been considered as included in a cellular detoxification system. Previous studies have highlighted the involvement of aryl hydrocarbon receptor (AHR) and Cyp1a in PAH-induced embryo toxicity. However, the response of other xenobiotic enzymes/transporters in PAH-mediated embryo toxicity is not fully characterized. In the present study, rare minnow embryos were exposed to 10 and 100µg/L BaP within 4h post-fertilization (hpf) up to 168 hpf. RNA was extracted at 24, 48, 96, and 168 hpf. The basal and BaP-induced expression of phase I enzyme genes (cyp1a, 1b1, and 1c1), phase II enzyme gene (gstm and ugt1a), and ABC transporter genes (abcb1, abcc1, abcc2, and abcg2) mRNA was determined using real-time PCR. Severe developmental defects (e.g., spinal deformities, pericardial and yolk-sac edema) were observed in the BaP treated groups. The basal expression showed that gstm was most strongly expressed, followed by abcb1, ugt1a, and abcc2, whereas cyp1a, 1b1, 1c1, and abcg2 showed weak expression. BaP significantly induced the mRNA expression of three CYP1s (cyp1a, 1b1, and 1c1) (p<0.05) and the ABC transporters (abcc1, abcc2, and abcg2) in a dose-dependent manner. However, the mRNA expression of Phase II enzymes (gstm, ugt1a) for the BaP treatments showed no significant difference with that of the controls. Furthermore, distinct induced patterns of these genes were observed during different exposure periods. Simultaneous up-regulation of the cyp and ABC transporter gene transcripts suggests that a possible involvement and cooperation in the detoxification process could provide protection against the BaP toxicity of rare minnows at the early life stage.

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

    PubMed

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

    2015-01-01

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

  2. Encapsulated Brucella ovis Lacking a Putative ATP-Binding Cassette TransporterabcBA) Protects against Wild Type Brucella ovis in Rams

    PubMed Central

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

    2015-01-01

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

  3. Axial ligand replacement mechanism in heme transfer from streptococcal heme-binding protein Shp to HtsA of the HtsABC transporter.

    PubMed

    Ran, Yanchao; Malmirchegini, G Reza; Clubb, Robert T; Lei, Benfang

    2013-09-17

    The heme-binding protein Shp of Group A Streptococcus rapidly transfers its heme to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. Heme axial residue-to-alanine replacement mutant proteins of Shp and HtsA (Shp(M66A), Shp(M153A), HtsA(M79A), and HtsA(H229A)) were used to probe the axial displacement mechanism of this heme transfer reaction. Ferric Shp(M66A) at high pH and Shp(M153A) have a pentacoordinate heme iron complex with a methionine axial ligand. ApoHtsA(M79A) efficiently acquires heme from ferric Shp but alters the reaction mechanism to two kinetic phases from a single phase in the wild-type protein reactions. In contrast, apoHtsA(H229A) cannot assimilate heme from ferric Shp. The conversion of pentacoordinate holoShp(M66A) into pentacoordinate holoHtsA(H229A) involves an intermediate, whereas holoHtsA(H229A) is directly formed from pentacoordinate holoShp(M153A). Conversely, apoHtsA(M79A) reacts with holoShp(M66A) and holoShp(M153A) in mechanisms with one and two kinetic phases, respectively. These results imply that the Met79 and His229 residues of HtsA displace the Met66 and Met153 residues of Shp, respectively. Structural docking analysis supports this mechanism of the specific axial residue displacement. Furthermore, the rates of the cleavage of the axial bond in Shp in the presence of a replacing HtsA axial residue are greater than that in the absence of a replacing HtsA axial residue. These findings reveal a novel heme transfer mechanism of the specific displacement of the Shp axial residues with the HtsA axial residues and the involvement of the HtsA axial residues in the displacement.

  4. A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins.

    PubMed

    Hung, Miao-Chiu; Humbert, María Victoria; Laver, Jay R; Phillips, Renee; Heckels, John E; Christodoulides, Myron

    2015-08-26

    The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre <4) in a human serum bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new

  5. 75 FR 11991 - ABC & D Recycling, Inc.-Lease and Operation Exemption-a Line of Railroad in Ware, MA

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-12

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF TRANSPORTATION Surface Transportation Board ABC & D Recycling, Inc.--Lease and Operation Exemption--a Line of Railroad in Ware, MA ABC & D Recycling, Inc. (ABC & D), a noncarrier, has filed a verified notice of...

  6. ABC proteins protect the human body and maintain optimal health.

    PubMed

    Ueda, Kazumitsu

    2011-01-01

    Human MDR1, a multi-drug transporter gene, was isolated as the first of the eukaryote ATP Binding Cassette (ABC) proteins from a multidrug-resistant carcinoma cell line in 1986. To date, over 25 years, many ABC proteins have been found to play important physiological roles by transporting hydrophobic compounds. Defects in their functions cause various diseases, indicating that endogenous hydrophobic compounds, as well as water-soluble compounds, are properly transported by transmembrane proteins. MDR1 transports a large number of structurally unrelated drugs and is involved in their pharmacokinetics, and thus is a key factor in drug interaction. ABCA1, an ABC protein, eliminates excess cholesterol in peripheral cells by generating HDL. Because ABCA1 is a key molecule in cholesterol homeostasis, its function and expression are highly regulated. Eukaryote ABC proteins function on the body surface facing the outside and in organ pathways to adapt to the extracellular environment and protect the body to maintain optimal health.

  7. Protective effect of a DNA vaccine containing an open reading frame with homology to an ABC-type transporter present in the genomic island 3 of Brucella abortus in BALB/c mice.

    PubMed

    Riquelme-Neira, Roberto; Retamal-Díaz, Angello; Acuña, Francisca; Riquelme, Pablo; Rivera, Alejandra; Sáez, Darwin; Oñate, Angel

    2013-08-12

    The immunogenicity of a DNA vaccine containing an open reading frame (ORF) of genomic island 3 (GI-3), specific for Brucella abortus and Brucella melitensis, has been examined. Intramuscular injection of plasmid DNA carrying the open reading frame with homology to an ABC-type transporter (pV278a) into BALB/c mice elicited both humoral and cellular immune responses. Mice injected with pV278a had a dominant immunoglobulin G2a (IgG2a) response. This DNA vaccine elicited a T-cell-proliferative response and induced significant levels of interferon gamma (INF-γ) upon restimulation with recombinant 278a protein. Upon stimulation with an appropriate recombinant protein or crude Brucella protein, the vaccine did not induce IL-4, suggesting a typical T-helper (TH1) response. Furthermore, the vaccine induced protection in BALB/c mice when challenged with the virulent strain Brucella abortus 2308. Taken together, these data suggest that DNA vaccination offers an improved delivery of the homologous of an ABC-type transporter antigen, and provides the first evidence of a protective effect of this antigen in the construction of vaccines against B. abortus.

  8. Role of an ABC importer in mycobacterial drug resistance.

    PubMed

    Chakraborti, P K; Bhatt, K; Banerjee, S K; Misra, P

    1999-08-01

    Phosphate specific transporter (Pst) in bacteria is involved in phosphate transport. Pst is a multisubunit system which belongs to the ABC family of transporters. The import function of this transporter is known to be operative at media phosphate concentrations below the millimolar range. However, we found amplification of this transporter in a laboratory generated ciprofloxacin resistant Mycobacterium smegmatis colony (CIPr) which was grown in a condition when phosphate scavenging function of this operon was inoperative. Our results therefore argue the role of this ABC importer in conferring high level of fluoroquinolone resistance in CIPr.

  9. ABC's of Being Smart

    ERIC Educational Resources Information Center

    Foster, Joanne

    2011-01-01

    Determining what giftedness is all about means focusing on many aspects of the individual. In this paper, the author focuses on letter D of the ABC's of being smart. She starts with specifics about giftedness (details), and then moves on to some ways of thinking (dispositions).

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

    PubMed Central

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

    2014-01-01

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

  11. 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

  12. A novel mode of ferric ion coordination by the periplasmic ferric ion-binding subunit FbpA of an ABC-type iron transporter from Thermus thermophilus HB8.

    PubMed

    Wang, Shipeng; Ogata, Misaki; Horita, Shoichiro; Ohtsuka, Jun; Nagata, Koji; Tanokura, Masaru

    2014-01-01

    Crystal structures of FbpA, the periplasmic ferric ion-binding protein of an iron-uptake ABC transporter, from Thermus thermophilus HB8 (TtFbpA) have been solved in apo and ferric ion-bound forms at 1.8 and 1.7 Å resolution, respectively. The latter crystal structure shows that the bound ferric ion forms a novel six-coordinated complex with three tyrosine side chains, two bicarbonates and a water molecule in the metal-binding site. The results of gel-filtration chromatography and dynamic light scattering show that TtFbpA exists as a monomer in solution regardless of ferric ion binding and that TtFbpA adopts a more compact conformation in the ferric ion-bound state than in the apo state in solution.

  13. Learning the ABC of oral fungal drug resistance.

    PubMed

    Cannon, R D; Holmes, A R

    2015-12-01

    ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals. PMID:26042641

  14. Learning the ABC of oral fungal drug resistance.

    PubMed

    Cannon, R D; Holmes, A R

    2015-12-01

    ATP-binding cassette (ABC) proteins are ubiquitous in prokaryotes and eukaryotes. They are involved in energy-dependent transport of molecules across membranes. ABC proteins are often promiscuous transporters that can translocate a variety of substrates. In oral fungi, especially in Candida species, they have been implicated as major contributors to the high-level azole resistance of clinical isolates from infections that do not respond to drug therapy. Although this is predominantly due to efflux of azoles from the cells, ABC proteins can contribute to fungal drug resistance in other ways as well. Cells in biofilms are notoriously resistant to antifungal agents. ABC proteins can contribute to this resistance through the efflux of drugs. Biofilms are complex communities of myriad microorganisms which, to survive in such a milieu, need to communicate with, and respond to, other microorganisms and their products. ABC proteins are involved in the secretion of fungal mating factors and quorum sensing molecules. These molecules affect biofilm structure and behavior that can result in increased drug resistance. Hence, ABC proteins make multiple contributions to oral fungal drug resistance through a variety of responses to environmental signals.

  15. Do You Know Your ABC?

    ERIC Educational Resources Information Center

    Neale, Claire

    2013-01-01

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

  16. Genome-wide comparative analysis of ABC systems in the Bdellovibrio-and-like organisms

    PubMed Central

    Li, Nan; Chen, Huan; Williams, Henry N.

    2015-01-01

    Bdellovibrio -and-like organisms (BALOs) are gram-negative, predatory bacteria with wide variations in genome sizes and GC content and ecological habitats. The ATP-binding cassette (ABC) systems have been identified in several prokaryotes, fungi and plants and have a role in transport of materials in and out of cells and in cellular processes. However, knowledge of the ABC systems of BALOs remains obscure. A total of 269 putative ABC proteins were identified in BALOs. The genes encoding these ABC systems occupy nearly 1.3% of the gene content in freshwater Bdellovibrio strains and about 0.7% in their saltwater counterparts. The proteins found belong to 25 ABC system families based on their structural characteristics and functions. Among these, 16 families function as importers, 6 as exporters and 3 are involved in various cellular processes. Eight of these 25 ABC system families were deduced to be the core set of ABC systems conserved in all BALOs. All Bacteriovorax strains have 28 or less ABC systems. On the contrary, the freshwater Bdellovibrio strains have more ABC systems, typically around 51. In the genome of Bdellovibrio exovorus JSS (CP003537.1), 53 putative ABC systems were detected, representing the highest number among all the BALO genomes examined in this study. Unexpected high numbers of ABC systems involved in cellular processes were found in all BALOs. Phylogenetic analysis suggests that the majority of ABC proteins can be assigned into many separate families with high bootstrap supports (>50%). In this study, a general framework of sequence–structure–function connections for the ABC systems in BALOs was revealed providing novel insights for future investigations. PMID:25707746

  17. Genome organisation and expression profiling of ABC protein-encoding genes in Heterobasidion annosum s.l. complex.

    PubMed

    Baral, Bikash; Kovalchuk, Andriy; Asiegbu, Fred O

    2016-03-01

    Members of Heterobasidion annosum species complex are widely regarded as the most destructive fungal pathogens of conifer trees in the boreal and temperate zones of Northern hemisphere. To invade and colonise their host trees, Heterobasidion fungi must overcome components of host chemical defence, including terpenoid oleoresin and phenolic compounds. ABC transporters may play an important role in this process participating in the export of toxic host metabolites and maintaining their intracellular concentration below the critical level. We have identified and phylogenetically classified Heterobasidion genes encoding ABC transporters and closely related ABC proteins. The number of ABC proteins in the Heterobasidion genome is one of the lowest among analysed species of Agaricomycotina. Using quantitative RT-PCR, we have analysed transcriptional response of Heterobasidion ABC transporter-encoding genes to monoterpenes as well as their expression profile during growth on pine wood in comparison to the growth on defined media. Several ABC transporters were up-regulated during growth on pine wood. The ABC-transporter encoding gene ABCG1.1 was induced both during growth of H. annosum on pine wood and upon exposure to monoterpenes. Our experimental data demonstrate the differential responses of Heterobasidion ABC genes to growth conditions and chemical stressors. The presented results suggest a potential role of Heterobasidion ABC-G transporters in the resistance to the components of conifer chemical defence. PMID:26895866

  18. Bacitracin and nisin resistance in Staphylococcus aureus: a novel pathway involving the BraS/BraR two-component system (SA2417/SA2418) and both the BraD/BraE and VraD/VraE ABC transporters.

    PubMed

    Hiron, Aurélia; Falord, Mélanie; Valle, Jaione; Débarbouillé, Michel; Msadek, Tarek

    2011-08-01

    Two-component systems (TCSs) are key regulatory pathways allowing bacteria to adapt their genetic expression to environmental changes. Bacitracin, a cyclic dodecylpeptide antibiotic, binds to undecaprenyl pyrophosphate, the lipid carrier for cell wall precursors, effectively inhibiting peptidoglycan biosynthesis. We have identified a novel and previously uncharacterized TCS in the major human pathogen Staphylococcus aureus that we show to be essential for bacitracin and nisin resistance: the BraS/BraR system (Bacitracin resistance associated; SA2417/SA2418). The braRS genes are located immediately upstream from genes encoding an ABC transporter, accordingly designated BraDE. We have shown that the BraSR/BraDE module is a key bacitracin and nisin resistance determinant in S. aureus. In the presence of low antibiotic concentrations, BraSR activate transcription of two operons encoding ABC transporters: braDE and vraDE. We identified a highly conserved imperfect palindromic sequence upstream from the braDE and vraDE promoter sequences, essential for their transcriptional activation by BraSR, suggesting it is the likely BraR binding site. We demonstrated that the two ABC transporters play distinct and original roles in antibiotic resistance: BraDE is involved in bacitracin sensing and signalling through BraSR, whereas VraDE acts specifically as a detoxification module and is sufficient to confer bacitracin and nisin resistance when produced on its own. We show that these processes require functional BraD and VraD nucleotide-binding domain proteins, and that the large extracellular loop of VraE confers its specificity in bacitracin resistance. This is the first example of a TCS associated with two ABC transporters playing separate roles in signal transduction and antibiotic resistance. PMID:21696458

  19. The ABCs of Sex Ed.

    ERIC Educational Resources Information Center

    Sroka, Stephen R.

    2002-01-01

    Cites statistics on extent of sexually transmitted diseases and pregnancies among adolescents; describes ideological dispute over how to teach sex education; advocates teaching the ABCs of sex education: Abstinence, Be Monogamous, and Condoms. (PKP)

  20. The ABC of Ribosome-Related Antibiotic Resistance.

    PubMed

    Wilson, Daniel N

    2016-01-01

    The increase in multidrug-resistant pathogenic bacteria is limiting the utility of our current arsenal of antimicrobial agents. Mechanistically understanding how bacteria obtain antibiotic resistance is a critical first step to the development of improved inhibitors. One common mechanism for bacteria to obtain antibiotic resistance is by employing ATP-binding cassette (ABC) transporters to actively pump the drug from the cell. The ABC-F family includes proteins conferring resistance to a variety of clinically important ribosome-targeting antibiotics; however, controversy remains as to whether resistance is conferred via efflux like other ABC transporters or whether another mechanism, such as ribosome protection, is at play. A recent study by Sharkey and coworkers (L. K. Sharkey, T. A. Edwards, and A. J. O'Neill, mBio 7:e01975-15, 2016, http://dx.doi.org/10.1128/mBio.01975-15) provides strong evidence that ABC-F proteins conferring antibiotic resistance utilize ribosome protection mechanisms, namely, by interacting with the ribosome and displacing the drug from its binding site, thus revealing a novel role for ABC-F proteins in antibiotic resistance. PMID:27143393

  1. The ABC of Ribosome-Related Antibiotic Resistance.

    PubMed

    Wilson, Daniel N

    2016-05-03

    The increase in multidrug-resistant pathogenic bacteria is limiting the utility of our current arsenal of antimicrobial agents. Mechanistically understanding how bacteria obtain antibiotic resistance is a critical first step to the development of improved inhibitors. One common mechanism for bacteria to obtain antibiotic resistance is by employing ATP-binding cassette (ABC) transporters to actively pump the drug from the cell. The ABC-F family includes proteins conferring resistance to a variety of clinically important ribosome-targeting antibiotics; however, controversy remains as to whether resistance is conferred via efflux like other ABC transporters or whether another mechanism, such as ribosome protection, is at play. A recent study by Sharkey and coworkers (L. K. Sharkey, T. A. Edwards, and A. J. O'Neill, mBio 7:e01975-15, 2016, http://dx.doi.org/10.1128/mBio.01975-15) provides strong evidence that ABC-F proteins conferring antibiotic resistance utilize ribosome protection mechanisms, namely, by interacting with the ribosome and displacing the drug from its binding site, thus revealing a novel role for ABC-F proteins in antibiotic resistance.

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

    PubMed Central

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

    2014-01-01

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

  3. The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA.

    PubMed

    Dalmas, Olivier; Orelle, Cédric; Foucher, Anne-Emmanuelle; Geourjon, Christophe; Crouzy, Serge; Di Pietro, Attilio; Jault, Jean-Michel

    2005-11-01

    The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, a multidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide-binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 Angstrom were added. Altogether, these results give support to the existence, in the resting state, of a monomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide-binding domain for the proper functioning of a multidrug ATP-binding cassette transporter. PMID:16107340

  4. Trehalose-recycling ABC transporter LpqY-SugA-SugB-SugC is essential for virulence of Mycobacterium tuberculosis

    PubMed Central

    Kalscheuer, Rainer; Weinrick, Brian; Veeraraghavan, Usha; Besra, Gurdyal S.; Jacobs, William R.

    2010-01-01

    Mycobacterium tuberculosis (Mtb) is an exclusively human pathogen that proliferates within phagosomes of host phagocytes. Host lipids are believed to provide the major carbon and energy sources for Mtb, with only limited availability of carbohydrates. There is an apparent paradox because five putative carbohydrate uptake permeases are present in Mtb, but there are essentially no host carbohydrates inside phagosomes. Nevertheless, carbohydrate transporters have been implicated in Mtb pathogenesis, suggesting that acquisition of host sugars is important during some stages of infection. Here we show, however, that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is highly specific for uptake of the disaccharide trehalose, a sugar not present in mammals, thus refuting a role in nutrient acquisition from the host. Trehalose release is known to occur as a byproduct of the biosynthesis of the mycolic acid cell envelope by Mtb’s antigen 85 complex. The antigen 85 complex constitutes a group of extracellular mycolyl transferases, which transfer the lipid moiety of the glycolipid trehalose monomycolate (TMM) to arabinogalactan or another molecule of TMM, yielding trehalose dimycolate. These reactions also lead to the concomitant extracellular release of the trehalose moiety of TMM. We found that the LpqY-SugA-SugB-SugC ATP-binding cassette transporter is a recycling system mediating the retrograde transport of released trehalose. Perturbations in trehalose recycling strongly impaired virulence of Mtb. This study reveals an unexpected accessory component involved in the formation of the mycolic acid cell envelope in mycobacteria and provides a previously unknown role for sugar transporters in bacterial pathogenesis. PMID:21118978

  5. Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli

    PubMed Central

    Ohtsu, Iwao; Kawano, Yusuke; Suzuki, Marina; Morigasaki, Susumu; Saiki, Kyohei; Yamazaki, Shunsuke; Nonaka, Gen; Takagi, Hiroshi

    2015-01-01

    Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (Km = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids. PMID:25837721

  6. Meningococcal internalization into human endothelial and epithelial cells is triggered by the influx of extracellular L-glutamate via GltT L-glutamate ABC transporter in Neisseria meningitidis.

    PubMed

    Takahashi, Hideyuki; Kim, Kwang Sik; Watanabe, Haruo

    2011-01-01

    Meningococcal internalization into human cells is likely to be a consequence of meningococcal adhesion to human epithelial and endothelial cells. Here, we identified three transposon mutants of Neisseria meningitidis that were primarily defective in the internalization of human brain microvascular endothelial cells (HBMEC), with insertions occurring in the gltT (a sodium-independent L-glutamate transporter) gene or its neighboring gene, NMB1964 (unknown function). NMB1964 was tentatively named gltM in this study because of the presence of a mammalian cell entry (MCE)-related domain in the deduced amino acid sequences. The null ΔgltT-ΔgltM N. meningitidis mutant was also defective in the internalization into human umbilical vein endothelial cells and the human lung carcinoma epithelial cell line A549, and the defect was suppressed by transcomplementation of the mutants with gltT(+)-gltM(+) genes. The intracellular survival of the ΔgltT-ΔgltM mutant in HBMEC was not largely different from that of the wild-type strain under our experimental conditions. Introduction of a1-bp deletion and amber or ochre mutations in gltT-gltM genes resulted in the loss of efficient internalization into HBMEC. The defect in meningococcal internalization into HBMEC and L-glutamate uptake in the ΔgltT-ΔgltM mutant were suppressed only in strains expressing both GltT and GltM proteins. The efficiency of meningococcal invasion to HBMEC decreased under L-glutamate-depleted conditions. Furthermore, ezrin, a key membrane-cytoskeleton linker, accumulated beneath colonies of the gltT(+)-gltM(+) N. meningitidis strain but not of the ΔgltT-ΔgltM mutant. These findings suggest that l-glutamate influx via the GltT-GltM L-glutamate ABC transporter serves as a cue for N. meningitidis internalization into host cells. PMID:20956569

  7. Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis.

    PubMed

    Brunetti, Patrizia; Zanella, Letizia; De Paolis, Angelo; Di Litta, Davide; Cecchetti, Valentina; Falasca, Giuseppina; Barbieri, Maurizio; Altamura, Maria Maddalena; Costantino, Paolo; Cardarelli, Maura

    2015-07-01

    The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.

  8. The Maize glossy13 Gene, Cloned via BSR-Seq and Seq-Walking Encodes a Putative ABC Transporter Required for the Normal Accumulation of Epicuticular Waxes

    PubMed Central

    Liu, Sanzhen; Ma, Xiaoli; Dietrich, Charles R.; Hu, Heng-Cheng; Zhang, Gaisheng; Liu, Zhiyong; Zheng, Jun; Wang, Guoying; Schnable, Patrick S.

    2013-01-01

    Aerial plant surfaces are covered by epicuticular waxes that among other purposes serve to control water loss. Maize glossy mutants originally identified by their “glossy” phenotypes exhibit alterations in the accumulation of epicuticular waxes. By combining data from a BSR-Seq experiment and the newly developed Seq-Walking technology, GRMZM2G118243 was identified as a strong candidate for being the glossy13 gene. The finding that multiple EMS-induced alleles contain premature stop codons in GRMZM2G118243, and the one knockout allele of gl13, validates the hypothesis that gene GRMZM2G118243 is gl13. Consistent with this, GRMZM2G118243 is an ortholog of AtABCG32 (Arabidopsis thaliana), HvABCG31 (barley) and OsABCG31 (rice), which encode ABCG subfamily transporters involved in the trans-membrane transport of various secondary metabolites. We therefore hypothesize that gl13 is involved in the transport of epicuticular waxes onto the surfaces of seedling leaves. PMID:24324772

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

    PubMed

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

    2013-06-21

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

  10. Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis.

    PubMed

    Brunetti, Patrizia; Zanella, Letizia; De Paolis, Angelo; Di Litta, Davide; Cecchetti, Valentina; Falasca, Giuseppina; Barbieri, Maurizio; Altamura, Maria Maddalena; Costantino, Paolo; Cardarelli, Maura

    2015-07-01

    The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2. PMID:25900618

  11. Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis

    PubMed Central

    Brunetti, Patrizia; Zanella, Letizia; De Paolis, Angelo; Di Litta, Davide; Cecchetti, Valentina; Falasca, Giuseppina; Barbieri, Maurizio; Altamura, Maria Maddalena; Costantino, Paolo; Cardarelli, Maura

    2015-01-01

    The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC–Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC–Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2. PMID:25900618

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

    PubMed Central

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

    2015-01-01

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

  13. Employment of a promoter-swapping technique shows that PhoU modulates the activity of the PstSCAB2 ABC transporter in Escherichia coli.

    PubMed

    Rice, Christopher D; Pollard, Jacob E; Lewis, Zachery T; McCleary, William R

    2009-02-01

    Expression of the Pho regulon in Escherichia coli is induced in response to low levels of environmental phosphate (P(i)). Under these conditions, the high-affinity PstSCAB(2) protein (i.e., with two PstB proteins) is the primary P(i) transporter. Expression from the pstSCAB-phoU operon is regulated by the PhoB/PhoR two-component regulatory system. PhoU is a negative regulator of the Pho regulon; however, the mechanism by which PhoU accomplishes this is currently unknown. Genetic studies of phoU have proven to be difficult because deletion of the phoU gene leads to a severe growth defect and creates strong selection for compensatory mutations resulting in confounding data. To overcome the instability of phoU deletions, we employed a promoter-swapping technique that places expression of the phoBR two-component system under control of the P(tac) promoter and the lacO(ID) regulatory module. This technique may be generally applicable for controlling expression of other chromosomal genes in E. coli. Here we utilized P(phoB)::P(tac) and P(pstS)::P(tac) strains to characterize phenotypes resulting from various DeltaphoU mutations. Our results indicate that PhoU controls the activity of the PstSCAB(2) transporter, as well as its abundance within the cell. In addition, we used the P(phoB)::P(tac) DeltaphoU strain as a platform to begin characterizing new phoU mutations in plasmids.

  14. 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.

  15. Synthesis of poly[N-(2-hydroxypropyl)methacrylamide] conjugates of inhibitors of the ABC transporter that overcome multidrug resistance in doxorubicin-resistant P388 cells in vitro.

    PubMed

    Subr, V; Sivák, L; Koziolová, E; Braunová, A; Pechar, M; Strohalm, J; Kabešová, M; Ríhová, B; Ulbrich, K; Kovář, M

    2014-08-11

    The effects of novel polymeric therapeutics based on water-soluble N-(2-hydroxypropyl)methacrylamide copolymers (P(HPMA)) bearing the anticancer drug doxorubicin (Dox), an inhibitor of ABC transporters, or both, on the viability and the proliferation of the murine monocytic leukemia cell line P388 (parental cell line) and its doxorubicin-resistant subline P388/MDR were studied in vitro. The inhibitor derivatives 5-methyl-4-oxohexanoyl reversin 121 (MeOHe-R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe-RIT), showing the highest inhibitory activities, were conjugated to the P(HPMA) via the biodegradable pH-sensitive hydrazone bond, and the ability of these conjugates to block the ATP driven P-glycoprotein (P-gp) efflux pump was tested. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121 showed a dose-dependent increase in the ability to sensitize the P388/MDR cells to Dox from 1.5 to 24 μM, and achieved an approximately 50-fold increase in sensitization at 24 μM. The P(HPMA) conjugate P-Ahx-NH-N═MeOHe-RIT showed moderate activity at 6 μM (∼10 times higher sensitization) and increased sensitization by 50-fold at 12 μM. The cytostatic activity of the P(HPMA) conjugate P-Ahx-NH-N═MeOHe-R121(Dox) containing Dox and the P-gp inhibitor MeOHe-R121, both bound via hydrazone bonds to the P(HPMA) carrier, was almost 30 times higher than that of the conjugate P-Ahx-NH-N═Dox toward the P388/MDR cells in vitro. A similar result was observed for P-Ahx-NH-N═MeOHe-RIT(Dox), which exhibited almost 10 times higher cytostatic activity than P-Ahx-NH-N═Dox.

  16. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein.

    PubMed

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V; Xia, Di

    2016-08-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space group P1), with unit-cell parameters a = 40.67, b = 44.91, c = 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  17. 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.

  18. The ABCs of Student Engagement

    ERIC Educational Resources Information Center

    Parsons, Seth A.; Nuland, Leila Richey; Parsons, Allison Ward

    2014-01-01

    Student engagement is an important consideration for teachers and administrators because it is explicitly associated with achievement. What the authors call the ABC's of engagement they outline as: Affective engagement, Behavioral engagement, and Cognitive engagement. They also present "Three Things Every Teacher Needs to Know about…

  19. The ABC transporter HrtAB confers resistance to hemin toxicity and is regulated in a hemin-dependent manner by the ChrAS two-component system in Corynebacterium diphtheriae.

    PubMed

    Bibb, Lori A; Schmitt, Michael P

    2010-09-01

    Corynebacterium diphtheriae, the causative agent of the severe respiratory disease diphtheria, utilizes hemin and hemoglobin as iron sources for growth in iron-depleted environments. Because of the toxicity of high levels of hemin and iron, these compounds are often tightly regulated in bacterial systems. In this report, we identify and characterize the C. diphtheriae hrtAB genes, which encode a putative ABC type transporter involved in conferring resistance to the toxic effects of hemin. Deletion of the hrtAB genes in C. diphtheriae produced increased sensitivity to hemin, which was complemented by a plasmid harboring the cloned hrtAB locus. The HrtAB system was not involved in the uptake and use of hemin as an iron source. The hrtAB genes are located on the C. diphtheriae genome upstream from the chrSA operon, which encodes a previously characterized two-component signal transduction system that regulates gene expression in a heme-dependent manner. The hrtB promoter is activated by the ChrAS system in the presence of hemin or hemoglobin, and mutations in the chrSA genes abolish heme-activated expression from the hrtB promoter. It was also observed that transcription from the hrtB promoter is reduced in a dtxR deletion mutant, suggesting that DtxR is required for optimal expression of hrtAB. Previous studies proposed that the ChrS sensor kinase may be responsive to an environmental signal, such as hemin. We show that specific point mutations in the ChrS N-terminal transmembrane domain result in a reduced ability to activate the hrtB promoter in the presence of a heme source, suggesting that this putative sensor region is essential for the detection of a signal produced in response to hemin exposure. This study shows that the HrtAB system is required for protection from hemin toxicity and that expression of the hrtAB genes is regulated by the ChrAS two-component system. This study demonstrates a direct correlation between the detection of heme or a heme

  20. The ABC daycare disaster of Hermosillo, Mexico.

    PubMed

    Greenhalgh, David G; Chang, Philip; Maguina, Pirko; Combs, Elena; Sen, Soman; Palmieri, Tina L

    2012-01-01

    On June 5, 2009, the ABC Daycare facility in Hermosillo, Mexico, caught on fire with an estimated 142 children and 6 adult caregivers inside. The purpose of this article is to describe the factors contributing to the disaster including care of the survivors, tertiary burn center triage, patient transport, and treatment for this international mass casualty event. Finally, the results of an investigation performed by the Mexican Government are reviewed. A summary of the Mexican Government's investigation of the circumstances of fire and an examination of prevention lapses in other Mexican daycare centers was obtained from their public Web site. The demographic and clinical characteristics of the children transported to the burn center were obtained from the patients' medical records and transport data sheets. The ABC Daycare had many fire safety breaches that contributed to the severity of the tragedy. Twenty-nine children died at the scene and more than 35 children were hospitalized throughout Mexico. A total of 12 children were transported to two Shriners Hospitals, 9 to Sacramento, and 3 to Cincinnati. The mean age of patients sent to the Shriners Hospitals was 2.9 ± 0.16 years (2-4 years), with 5 being male and 7 female. The mean duration between injury and arrival was 9.2 ± 2.1 days, the burn size was 43.0 ± 6.8% TBSA (6.5-80%), and there were 3.75 operations per patient. Four had fourth-degree burns requiring finger amputations (2), flaps to cover bone (1), or a through-knee amputation (1). Ten patients were admitted to the intensive care unit, and nine patients (seven with inhalation injury) required mechanical ventilation for a mean of 23.6 ± 10.3 days. All the surviving children were discharged after a mean length of stay of 45.9 ± 8.7 days. In the first year postinjury, seven children were readmitted a total of 11 times for reconstructive surgery, wound care, or rehabilitation. Ultimately, a total of 49 children died. A review of other daycare centers

  1. 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

  2. An ABC for decision making.

    PubMed

    Garcia, Luiz Henrique Costa; Ferreira, Bruna Cortez

    2015-01-01

    The present study was aimed at proposing a systematic evaluation of cranial computed tomography, identifying the main aspects to be analyzed in order to facilitate the decision making process regarding diagnosis and management in emergency settings. The present descriptive study comprised a literature review at the following databases: Access Medicine and Access Emergency Medicine (McGraw- Hill Education); British Medical Journal Evidence Center; UptoDate; Bireme; PubMed; Lilacs; SciELO; ProQuest; Micromedex (Thomson Reuters); Embase. Once the literature review was completed, the authors identified the main diseases with tomographic repercussions and proposed the present system to evaluate cranial computed tomography images. An easy-to-memorize ABC system will facilitate the decision making in emergency settings, as it covers the main diseases encountered by intensivists and emergency physicians, and provides a sequential guidance about anatomical structures to be investigated as well as their respective alterations.

  3. An ABC for decision making*

    PubMed Central

    Garcia, Luiz Henrique Costa; Ferreira, Bruna Cortez

    2015-01-01

    The present study was aimed at proposing a systematic evaluation of cranial computed tomography, identifying the main aspects to be analyzed in order to facilitate the decision making process regarding diagnosis and management in emergency settings. The present descriptive study comprised a literature review at the following databases: Access Medicine and Access Emergency Medicine (McGraw- Hill Education); British Medical Journal Evidence Center; UptoDate; Bireme; PubMed; Lilacs; SciELO; ProQuest; Micromedex (Thomson Reuters); Embase. Once the literature review was completed, the authors identified the main diseases with tomographic repercussions and proposed the present system to evaluate cranial computed tomography images. An easy-to-memorize ABC system will facilitate the decision making in emergency settings, as it covers the main diseases encountered by intensivists and emergency physicians, and provides a sequential guidance about anatomical structures to be investigated as well as their respective alterations. PMID:25987751

  4. Applying the ABCs in provider organizations.

    PubMed

    Pandey, Seema

    2012-11-01

    Activity-based costing (ABC) is an accounting technique designed to guard against potentially serious financial problems that can arise when an organization's accounting costs deviate significantly from its actual costs. In general, an ABC analysis considers two factors: a cost element (a directly measurable unit of cost, such as the cost of an item) and a cost driver (a directly measurable feature of the service, such as how often the item is used). ABC is best applied to specific service areas, orservice packages, for which consumption of resources is largely predictable and atomic units of services can be accurately identified. PMID:23173369

  5. Applying the ABCs in provider organizations.

    PubMed

    Pandey, Seema

    2012-11-01

    Activity-based costing (ABC) is an accounting technique designed to guard against potentially serious financial problems that can arise when an organization's accounting costs deviate significantly from its actual costs. In general, an ABC analysis considers two factors: a cost element (a directly measurable unit of cost, such as the cost of an item) and a cost driver (a directly measurable feature of the service, such as how often the item is used). ABC is best applied to specific service areas, orservice packages, for which consumption of resources is largely predictable and atomic units of services can be accurately identified.

  6. Synthesis and modulation properties of imidazo[4,5-b]pyridin-7-one and indazole-4,7-dione derivatives towards the Cryptosporidium parvum CpABC3 transporter.

    PubMed

    Zeinyeh, Waël; Xia, Hexue; Lawton, Philippe; Radix, Sylvie; Marminon, Christelle; Nebois, Pascal; Walchshofer, Nadia

    2010-06-01

    The syntheses of new N-polysubstituted imidazo[4,5-b]pyridine-7-one (IP, 5 and 8a-8f) and indazole-4,7-dione (ID, 9 and 10) derivatives are described. The binding affinity of IP and ID towards the recombinant Nucleotide Binding Domain NBD1 of Cryptosporidium parvum CpABC3 was evaluated by intrinsic fluorescence quenching. IP induced a moderate quenching of the intrinsic fluorescence of H6-NBD1 whereas IDs 9 and 10 showed a binding affinity comparable to the ATP analogue TNP-ATP. In addition, 8d, 8e and 10 were shown to be competitive inhibitors of the ATPase activity, but with low affinity. These compounds could thus act like some flavonoid derivatives, which can partly overlap both the nucleotide-binding site and the adjacent hydrophobic steroid-binding region of mammalian P-glycoproteins.

  7. 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

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

    PubMed Central

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

    2015-01-01

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

  9. Communicating a New ABC: Advocacy, Partnerships, and Implementing Change.

    ERIC Educational Resources Information Center

    Ryan, Cathy

    2001-01-01

    Reiterates some key points and responds to challenges issued by the speakers to the Association for Business Communication (ABC) conference. Notes that Paula Pomerenke spoke about formalizing support of Plain Language across the continents and about ABC's continuing need to strengthen relationships with practitioners. Suggests the ABC build…

  10. The New ABC of the Arts

    ERIC Educational Resources Information Center

    Tusa, John

    2007-01-01

    This article presents a new alphabet of the arts that shows how the arts world has been transformed the past five years. Beginning with "A" for assessment and continuing through "Y" for year end, the ABC of the arts illustrates the ways in which the arts world is judged, managed, and evaluated, and shows that the skill of arts management is to…

  11. Calculus ABCs: A Gateway for Freshman Calculus

    ERIC Educational Resources Information Center

    Fulton, Scott R.

    2003-01-01

    This paper describes a gateway testing program designed to ensure that students acquire basic skills in freshman calculus. Students must demonstrate they have mastered standards for "Absolutely Basic Competency"--the Calculus ABCs--in order to pass the course with a grade of C or better. We describe the background, standards, and testing program.…

  12. The ABC's of Learning in Infancy.

    ERIC Educational Resources Information Center

    Saunders, Minta M.

    Learning in infancy is based on activity, beginnings, and curiosity, the so-called ABC's. Earliest behavior consists of mass activity, the period from birth to 24 months of sensory-motor development which provides the foundation for all future learning. Adults must provide space, toys, and affectionate care to help infants proceed through…

  13. The ABCs of Managing Teacher Stress.

    ERIC Educational Resources Information Center

    Nagel, Liza; Brown, Sheri

    2003-01-01

    Describes stress management for teachers and presents strategies that teachers can use to lessen the impact of stress. Outlines the ABCs of stress: Acknowledge, Behavior Modification, and Communication. Notes that stress can motivate teachers to explore new instructional strategies, adopt innovative approaches to increasing student motivation, and…

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

    PubMed

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  16. Nanostructured assemblies from amphiphilic ABC multiblock polymers

    NASA Astrophysics Data System (ADS)

    Hillmyer, Marc A.

    2012-02-01

    Amphiphilic AB diblock copolymers containing a water compatible segment can self-assemble in aqueous media to give supramolecular structures that include simple spherical micelles and macromolecular vesicles termed polymersomes. Amphiphilic ABA triblocks with hydrophobic end blocks can adopt analogous structures but can also form gels at high polymer concentrations. The structural and chemical diversity demonstrated in block copolymer micelles and gels makes them attractive for applications ranging from drug delivery to personal care products to nanoreactors. The inclusion of a third block in amphiphilic ABC triblock systems can lead to a much wider array of self-assembled structures that depend not only on composition but also on block sequence, architecture and incompatibility considerations. I will present our recent efforts on tuning micelle and gel structure and behavior using controlled architecture ABC triblocks. The combination of diverse polymer segments into a single macromolecule is a powerful method for development of self-assembled structures with both new form and new function.

  17. Three ways to learn the ABCs.

    PubMed

    Ng, M; Yanofsky, M F

    2000-02-01

    The ABC model of flower development represents a milestone in explaining how the fate of emerging floral organ primordia is specified. This model states that organ identity is specified by different combinations of the activities of the A, B and C class homeotic genes. In spite of the remarkable simplicity of this model, the complex regulatory interactions that establish the initial pattern of A, B and C gene activity have yet to be fully explained. It has been shown that the LEAFY gene functions early to promote flower meristem identity, and that it is subsequently required for the normal expression of the ABC genes. Recently, LEAFY has been identified as an immediate upstream regulator of the floral homeotic genes, thus opening up an avenue to examine the transcriptional interactions that underlie floral patterning. PMID:10679448

  18. Pharmacological correction of misfolding of ABC proteins☆

    PubMed Central

    Rudashevskaya, Elena L.; Stockner, Thomas; Trauner, Michael; Freissmuth, Michael; Chiba, Peter

    2014-01-01

    The endoplasmic reticulum (ER) quality control system distinguishes between correctly and incorrectly folded proteins to prevent processing of aberrantly folded conformations along the secretory pathway. Non-synonymous mutations can lead to misfolding of ABC proteins and associated disease phenotypes. Specific phenotypes may at least partially be corrected by small molecules, so-called pharmacological chaperones. Screening for folding correctors is expected to open an avenue for treatment of diseases such as cystic fibrosis and intrahepatic cholestasis. PMID:25027379

  19. Adhesion to chondroitinase ABC treated dentin

    PubMed Central

    Mazzoni, Annalisa; Pashley, David H.; Ruggeri, Alessandra; Vita, Francesca; Falconi, Mirella; Di Lenarda, Roberto; Breschi, Lorenzo

    2013-01-01

    Dentin bonding relies on complete resin impregnation throughout the demineralised hydrophilic collagen mesh. Chondroitin sulphate-glycosaminoglycans are claimed to regulate the three-dimensional arrangement of the dentin organic matrix and its hydrophilicity. The aim of this study was to investigate bond strength of two etch-and-rinse adhesives to chondroitinase ABC treated dentin. Human extracted molars were treated with chondroitinase ABC and a double labelling immunohistochemical technique was applied to reveal type I collagen and chondroitin 4/6 sulphate distribution under field emission in-lens scanning electron microscope. The immunohistochemical technique confirmed the effective removal of chondroitin 4/6 sulphate after the enzymatic treatment. Dentin surfaces exposed to chondroitinase ABC and untreated specimens prepared on untreated acid-etched dentin were bonded with Adper Scotchbond Multi-Purpose or Prime & Bond NT. Bonded specimens were submitted to microtensile testing and nanoleakage interfacial analysis under transmission electron microscope. Increased mean values of microtensile bond strength and reduced nanoleakage expression were found for both adhesives after chondroitinase ABC treatment of the dentin surface. Adper Scotchbond Multi-Purpose increased its bond strength about 28%, while bonding made with Prime & Bond NT almost doubled (92% increase) compared to untreated specimens. This study supports the hypothesis that adhesion can be enhanced by removal of chondroitin 4/6 sulphate and dermatan sulphate, probably due to a reduced amount of water content and enlarged interfibrillar spaces. Further studies should validate this hypothesis investigating the stability of chondroitin 4/6 and dermatan sulphate-depleted dentin bonded interface over time. PMID:18161809

  20. Analyzing health care operations using ABC.

    PubMed

    Ross, Thomas K

    2004-01-01

    The evolution of health care created a climate in which cost was subordinate to medical treatment. Current reimbursement constraints have increased the need for providers to be cost conscious, but they have discovered that current accounting practices do not provide the appropriate information to determine the cost of service or make decisions. This article argues that activity-based costing (ABC) can bridge the gap between the medical and financial communities and provide a foundation for performance improvement. PMID:15151193

  1. Pharmacological correction of misfolding of ABC proteins.

    PubMed

    Rudashevskaya, Elena L; Stockner, Thomas; Trauner, Michael; Freissmuth, Michael; Chiba, Peter

    2014-06-01

    The endoplasmic reticulum (ER) quality control system distinguishes between correctly and incorrectly folded proteins to prevent processing of aberrantly folded conformations along the secretory pathway. Non-synonymous mutations can lead to misfolding of ABC proteins and associated disease phenotypes. Specific phenotypes may at least partially be corrected by small molecules, so-called pharmacological chaperones. Screening for folding correctors is expected to open an avenue for treatment of diseases such as cystic fibrosis and intrahepatic cholestasis. PMID:25027379

  2. Conformational plasticity of the type I maltose ABC importer.

    PubMed

    Böhm, Simon; Licht, Anke; Wuttge, Steven; Schneider, Erwin; Bordignon, Enrica

    2013-04-01

    ATP-binding cassette (ABC) transporters couple the translocation of solutes across membranes to ATP hydrolysis. Crystal structures of the Escherichia coli maltose importer (MalFGK2) in complex with its substrate binding protein (MalE) provided unprecedented insights in the mechanism of substrate translocation, leaving the MalE-transporter interactions still poorly understood. Using pulsed EPR and cross-linking methods we investigated the effects of maltose and MalE on complex formation and correlated motions of the MalK2 nucleotide-binding domains (NBDs). We found that both substrate-free (open) and liganded (closed) MalE interact with the transporter with similar affinity in all nucleotide states. In the apo-state, binding of open MalE occurs via the N-lobe, leaving the C-lobe disordered, but upon maltose binding, closed MalE associates tighter to the transporter. In both cases the NBDs remain open. In the presence of ATP, the transporter binds both substrate-free and liganded MalE, both inducing the outward-facing conformation trapped in the crystal with open MalE at the periplasmic side and NBDs tightly closed. In contrast to ATP, ADP-Mg(2+) alone is sufficient to induce a semiopen conformation in the NBDs. In this nucleotide-driven state, the transporter binds both open and closed MalE with slightly different periplasmic configurations. We also found that dissociation of MalE is not a required step for substrate translocation since a supercomplex with MalE cross-linked to MalG retains the ability to hydrolyze ATP and to transport maltose. These features of MalE-MalFGK2 interactions highlight the conformational plasticity of the maltose importer, providing insights into the ATPase stimulation by unliganded MalE.

  3. Phase behavior of model ABC triblock copolymers

    NASA Astrophysics Data System (ADS)

    Chatterjee, Joon

    The phase behavior of poly(isoprene-b-styrene- b-ethylene oxide) (ISO), a model ABC triblock copolymer has been studied. This class of materials exhibit self-assembly, forming a large array of ordered morphologies at length scales of 5-100 nm. The formation of stable three-dimensionally continuous network morphologies is of special interest in this study. Since these nanostructures considerably impact the material properties, fundamental knowledge for designing ABC systems have high technological importance for realizing applications in the areas of nanofabrication, nanoporous media, separation membranes, drug delivery and high surface area catalysts. A comprehensive framework was developed to describe the phase behavior of the ISO triblock copolymers at weak to intermediate segregation strengths spanning a wide range of composition. Phases were characterized through a combination of characterization techniques, including small angle x-ray scattering, dynamic mechanical spectroscopy, transmission electron microscopy, and birefringence measurements. Combined with previous investigations on ISO, six different stable ordered state symmetries have been identified: lamellae (LAM), Fddd orthorhombic network (O70), double gyroid (Q230), alternating gyroid (Q214), hexagonal (HEX), and body-centered cubic (BCC). The phase map was found to be somewhat asymmetric around the fI = fO isopleth. This work provides a guide for theoretical studies and gives insight into the intricate effects of various parameters on the self-assembly of ABC triblock copolymers. Experimental SAXS data evaluated with a simple scattering intensity model show that local mixing varies continuously across the phase map between states of two- and three-domain segregation. Strategies of blending homopolymers with ISO triblock copolymer were employed for studying the swelling properties of a lamellar state. Results demonstrate that lamellar domains swell or shrink depending upon the type of homopolymer that

  4. Sequence analysis of a near-subtelomeric 35.4 kb DNA segment on the right arm of chromosome VII from Saccharomyces cerevisiae carrying the MAL1 locus reveals 15 complete open reading frames, including ZUO1, BGL2 and BIO2 genes and an ABC transporter gene.

    PubMed

    Volckaert, G; Voet, M; Robben, J

    1997-03-15

    The nucleotide sequence of 35,400 bp at approximately 10 kb from the right telomere of chromosome VII was determined. The segment contains the MAL1 locus, one of the five unlinked loci sufficient for maltose utilization. Until now, each of these loci was considered to contain three genes (for regulator, permease and alpha-glucosidase), but a fourth gene, presumably an extra alpha-glucosidase gene, was found at MAL1 adjacent to the usual cluster of three genes. The two glucosidase genes are present in opposite orientation, forming an inverted repeat structure. In addition to the four genes at MAL1, there are 11 complete, non-overlapping open reading frames (ORFs) longer than 300 bp in the sequence presented here. A new ABC transporter gene (YGR281w), required for oligomycin resistance was found (YOR1; Katzman et al., 1995), and the previously sequenced BGL2 (YGR282c), ZUO1 (YGR285c) and BIO2 (YGR286c) genes were located. The sequence of BIO2, a biotin synthetase gene, required substantial correction and the size of Bio2p is 375, rather than 356, amino acids. Two ORFs show rather weak similarities to animal genes: YGR278w to an unknown ORF of Caenorhabditis elegans and YGR284c to the murine Surf-4, a member of a cluster of at least four housekeeping genes. The remaining five ORFs do not encode known functions, but three of these show weak to high similarities to other ORFs in the Saccharomyces cerevisiae genome and one (YGR280c) codes for a particularly lysine-rich protein.

  5. Abc3-Mediated Efflux of an Endogenous Digoxin-like Steroidal Glycoside by Magnaporthe oryzae Is Necessary for Host Invasion during Blast Disease

    PubMed Central

    Patkar, Rajesh N.; Xue, Yang Kui; Shui, Guanghou; Wenk, Markus R.; Naqvi, Naweed I.

    2012-01-01

    Magnaporthe oryzae, which causes the devastating rice-blast disease, invades its host plants via a specialized infection structure called the appressorium. Previously, we showed that the ATP-Binding Cassette 3 transporter is necessary for appressorial function (host penetration) in M. oryzae. However, thus far, the molecular basis underlying impaired appressorial function in the abc3Δ remains elusive. We hypothesized that the abc3Δ appressoria accumulate excessive amounts of specific efflux substrate(s) of the Abc3 transporter in M. oryzae. We devised an innovative yeast-based strategy and identified Abc3 Transporter efflux Substrate (ATS) to be a digoxin-like endogenous steroidal glycoside that accumulates to inhibitory levels in M. oryzae abc3Δ appressoria. Exogenous ATS altered cell wall biogenesis and viability in wild-type Schizosaccharomyces pombe, but not in S. pombe expressing M. oryzae Abc3. We show that ATS associates with the Translation Elongation factor Tef2 in M. oryzae, and propose that ATS regulates ion homeostasis during pathogenesis. Excessive ATS accumulation, either intracellularly due to impaired efflux in the abc3Δ or when added exogenously to the wild type, renders M. oryzae nonpathogenic. Furthermore, we demonstrate that the host penetration defects in the abc3Δ are due to aberrant F-actin dynamics as a result of altered Tef2 function and/or ion homeostasis defects caused by excess accumulation of ATS therein. Rather surprisingly, excessive exogenous ATS or digoxin elicited the hypersensitive response in rice, even in the absence of the blast fungus. Lastly, reduced disease symptoms in the inoculated host plants in the presence of excessive digoxin suggest a potential use for such related steroidal glycosides in controlling rice-blast disease. PMID:22927822

  6. The Role of the Atypical Kinases ABC1K7 and ABC1K8 in Abscisic Acid Responses

    PubMed Central

    Manara, Anna; DalCorso, Giovanni; Furini, Antonella

    2016-01-01

    The ABC1K family of atypical kinases (activity of bc1 complex kinase) is represented in bacteria, archaea, and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA)-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement, and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling. PMID:27047531

  7. The Role of the Atypical Kinases ABC1K7 and ABC1K8 in Abscisic Acid Responses.

    PubMed

    Manara, Anna; DalCorso, Giovanni; Furini, Antonella

    2016-01-01

    The ABC1K family of atypical kinases (activity of bc1 complex kinase) is represented in bacteria, archaea, and eukaryotes. In plants they regulate diverse physiological processes in the chloroplasts and mitochondria, but their precise functions are poorly defined. ABC1K7 and ABC1K8 are probably involved in oxidative stress responses, isoprenyl lipid synthesis and distribution of iron within chloroplasts. Because reactive oxygen species take part in abscisic acid (ABA)-mediated processes, we investigated the functions of ABC1K7 and ABC1K8 during germination, stomatal movement, and leaf senescence. Both genes were upregulated by ABA treatment and some ABA-responsive physiological processes were affected in abc1k7 and abc1k8 mutants. Germination was more severely affected by ABA, osmotic stress and salt stress in the single and double mutants; the stomatal aperture was smaller in the mutants under standard growth conditions and was not further reduced by exogenous ABA application; ABA-induced senescence symptoms were more severe in the leaves of the single and double mutants compared to wild type leaves. Taken together, our results suggest that ABC1K7 and ABC1K8 might be involved in the cross-talk between ABA and ROS signaling. PMID:27047531

  8. Control of zinc homeostasis in Agrobacterium tumefaciens via zur and the zinc uptake genes znuABC and zinT.

    PubMed

    Bhubhanil, Sakkarin; Sittipo, Panida; Chaoprasid, Paweena; Nookabkaew, Sumontha; Sukchawalit, Rojana; Mongkolsuk, Skorn

    2014-11-01

    The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein. The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens. PMID:25227896

  9. Rapid quantification of murine ABC mRNAs by real time reverse transcriptase-polymerase chain reaction.

    PubMed

    Su, Yan Ru; Linton, MacRae F; Fazio, Sergio

    2002-12-01

    Several ATP-binding cassette (ABC) transporters are critically involved in cholesterol and phospholipid efflux, reverse cholesterol transport, and play an important role in the development of atherosclerosis. Quantification of ABC mRNA is important in studying the regulation of cellular cholesterol homeostasis and mechanisms related to the pathogenesis of atherosclerosis. We have developed a one-step real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method for measuring mRNA levels of ABCA1, ABCG1, and ABCA2 in murine tissues using the TaqMan(TM) technology. It has significant methodological benefits when compared to classic Northern blotting or semi-quantitative RT-PCR analysis. Using this method, we found high expression levels of ABCA1 in liver and macrophages, and of ABCG1 in the brain and macrophages. The expression of ABCA1 and ABCG1 were further induced in macrophages loaded with acLDL. In contrast, ABCA2 was expressed exclusively in the brain with low expression levels in the macrophages. This method provides a rapid, highly sensitive, specific, and reproducible quantification of ABC mRNA, and can be performed with nanograms of total RNA sample, thus making it a superior method for studying the regulation of ABC transporters in cholesterol efflux and its role in the pathogenesis of atherosclerosis in murine models.

  10. APOLLO 13: A News Bulletin from ABC

    NASA Technical Reports Server (NTRS)

    1974-01-01

    APOLLO 13: ABC breaks the news of a mishap aboard the spacecraft From the film documentary 'APOLLO 13: 'Houston, We've got a problem'', part of a documentary series on the APOLLO missions made in the early '70's and narrated by Burgess Meredith. APOLO 13 : Third manned lunar landing attempt with James A. Lovell, Jr., John L. Swigert, Jr., and Fred W. Haise, Jr. Pressure lost in SM oxygen system; mission aborted; LM used for life support. Mission Duration 142hrs 54mins 41sec

  11. ABC makes the switch to digital

    NASA Astrophysics Data System (ADS)

    Pearl, R. G.

    1984-05-01

    The digital satellite-distribution system being completed for the ABC radio network is briefly characterized. Audio programming on 19 channels at 15 kHz is digitized at the studio and transmitted by microwave to the uplink facility for distribution via Satcom I-R to 1800 affiliates with affiliate-owned receivers. A data channel comprising several 32-kbit/sec subchannels operates through network-owned data cards and printers installed in the affiliate ground stations to provide internal communication (to all affiliates, a selected group, or a single station) using soft addresses, data-distribution services for station customers, or the proposed 900-MHz nationwide paging service.

  12. Membrane Receptors and Transporters Involved in the Function and Transport of Vitamin A and Its Derivatives

    PubMed Central

    Sun, Hui

    2011-01-01

    The eye is the human organ most sensitive to vitamin A deficiency because of vision's absolute and heavy dependence on vitamin A for light perception. Studies of the molecular basis of vision have provided important insights into the intricate mechanistic details of the function, transport and recycling of vitamin A and its derivatives (retinoid). This review focuses on retinoid-related membrane receptors and transporters. Three kinds of mammalian membrane receptors and transporters are discussed: opsins, best known as vitamin A-based light sensors in vision; ABCA4, an ATP-dependent transporter specializes in the transport of vitamin A derivative; and STRA6, a recently identified membrane receptor that mediates cellular uptake of vitamin A. The evolutionary driving forces for their existence and the wide spectrum of human diseases associated with these proteins are discussed. Lessons learned from the study of the visual system might be useful for understanding retinoid biology and retinoid-related diseases in other organ systems as well. PMID:21704730

  13. The Reign of Confusion: ABC and the "Crisis in Iran."

    ERIC Educational Resources Information Center

    Palmerton, Patricia R.

    A study examined reports broadcast by ABC News between November 8, 1979 and December 7, 1979 in its series entitled "Crisis in Iran: America Held Hostage." Transcripts of approximately 50% of actual broadcasts were subjected to rhetorical critical analysis, from which the finding emerged that confusion was the predominant characteristic in ABC's…

  14. The ABCs of School Choice, 2009-2010 Edition

    ERIC Educational Resources Information Center

    Friedman Foundation for Educational Choice, 2010

    2010-01-01

    This publication presents the 2009-2010 edition of the Friedman Foundation for Educational Choice's "ABCs of School Choice". The "ABCs of School Choice" provides the latest in up-to-date and accurate information about the many school choice success stories taking place throughout the country. Readers will find this guide an essential resource on…

  15. To What Extent Does Attention Affect K-ABC Scores?

    ERIC Educational Resources Information Center

    Gordon, Michael; And Others

    1990-01-01

    Analyzed the protocols of 52 clinic-referred children who were administered the Kaufman Assessment Battery for Children (K-ABC) as well as version of the Continuous Performance Test (CPT), a laboratory measure of attention. Results demonstrated significant interrelationships among K-ABC and CPT scores. (Author/ABL)

  16. ABCs of Being Smart: S Is for Supporting

    ERIC Educational Resources Information Center

    Foster, Joanne

    2014-01-01

    Joanne Foster's article "R We There Yet?" was first published in "Parenting for High Potential" ("PHP") in 2006, which became the springboard for the "ABCs of Being Smart" series of columns. At that time, Foster invited "PHP" readers to think about their own versions of the "ABCs of Being…

  17. Measuring Academic Behavioural Confidence: The ABC Scale Revisited

    ERIC Educational Resources Information Center

    Sander, Paul; Sanders, Lalage

    2009-01-01

    The Academic Behavioural Confidence (ABC) scale has been shown to be valid and can be useful to teachers in understanding their students, enabling the design of more effective teaching sessions with large cohorts. However, some of the between-group differences have been smaller than expected, leading to the hypothesis that the ABC scale many not…

  18. Molecular examination of bone marrow stromal cells and chondroitinase ABC-assisted acellular nerve allograft for peripheral nerve regeneration

    PubMed Central

    Wang, Ying; Jia, Hua; Li, Wen-Yuan; Guan, Li-Xin; Deng, Lingxiao; Liu, Yan-Cui; Liu, Gui-Bo

    2016-01-01

    The present study aimed to evaluate the molecular mechanisms underlying combinatorial bone marrow stromal cell (BMSC) transplantation and chondroitinase ABC (Ch-ABC) therapy in a model of acellular nerve allograft (ANA) repair of the sciatic nerve gap in rats. Sprague Dawley rats (n=24) were used as nerve donors and Wistar rats (n=48) were randomly divided into the following groups: Group I, Dulbecco's modified Eagle's medium (DMEM) control group (ANA treated with DMEM only); Group II, Ch-ABC group (ANA treated with Ch-ABC only); Group III, BMSC group (ANA seeded with BMSCs only); Group IV, Ch-ABC + BMSCs group (Ch-ABC treated ANA then seeded with BMSCs). After 8 weeks, the expression of nerve growth factor, brain-derived neurotrophic factor and vascular endothelial growth factor in the regenerated tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Axonal regeneration, motor neuron protection and functional recovery were examined by immunohistochemistry, horseradish peroxidase retrograde neural tracing and electrophysiological and tibialis anterior muscle recovery analyses. It was observed that combination therapy enhances the growth response of the donor nerve locally as well as distally, at the level of the spinal cord motoneuron and the target muscle organ. This phenomenon is likely due to the propagation of retrograde and anterograde transport of growth signals sourced from the graft site. Collectively, growth improvement on the donor nerve, target muscle and motoneuron ultimately contribute to efficacious axonal regeneration and functional recovery. Thorough investigation of molecular peripheral nerve injury combinatorial strategies are required for the optimization of efficacious therapy and full functional recovery following ANA. PMID:27698684

  19. Molecular examination of bone marrow stromal cells and chondroitinase ABC-assisted acellular nerve allograft for peripheral nerve regeneration

    PubMed Central

    Wang, Ying; Jia, Hua; Li, Wen-Yuan; Guan, Li-Xin; Deng, Lingxiao; Liu, Yan-Cui; Liu, Gui-Bo

    2016-01-01

    The present study aimed to evaluate the molecular mechanisms underlying combinatorial bone marrow stromal cell (BMSC) transplantation and chondroitinase ABC (Ch-ABC) therapy in a model of acellular nerve allograft (ANA) repair of the sciatic nerve gap in rats. Sprague Dawley rats (n=24) were used as nerve donors and Wistar rats (n=48) were randomly divided into the following groups: Group I, Dulbecco's modified Eagle's medium (DMEM) control group (ANA treated with DMEM only); Group II, Ch-ABC group (ANA treated with Ch-ABC only); Group III, BMSC group (ANA seeded with BMSCs only); Group IV, Ch-ABC + BMSCs group (Ch-ABC treated ANA then seeded with BMSCs). After 8 weeks, the expression of nerve growth factor, brain-derived neurotrophic factor and vascular endothelial growth factor in the regenerated tissues were detected by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry. Axonal regeneration, motor neuron protection and functional recovery were examined by immunohistochemistry, horseradish peroxidase retrograde neural tracing and electrophysiological and tibialis anterior muscle recovery analyses. It was observed that combination therapy enhances the growth response of the donor nerve locally as well as distally, at the level of the spinal cord motoneuron and the target muscle organ. This phenomenon is likely due to the propagation of retrograde and anterograde transport of growth signals sourced from the graft site. Collectively, growth improvement on the donor nerve, target muscle and motoneuron ultimately contribute to efficacious axonal regeneration and functional recovery. Thorough investigation of molecular peripheral nerve injury combinatorial strategies are required for the optimization of efficacious therapy and full functional recovery following ANA.

  20. Transportation.

    ERIC Educational Resources Information Center

    Crank, Ron

    This instructional unit is one of 10 developed by students on various energy-related areas that deals specifically with transportation and energy use. Its objective is for the student to be able to discuss the implication of energy usage as it applies to the area of transportation. Some topics covered are efficiencies of various transportation…

  1. ABC's of Being Smart: I Can "C" Clearly Now

    ERIC Educational Resources Information Center

    Foster, Joanne

    2011-01-01

    In this paper, the author focuses on C of the ABC's of being smart. She continues to categorize the points for readers. These categories include the following: (1) being; (2) doing; and (3) stretching.

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

    PubMed Central

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

    2014-01-01

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

  3. ABC and IFC: Modules Detection Method for PPI Network

    PubMed Central

    Lei, Xiujuan; Tian, Jianfang

    2014-01-01

    Many clustering algorithms are unable to solve the clustering problem of protein-protein interaction (PPI) networks effectively. A novel clustering model which combines the optimization mechanism of artificial bee colony (ABC) with the fuzzy membership matrix is proposed in this paper. The proposed ABC-IFC clustering model contains two parts: searching for the optimum cluster centers using ABC mechanism and forming clusters using intuitionistic fuzzy clustering (IFC) method. Firstly, the cluster centers are set randomly and the initial clustering results are obtained by using fuzzy membership matrix. Then the cluster centers are updated through different functions of bees in ABC algorithm; then the clustering result is obtained through IFC method based on the new optimized cluster center. To illustrate its performance, the ABC-IFC method is compared with the traditional fuzzy C-means clustering and IFC method. The experimental results on MIPS dataset show that the proposed ABC-IFC method not only gets improved in terms of several commonly used evaluation criteria such as precision, recall, and P value, but also obtains a better clustering result. PMID:24991575

  4. ABCE1 Is a Highly Conserved RNA Silencing Suppressor

    PubMed Central

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

    2015-01-01

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

  5. Phase transition in the ABC model.

    PubMed

    Clincy, M; Derrida, B; Evans, M R

    2003-06-01

    Recent studies have shown that one-dimensional driven systems can exhibit phase separation even if the dynamics is governed by local rules. The ABC model, which comprises three particle species that diffuse asymmetrically around a ring, shows anomalous coarsening into a phase separated steady state. In the limiting case in which the dynamics is symmetric and the parameter q describing the asymmetry tends to one, no phase separation occurs and the steady state of the system is disordered. In the present work, we consider the weak asymmetry regime q=exp(-beta/N), where N is the system size, and study how the disordered state is approached. In the case of equal densities, we find that the system exhibits a second-order phase transition at some nonzero beta(c). The value of beta(c)=2pi square root 3 and the optimal profiles can be obtained by writing the exact large deviation functional. For nonequal densities, we write down mean-field equations and analyze some of their predictions. PMID:16241312

  6. Optimal ABC inventory classification using interval programming

    NASA Astrophysics Data System (ADS)

    Rezaei, Jafar; Salimi, Negin

    2015-08-01

    Inventory classification is one of the most important activities in inventory management, whereby inventories are classified into three or more classes. Several inventory classifications have been proposed in the literature, almost all of which have two main shortcomings in common. That is, the previous methods mainly rely on an expert opinion to derive the importance of the classification criteria which results in subjective classification, and they need precise item parameters before implementing the classification. While the problem has been predominantly considered as a multi-criteria, we examine the problem from a different perspective, proposing a novel optimisation model for ABC inventory classification in the form of an interval programming problem. The proposed interval programming model has two important features compared to the existing methods: it provides optimal results instead of an expert-based classification and it does not require precise values of item parameters, which are not almost always available before classification. Finally, by illustrating the proposed classification model in the form of numerical example, conclusion and suggestions for future works are presented.

  7. Metal Binding Specificity of the MntABC Permease of Neisseria gonorrhoeae and Its Influence on Bacterial Growth and Interaction with Cervical Epithelial Cells▿ †

    PubMed Central

    Lim, Karen H. L.; Jones, Christopher E.; vanden Hoven, Rachel N.; Edwards, Jennifer L.; Falsetta, Megan L.; Apicella, Michael A.; Jennings, Michael P.; McEwan, Alastair G.

    2008-01-01

    mntABC from Neisseria gonorrhoeae encodes an ABC permease which includes a periplasmic divalent cation binding receptor protein of the cluster IX family, encoded by mntC. Analysis of an mntC mutant showed that growth of N. gonorrhoeae could be stimulated by addition of either manganese(II) or zinc(II) ions, suggesting that the MntABC system could transport both ions. In contrast, growth of the mntAB mutant in liquid culture was possible only when the medium was supplemented with an antioxidant such as mannitol, consistent with the view that ion transport via MntABC is essential for protection of N. gonorrhoeae against oxidative stress. Using recombinant MntC, we determined that MntC binds Zn2+ and Mn2+ with almost equal affinity (dissociation constant of ∼0.1 μM). Competition assays with the metallochromic zinc indicator 4-(2-pyridylazo)resorcinol showed that MntC binds Mn2+ and Zn2+ at the same binding site. Analysis of the N. gonorrhoeae genome showed that MntC is the only Mn/Zn metal binding receptor protein cluster IX in this bacterium, in contrast to the situation in many other bacteria which have systems with dedicated Mn and Zn binding proteins as part of distinctive ABC cassette permeases. Both the mntC and mntAB mutants had reduced intracellular survival in a human cervical epithelial cell model and showed reduced ability to form a biofilm. These data suggest that the MntABC transporter is of importance for survival of Neisseria gonorrhoeae in the human host. PMID:18426887

  8. Functional Dynamics Revealed by the Structure of the SufBCD Complex, a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis.

    PubMed

    Hirabayashi, Kei; Yuda, Eiki; Tanaka, Naoyuki; Katayama, Sumie; Iwasaki, Kenji; Matsumoto, Takashi; Kurisu, Genji; Outten, F Wayne; Fukuyama, Keiichi; Takahashi, Yasuhiro; Wada, Kei

    2015-12-11

    ATP-binding cassette (ABC)-type ATPases are chemomechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.

  9. Effects of lipid environment on the conformational changes of an ABC importer.

    PubMed

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

    2014-01-01

    In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.

  10. ABC estimation of unit costs for emergency department services.

    PubMed

    Holmes, R L; Schroeder, R E

    1996-04-01

    Rapid evolution of the health care industry forces managers to make cost-effective decisions. Typical hospital cost accounting systems do not provide emergency department managers with the information needed, but emergency department settings are so complex and dynamic as to make the more accurate activity-based costing (ABC) system prohibitively expensive. Through judicious use of the available traditional cost accounting information and simple computer spreadsheets. managers may approximate the decision-guiding information that would result from the much more costly and time-consuming implementation of ABC. PMID:10156656

  11. 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

  12. Migration of Adipose-derived Mesenchymal Stem Cells Stably Expressing Chondroitinase ABC In vitro

    PubMed Central

    Wu, Jian-Huang; Li, Miao; Liang, Yan; Lu, Tao; Duan, Chun-Yue

    2016-01-01

    Background: Several studies have revealed that adipose-derived mesenchymal stem cells (ADSCs) can be used as seed cells for the treatment of spinal cord injury (SCI). Chondroitinase ABC (ChABC) decomposes chondroitin sulfate proteoglycans in the glial scar that forms following SCI, allowing stem cells to penetrate through the scar and promote recovery of nerve function. This study aimed to establish ADSCs that stably express ChABC (ChABC-ADSCs) and evaluate the migratory capability of ChABC-ADSCs in vitro. Methods: ADSCs were obtained from Sprague-Dawley rats using secondary collagenase digestion. Their phenotypes were characterized using flow cytometry detection of cell surface antigens and their stem cell properties were confirmed by induction of differentiation. After successful culture, ADSCs were transfected with lentiviral vectors and ChABC-ADSCs were obtained. Proliferation curves of ChABC-ADSCs were determined using the Cell Counting Kit-8 method, ChABC expression was verified using Western blotting, and the migration of ChABC-ADSCs was analyzed using the transwell assay. Results: Secondary collagenase digestion increased the isolation efficiency of primary ADSCs. Following transfection using lentiviral vectors, the proliferation of ChABC-ADSCs was reduced in comparison with control ADSCs at 48 h (P < 0.05). And the level of ChABC expression in the ChABC-ADSC group was significantly higher than that of the ADSC group (P < 0.05). Moreover, ChABC-ADSC migration in matrigel was significantly enhanced in comparison with the control (P < 0.05). Conclusions: Secondary collagenase digestion can be used to effectively isolate ADSCs. ChABC-ADSCs constructed using lentiviral vector transfection stably express ChABC, and ChABC expression significantly enhances the migratory capacity of ADSCs. PMID:27364797

  13. The A-B-C of Desalting.

    ERIC Educational Resources Information Center

    Department of the Interior, Washington, DC. Office of Water Research and Technology.

    This publication provides a simple explanation of how various processes convert sea or brackish water to fresh water. Included are descriptions of the membrane processes (reverse osmosis, electrodialysis, transport depletion, and piezodialysis); the distillation processes (multistage flash distillation, vertical tube distillation, multieffect…

  14. Dissociations among ABA, ABC, and AAB Recovery Effects

    ERIC Educational Resources Information Center

    Ungor, Metin; Lachnit, Harald

    2008-01-01

    In a human predictive learning experiment, the strengths of ABA, ABC, and AAB recovery effects after discrimination reversal learning were compared. Initially, a discrimination between two stimuli (X+, Y-) was trained in Context A. During Phase 2, participants received discrimination reversal training (X-, Y+) either in Context A (Group AAB) or in…

  15. The Value of Green Technology at ABC Community College

    ERIC Educational Resources Information Center

    McAllister, Bernadette

    2012-01-01

    A challenge facing community colleges nationwide is to reduce the carbon footprint of campuses by initiating green technology initiatives. This case study assessed the effect of switching from paper assignments to a learning management system at ABC Community College. The topic is important because federal and state funding, as well as…

  16. Electronic excitation spectrum of ABC-stacked multilayer graphene

    NASA Astrophysics Data System (ADS)

    Henni, Y.; Nogajewski, K.; Ojeda Collado, H. P.; Usaj, G.; Balseiro, C. A.; Potemski, M.; Faugeras, C.

    The electronic properties of ABC graphene trilayers has attracted lot of attention recently due to their potential applications in engineering carbon-based devices with gate tunable electrical conductivity. Morever,ABC-stacked thin layers of graphite are predicted to host peculiar surface electronic states, with a flat dispersion over most of the Brillouin zone. The associated high density of states is likely to favour the emergence of exotic electronic phases, such as charge density waves or even superconductivity. We present a micro-magneto-Raman scattering study of a thin graphite flake produced by exfoliation of natural graphite, composed of ~15graphene layers, and including a large ABC-stacked domain. Exploring the low temperature Raman scattering spectrum of this domain up to B=29T,we identify inter Landau level electronic excitations within the surface flat bands,together with electronic excitations involving the gapped states in the bulk. This interband electronic excitation at B=0T can be observed,up to room temperature, directly in the Raman scattering spectrum as a broad(~ 180 cm-1) feature. Because the energy gap strongly depends on the number of layers,this electronic excitation can be used to identify and characterize ABC-stacked graphite thin layers.

  17. Selections from the ABC 2009 Annual Convention, Portsmouth, Virginia

    ERIC Educational Resources Information Center

    Whalen, D. Joel

    2010-01-01

    The "My Favorite Assignment" Session at the 2009 Association for Business Communication (ABC) annual convention in Portsmouth, Virginia, featured over a dozen teachers sharing pedagogical innovations in a fast-paced, 4-minute format designed by Dan Dietrich. The wide variety of ideas and techniques presented makes these sessions popular ABC…

  18. The ABCs for Pre-Service Teacher Cultural Competency Development

    ERIC Educational Resources Information Center

    He, Ye; Cooper, Jewell E.

    2009-01-01

    In an effort to combine pre-service teachers' self-reflection with their field experiences to enhance their cultural competency, this study adopted Schmidt's ABC's (Autobiography, Biography, and Cross-cultural Comparison) Model in two courses in a pre-service teacher education program. Through group comparisons, this study measured the impact that…

  19. The Library ABC's Game: Sneaking in Learning through Gaming

    ERIC Educational Resources Information Center

    Maxwell, D. Jackson

    2007-01-01

    Teaching library terminology and definitions can be a real bore. Unfortunately, no matter how one looks at it, students need to learn a set of basic library words and their meanings. The Library ABC's game teaches elementary age students library terms and definitions, and it is effective, efficient, easy, exciting, and fun. Introduce the Library…

  20. The K-ABC in Historical and Contemporary Perspective.

    ERIC Educational Resources Information Center

    Anastasi, Anne

    1984-01-01

    The Kaufman Assessment Battery for Children is examined with particular attention to evolution of current psychometric concepts and methods, as well as the historical sources of popular misconceptions. The K-ABC reveals sophisticated applications of current test construction methodology but requires knowledgeable examiners. (Author/CL)

  1. Selections from the ABC 2012 Annual Convention, Honolulu, Hawaii

    ERIC Educational Resources Information Center

    Whalen, D. Joel

    2013-01-01

    The 13 Favorite Assignments featured here were presented at the 2012 Association for Business Communication (ABC) Annual Convention, Honolulu, Hawaii. A variety of learning objectives are featured, including the following: enhancing resume's visual impact, interpersonal skills, social media, team building, web design, community service…

  2. Selections from the ABC 2011 Annual Convention, Montreal, Canada

    ERIC Educational Resources Information Center

    Whalen, D. Joel; Andersen, Ken; Campbell, Gloria; Crenshaw, Cheri; Cross, Geoffrey A.; Grinols, Anne Bradstreet; Hildebrand, John; Newman, Amy; Ortiz, Lorelei A.; Paulson, Edward; Phillabaum, Melinda; Powell, Elizabeth A.; Sloan, Ryan

    2012-01-01

    The 12 Favorite Assignments featured in this article were presented at the 2011 Annual Convention of the Association for Business Communication (ABC), Montreal, Canada. A variety of learning objectives are featured: delivering bad news, handling difficult people, persuasion, reporting financial analysis, electronic media, face-to-face…

  3. Beyond the ABCs: The Pleasures of the Alphabet Book.

    ERIC Educational Resources Information Center

    Thatcher, Debra H.

    2002-01-01

    Identifies seven types of alphabet books: letter shapes, word play, art play, topical/thematic, multicultural, narrative, and puzzles. Presents annotations of around 30 titles. Suggests that alphabet books are not intended just for the emergent reader--there is a wide range of ABC books with intriguing stories, captivating illustrations, playful…

  4. ABCs of Being Smart... G Is for Gifted!

    ERIC Educational Resources Information Center

    Foster, Joanne

    2012-01-01

    Giftedness can generate speculation, misconceptions, expectations, pride, innuendo, apprehension, puzzlement--and the list goes on. What does it mean to be a gifted learner? In this installment of the series "ABCs of Being Smart," the author grapples with the term gifted, giving a glimpse into giftedness, along with some general guidelines for…

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

    PubMed Central

    2014-01-01

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

  6. Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes.

    PubMed

    Schneider, Erwin; Eckey, Viola; Weidlich, Daniela; Wiesemann, Nicole; Vahedi-Faridi, Ardeshir; Thaben, Paul; Saenger, Wolfram

    2012-04-01

    ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium. PMID:21561685

  7. Receptor-transporter interactions of canonical ATP-binding cassette import systems in prokaryotes.

    PubMed

    Schneider, Erwin; Eckey, Viola; Weidlich, Daniela; Wiesemann, Nicole; Vahedi-Faridi, Ardeshir; Thaben, Paul; Saenger, Wolfram

    2012-04-01

    ATP-binding cassette (ABC) transport systems mediate the translocation of solutes across biological membranes at the expense of ATP. They share a common modular architecture comprising two pore-forming transmembrane domains and two nucleotide binding domains. In prokaryotes, ABC transporters are involved in the uptake of a large variety of chemicals, including nutrients, osmoprotectants and signal molecules. In pathogenic bacteria, some ABC importers are virulence factors. Canonical ABC import systems require an additional component, a substrate-specific receptor or binding protein for function. Interaction of the liganded receptor with extracytoplasmic loop regions of the transmembrane domains initiate the transport cycle. In this review we summarize the current knowledge on receptor-transporter interplay provided by crystal structures as well as by biochemical and biophysical means. In particular, we focus on the maltose/maltodextrin transporter of enterobacteria and the transporters for positively charged amino acids from the thermophile Geobacillus stearothermophilus and Salmonella enterica serovar Typhimurium.

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

    PubMed Central

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

    2013-01-01

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

  9. 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 (...

  10. Transmembrane gate movements in the type II ATP-binding cassette (ABC) importer BtuCD-F during nucleotide cycle.

    PubMed

    Joseph, Benesh; Jeschke, Gunnar; Goetz, Birke A; Locher, Kaspar P; Bordignon, Enrica

    2011-11-25

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.

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

    PubMed

    Lu, Hong; Xu, Yongyu; Cui, Feng

    2016-09-01

    The ATP-binding cassette (ABC) transporter family functions in the ATP-dependent transportation of various substrates across biological membranes. ABC proteins participate in various biological processes and insecticide resistance in insects, and are divided into eight subfamilies (A-H). Mosquitoes are important vectors of human diseases, but the mechanism by which the ABC transporter family evolves in mosquitoes is unknown. In this study, we classified and compared the ABC transporter families of three mosquitoes, namely, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus. The three mosquitoes have 55, 69, and 70 ABC genes, respectively. The C. p. quinquefasciatus had approximately 40% and 65% expansion in the ABCG subfamily, mainly in ABCG1/G4, compared with the two other mosquito species. The ABCB, ABCD, ABCE, and ABCF subfamilies were conserved in the three mosquito species. The C. p. quinquefasciatus transcriptomes during development showed that the ABCG and ABCC genes were mainly highly expressed at the egg and pupal stages. The pigment-transport relative brown, white, and scarlet, as well as the ABCF subfamily, were highly expressed at the egg stage. The highly expressed genes in larvae included three ABCA3 genes. The majority of the highly expressed genes in adults were ABCG1/4 genes. These results provided insights into the evolution of the ABC transporter family in mosquitoes. PMID:27521922

  12. Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa.

    PubMed

    Weeks, Jon W; Nickels, Logan M; Ntreh, Abigail T; Zgurskaya, Helen I

    2015-10-01

    In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role. PMID:26193906

  13. Non-equivalent roles of two periplasmic subunits in the function and assembly of triclosan pump TriABC from Pseudomonas aeruginosa.

    PubMed

    Weeks, Jon W; Nickels, Logan M; Ntreh, Abigail T; Zgurskaya, Helen I

    2015-10-01

    In Gram-negative bacteria, multidrug efflux transporters function in complexes with periplasmic membrane fusion proteins (MFPs) that enable antibiotic efflux across the outer membrane. In this study, we analyzed the function, composition and assembly of the triclosan efflux transporter TriABC-OpmH from Pseudomonas aeruginosa. We report that this transporter possesses a surprising substrate specificity that encompasses not only triclosan but the detergent SDS, which are often used together in antibacterial soaps. These two compounds interact antagonistically in a TriABC-dependent manner and negate antibacterial properties of each other. Unlike other efflux pumps that rely on a single MFP for their activities, two different MFPs, TriA and TriB, are required for triclosan/SDS resistance mediated by TriABC-OpmH. We found that analogous mutations in the α-helical hairpin and membrane proximal domains of TriA and TriB differentially affect triclosan efflux and assembly of the complex. Furthermore, our results show that TriA and TriB function as a dimer, in which TriA is primarily responsible for stabilizing interactions with the outer membrane channel, whereas TriB is important for the stimulation of the transporter. We conclude that MFPs are engaged into complexes as asymmetric dimers, in which each protomer plays a specific role.

  14. TaAbc1, a member of Abc1-like family involved in hypersensitive response against the stripe rust fungal pathogen in wheat.

    PubMed

    Wang, Xiaojing; Wang, Xiaojie; Duan, Yinghui; Yin, Shuining; Zhang, Hongchang; Huang, Li; Kang, Zhensheng

    2013-01-01

    To search for genes involved in wheat (Triticum aestivum L.) defense response to the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned a new wheat gene similar to the genes in the Abc1-like gene family. The new gene, designated as TaAbc1, encodes a 717-amino acid, 80.35 kD protein. The TaAbc1 protein contains two conserved domains shared by Abc1-like proteins, two trans-membrane domains at the C-terminal, and a 36-amino acid chloroplast targeting presequence at the N-terminal. Characterization of TaAbc1 expression revealed that gene expression was tissue-specific and could be up-regulated by biotic agents (e.g., stripe rust pathogen) and/or by an abiotic stress like wounding. High-fold induction was associated with the hypersensitive response (HR) triggered only by avirulent stripe rust pathotypes, suggesting that TaAbc1 is a rust-pathotype specific HR-mediator. Down-regulating TaAbc1 reduced HR but not the overall resistance level in Suwon11 to CYR23, suggesting TaAbc1 was involved in HR against stripe rust, but overall host resistance is not HR-dependent.

  15. Goethe and the ABC model of flower development.

    PubMed

    Coen, E

    2001-06-01

    About 10 years ago, the ABC model for the genetic control of flower development was proposed. This model was initially based on the analysis of mutant flowers but has subsequently been confirmed by molecular analysis. This paper describes the 200-year history behind this model, from the late 18th century when Goethe arrived at his idea of plant metamorphosis, to the genetic studies on flower mutants carried out on Arabidopsis and Antirrhinum in the late 20th century.

  16. Second-order nonlinear optical metamaterials: ABC-type nanolaminates

    SciTech Connect

    Alloatti, L. Kieninger, C.; Lauermann, M.; Köhnle, K.; Froelich, A.; Wegener, M.; Frenzel, T.; Freude, W.; Leuthold, J.; Koos, C.

    2015-09-21

    We demonstrate a concept for second-order nonlinear metamaterials that can be obtained from non-metallic centrosymmetric constituents with inherently low optical absorption. The concept is based on iterative atomic-layer deposition of three different materials, A = Al{sub 2}O{sub 3}, B = TiO{sub 2}, and C = HfO{sub 2}. The centrosymmetry of the resulting ABC stack is broken since the ABC and the inverted CBA sequences are not equivalent—a necessary condition for non-zero second-order nonlinearity. In our experiments, we find that the bulk second-order nonlinear susceptibility depends on the density of interfaces, leading to a nonlinear susceptibility of 0.26 pm/V at a wavelength of 800 nm. ABC-type nanolaminates can be deposited on virtually any substrate and offer a promising route towards engineering of second-order optical nonlinearities at both infrared and visible wavelengths.

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

    PubMed Central

    Sharkey, Liam K. R.; Edwards, Thomas A.

    2016-01-01

    ABSTRACT Members of the ABC-F subfamily of ATP-binding cassette proteins mediate resistance to a broad array of clinically important antibiotic classes that target the ribosome of Gram-positive pathogens. The mechanism by which these proteins act has been a subject of long-standing controversy, with two competing hypotheses each having gained considerable support: antibiotic efflux versus ribosomal protection. Here, we report on studies employing a combination of bacteriological and biochemical techniques to unravel the mechanism of resistance of these proteins, and provide several lines of evidence that together offer clear support to the ribosomal protection hypothesis. Of particular note, we show that addition of purified ABC-F proteins to an in vitro translation assay prompts dose-dependent rescue of translation, and demonstrate that such proteins are capable of displacing antibiotic from the ribosome in vitro. To our knowledge, these experiments constitute the first direct evidence that ABC-F proteins mediate antibiotic resistance through ribosomal protection. PMID:27006457

  18. High-level assessment of LANL ABC Design

    SciTech Connect

    Not Available

    1994-04-15

    An annual weapon`s grade Pu disposition goal should be stated and related to the amount of Pu that needs to be disposed of. It needs to be determined to what extent it is possible to destroy Pu without building up any new Pu, i.e., how realistic this goal is. The strong positive Doppler coefficient for a Pu core might require the addition of some fertile material to ensure a negative Doppler coefficient. This in turn will affect the net Pu disposition rate. If a fertile material is required throughout the life of the ABC to ensure a negative Doppler coefficient, the difference between the molten salt ABC and other reactors in regard to Pu disposition is not a principled difference anymore but one of degree. A rationale has then to be developed that explains why {open_quotes}x{close_quotes} kg production of fissile material are acceptable but {open_quotes}y{close_quotes} kg are not. It is important to determine how a requirement for electricity production will impact on the ABC design choices. It is conceivable that DOE will not insist on electricity generation. In this case advantage has to be taken in terms of design simplifications and relaxed operating conditions.

  19. Recent advances regarding the role of ABC subfamily C member 10 (ABCC10) in the efflux of antitumor drugs

    PubMed Central

    Kathawala, Rishil J.; Wang, Yi-Jun; Ashby, Charles R.; Chen, Zhe-Sheng

    2014-01-01

    ABCC10, also known as multidrug-resistant protein 7 (MRP7), is the tenth member of the C subfamily of the ATP-binding cassette (ABC) superfamily. ABCC10 mediates multidrug resistance (MDR) in cancer cells by preventing the intracellular accumulation of certain antitumor drugs. The ABCC10 transporter is a 171-kDa protein that is localized on the basolateral cell membrane. ABCC10 is a broad-specificity transporter of xenobiotics, including antitumor drugs, such as taxanes, epothilone B, vinca alkaloids, and cytarabine, as well as modulators of the estrogen pathway, such as tamoxifen. In recent years, ABCC10 inhibitors, including cepharanthine, lapatinib, erlotinib, nilotinib, imatinib, sildenafil, and vardenafil, have been reported to overcome ABCC10-mediated MDR. This review discusses some recent and clinically relevant aspects of the ABCC10 drug efflux transporter from the perspective of current chemotherapy, particularly its inhibition by tyrosine kinase inhibitors and phosphodiesterase type 5 inhibitors. PMID:24103790

  20. Recent advances regarding the role of ABC subfamily C member 10 (ABCC10) in the efflux of antitumor drugs.

    PubMed

    Kathawala, Rishil J; Wang, Yi-Jun; Ashby, Charles R; Chen, Zhe-Sheng

    2014-05-01

    ABCC10, also known as multidrug-resistant protein 7 (MRP7), is the tenth member of the C subfamily of the ATP-binding cassette (ABC) superfamily. ABCC10 mediates multidrug resistance (MDR) in cancer cells by preventing the intracellular accumulation of certain antitumor drugs. The ABCC10 transporter is a 171-kDa protein that is localized on the basolateral cell membrane. ABCC10 is a broad-specificity transporter of xenobiotics, including antitumor drugs, such as taxanes, epothilone B, vinca alkaloids, and cytarabine, as well as modulators of the estrogen pathway, such as tamoxifen. In recent years, ABCC10 inhibitors, including cepharanthine, lapatinib, erlotinib, nilotinib, imatinib, sildenafil, and vardenafil, have been reported to overcome ABCC10-mediated MDR. This review discusses some recent and clinically relevant aspects of the ABCC10 drug efflux transporter from the perspective of current chemotherapy, particularly its inhibition by tyrosine kinase inhibitors and phosphodiesterase type 5 inhibitors. PMID:24103790

  1. Altered Profile of Secondary Metabolites in the Root Exudates of Arabidopsis ATP-Binding Cassette Transporter Mutants1[C][W][OA

    PubMed Central

    Badri, Dayakar V.; Loyola-Vargas, Victor M.; Broeckling, Corey D.; De-la-Peña, Clelia; Jasinski, Michal; Santelia, Diana; Martinoia, Enrico; Sumner, Lloyd W.; Banta, Lois M.; Stermitz, Frank; Vivanco, Jorge M.

    2008-01-01

    Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters. PMID:18065561

  2. ABC1K atypical kinases in plants; filling the organellar kinase void

    PubMed Central

    Lundquist, Peter K.; Davis, Jerrold I.; van Wijk, Klaas J.

    2014-01-01

    Surprisingly few protein kinases have been demonstrated in chloroplasts or mitochondria. Here we discuss the “activity of bc1 complex kinase” (ABC1K) protein family which we suggest locate in mitochondria and plastids, thus filling the kinase void. The ABC1Ks are atypical protein kinases and their ancestral function is the regulation of quinone synthesis. ABC1Ks have proliferated from 1–2 members in non-photosynthetic organisms to more than 16 members in algae and higher plants. In this review we reconstruct the evolutionary history of the ABC1K family, provide a functional domain analysis for angiosperms and a nomenclature for ABC1Ks in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and maize (Zea mays). Finally, we hypothesize that targets of ABC1Ks include enzymes of prenyl-lipid metabolism as well as components of the organellar gene expression machineries. PMID:22694836

  3. Creating an iPhone application for collecting continuous ABC data.

    PubMed

    Whiting, Seth W; Dixon, Mark R

    2012-01-01

    This paper provides an overview and task analysis for creating a continuous ABC data-collection application using Xcode on a Mac computer. Behavior analysts can program an ABC data collection system, complete with a customized list of target clients, antecedents, behaviors, and consequences to be recorded, and have the data automatically sent to an e-mail account after observations have concluded. Further suggestions are provided to customize the ABC data- collection system for individual preferences and clinical needs.

  4. Improvement of proteolytic and oxidative stability of Chondroitinase ABC I by cosolvents.

    PubMed

    Nazari-Robati, Mahdieh; Golestani, Abolfazl; Asadikaram, GholamReza

    2016-10-01

    Recently, utilization of the enzyme Chondroitinase ABC I (cABC I) has received considerable attention in treatment of spinal cord injury. cABC I removes chondroitin sulfate proteoglycans which are inhibitory to axon growth and enhances nerve regeneration. Therefore, determination of cABC I resistance to proteolysis and oxidation provides valuable information for optimizing its clinical application. In this work, proteolytic stability of cABC I to trypsin and chymotrypsin as well as its oxidative resistance to H2O2 was measured. Moreover, the effect of cosolvents glycerol, sorbitol and trehalose on cABC I proteolytic and oxidative stability was determined. The results indicated that cABC I is highly susceptible to proteolysis and oxidation. Comparison of proteolytic patterns demonstrated a high degree of similarity which confirmed the exposure of specific regions of cABC I to proteolysis. However, proteolytic degradation was significantly reduced in the presence of cosolvents. In addition, cosolvents decreased the rate of both cABC I proteolytic and oxidative inactivation. Notably, the degree of stabilization provided by these cosolvents varied greatly. These findings indicated the high potential of cosolvents in protein stabilization to proteolysis and oxidative inactivation.

  5. MdsABC-Mediated Pathway for Pathogenicity in Salmonella enterica Serovar Typhimurium.

    PubMed

    Song, Saemee; Lee, Boeun; Yeom, Ji-Hyun; Hwang, Soonhye; Kang, Ilnam; Cho, Jang-Cheon; Ha, Nam-Chul; Bae, Jeehyeon; Lee, Kangseok; Kim, Yong-Hak

    2015-11-01

    MdsABC is a Salmonella-specific tripartite efflux pump that has been implicated in the virulence of Salmonella enterica serovar Typhimurium; however, little is known about the virulence factors associated with this pump. We observed MdsABC expression-dependent alterations in the degree of resistance to extracellular oxidative stress and macrophage-mediated killing. Thin-layer chromatography and tandem mass spectrometry analyses revealed that overexpression of MdsABC led to increased secretion of 1-palmitoyl-2-stearoyl-phosphatidylserine (PSPS), affecting the ability of the bacteria to invade and survive in host cells. Overexpression of MdsABC and external addition of PSPS similarly rendered the mdsABC deletion strain resistant to diamide. Diagonal gel analysis showed that PSPS treatment reduced the diamide-mediated formation of disulfide bonds, particularly in the membrane fraction of the bacteria. Salmonella infection of macrophages induced the upregulation of MdsABC expression and led to an increase of intracellular bacterial number and host cell death, similar to the effects of MdsABC overexpression and PSPS pretreatment on the mdsABC deletion strain. Our study shows that MdsABC mediates a previously uncharacterized pathway that involves PSPS as a key factor for the survival and virulence of S. Typhimurium in phagocytic cells.

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

    SciTech Connect

    Zhang, Han; Rahman, Sadia; Li, Wen; Fu, Guoxing; Kaur, Parjit

    2015-03-27

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

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

    PubMed

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

    2015-02-01

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

  8. Improved avidin-biotin-peroxidase complex (ABC) staining.

    PubMed

    Cattoretti, G; Berti, E; Schiró, R; D'Amato, L; Valeggio, C; Rilke, F

    1988-02-01

    A considerable intensification of the avidin-biotin-peroxidase complex staining system (ABC) was obtained by sequentially overlaying the sections to be immunostained with an avidin-rich and a biotin-rich complex. Each sequential addition contributed to the deposition of horseradish peroxidase on the immunostained site and allowed the subsequent binding of a complementary complex. With this technique a higher dilution of the antisera could be used and minute amounts of antigen masked by the fixative could be demonstrated on paraffin sections.

  9. Design of the storage location based on the ABC analyses

    NASA Astrophysics Data System (ADS)

    Jemelka, Milan; Chramcov, Bronislav; Kříž, Pavel

    2016-06-01

    The paper focuses on process efficiency and saving storage costs. Maintaining inventory through putaway strategy takes personnel time and costs money. The aim is to control inventory in the best way. The ABC classification based on Villefredo Pareto theory is used for a design of warehouse layout. New design of storage location reduces the distance of fork-lifters, total costs and it increases inventory process efficiency. The suggested solutions and evaluation of achieved results are described in detail. Proposed solutions were realized in real warehouse operation.

  10. SU-E-T-401: Feasibility Study of Using ABC to Gate Lung SBRT Treatment

    SciTech Connect

    Cao, D; Xie, X; Shepard, D

    2014-06-01

    Purpose: The current SBRT treatment techniques include free breathing (FB) SBRT and gated FB SBRT. Gated FB SBRT has smaller target and less lung toxicity with longer treatment time. The recent development of direct connectivity between the ABC and linac allowing for automated beam gating. In this study, we have examined the feasibility of using ABC system to gate the lung SBRT treatment. Methods: A CIRS lung phantom with a 3cm sphere-insert and a moving chest plate was used in this study. Sinusoidal motion was used for the FB pattern. An ABC signal was imported to simulate breath holds. 4D-CT was taken in FB mode and average-intensity-projection (AIP) was used to create FB and 50% gated FB SBRT planning CT. A manually gated 3D CT scan was acquired for ABC gated SBRT planning.An SBRT plan was created for each treatment option. A surface-mapping system was used for 50% gating and ABC system was used for ABC gating. A manually gated CBCT scan was also performed to verify setup. Results: Among three options, the ABC gated plan has the smallest PTV of 35.94cc, which is 35% smaller comparing to that of the FB plan. Consequently, the V20 of the left lung reduced by 15% and 23% comparing to the 50% gated FB and FB plans, respectively. The FB plan took 4.7 minutes to deliver, while the 50% gated FB plan took 18.5 minutes. The ABC gated plan delivery took only 10.6 minutes. A stationary target with 3cm diameter was also obtained from the manually gated CBCT scan. Conclusion: A strategy for ABC gated lung SBRT was developed. ABC gating can significantly reduce the lung toxicity while maintaining the target coverage. Comparing to the 50% gated FB SBRT, ABC gated treatment can also provide less lung toxicity as well as improved delivery efficiency. This research is funded by Elekta.

  11. Dual Zinc Transporter Systems in Vibrio cholerae Promote Competitive Advantages over Gut Microbiome.

    PubMed

    Sheng, Ying; Fan, Fenxia; Jensen, Owen; Zhong, Zengtao; Kan, Biao; Wang, Hui; Zhu, Jun

    2015-10-01

    Zinc is an essential trace metal required for numerous cellular processes in all forms of life. In order to maintain zinc homeostasis, bacteria have developed several transport systems to regulate its uptake. In this study, we investigated zinc transport systems in the enteric pathogen Vibrio cholerae, the causative agent of cholera. Bioinformatic analysis predicts that two gene clusters, VC2081 to VC2083 (annotated as zinc utilization genes znuABC) and VC2551 to VC2555 (annotated as zinc-regulated genes zrgABCDE), are regulated by the putative zinc uptake regulator Zur. Using promoter reporter and biochemical assays, we confirmed that Zur represses znuABC and zrgABCDE promoters in a Zn(2+)-dependent manner. Under Zn(2+)-limiting conditions, we found that mutations in either the znuABC or zrgABCDE gene cluster affect bacterial growth, with znuABC mutants displaying a more severe growth defect, suggesting that both ZnuABC and ZrgABCDE are involved in Zn(2+) uptake and that ZnuABC plays the predominant role. Furthermore, we reveal that ZnuABC and ZrgABCDE are important for V. cholerae colonization in both infant and adult mouse models, particularly in the presence of other intestinal microbiota. Collectively, our studies indicate that these two zinc transporter systems play vital roles in maintaining zinc homeostasis during V. cholerae growth and pathogenesis.

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

    PubMed

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

    2015-01-01

    Over-expression of ATP-binding cassette (ABC) transporters, a large family of integral membrane proteins that decrease cellular drug uptake and accumulation by active extrusion, is one of the major causes of cancer multi-drug resistance (MDR) that frequently leads to failure of chemotherapy. Carbon nanotubes (CNTs)-based drug delivery devices hold great promise in enhancing the efficacy of cancer chemotherapy. However, CNTs' effects on the ABC transporters remain under-investigated. In this study, we found that multiwalled carbon nanotubes (MWCNTs) reduced transport activity and expression of ABC transporters including ABCB1/Pgp and ABCC4/MRP4 in human colon adenocarcinoma Caco-2 cells. Proto-oncogene c-Myc, which directly regulates ABC gene expression, was concurrently decreased in MWCNT-treated cells and forced over-expression of c-Myc reversed MWCNTs' inhibitory effects on ABCB1 and ABCC4 expression. MWCNT-cell membrane interaction and cell membrane oxidative damage were observed. However, antioxidants such as vitamin C, β-mecaptoethanol and dimethylthiourea failed to antagonize MWCNTs' down-regulation of ABC transporters. These data suggest that MWCNTs may act on c-Myc, but not through oxidative stress, to down-regulate ABC transporter expression. Our findings thus shed light on CNTs' novel cellular effects that may be utilized to develop CNTs-based drug delivery devices to overcome ABC transporter-mediated cancer chemoresistance. PMID:25461681

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

    SciTech Connect

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

    2015-01-01

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

  14. What do drug transporters really do?

    PubMed Central

    Nigam, Sanjay K.

    2016-01-01

    Potential drug–drug interactions mediated by the ATP-binding cassette (ABC) transporter and solute carrier (SLC) transporter families are of clinical and regulatory concern. However, the endogenous functions of these drug transporters are not well understood. Discussed here is evidence for the roles of ABC and SLC transporters in the handling of diverse substrates, including metabolites, antioxidants, signalling molecules, hormones, nutrients and neurotransmitters. It is suggested that these transporters may be part of a larger system of remote communication (‘remote sensing and signalling’) between cells, organs, body fluid compartments and perhaps even separate organisms. This broader view may help to clarify disease mechanisms, drug–metabolite interactions and drug effects relevant to diabetes, chronic kidney disease, metabolic syndrome, hypertension, gout, liver disease, neuropsychiatric disorders, inflammatory syndromes and organ injury, as well as prenatal and postnatal development. PMID:25475361

  15. Sugar transport in (hyper)thermophilic archaea.

    PubMed

    Koning, Sonja M; Albers, Sonja-Verena; Konings, Wil N; Driessen, Arnold J M

    2002-03-01

    Hyperthermophilic archaea show important metabolic adaptations for growth on carbohydrates under hostile conditions. For carbohydrate uptake so far only ABC-type transporters have been described that are equipped with a uniquely high affinity as compared to mesophilic bacterial systems. This allows these organisms to efficiently scavenge all available carbohydrates from the extreme environment.

  16. ABCs of Public Education in North Carolina: A Journey toward Excellence.

    ERIC Educational Resources Information Center

    North Carolina State Board of Education, Raleigh.

    In 1995, the North Carolina General Assembly directed the North Carolina State Board of Education to develop a plan to bolster student growth and performance in grades 4-8 throughout the state. In response, the board developed the ABCs of Public Education. (ABC stands for Accountability; teaching the Basics of reading, writing, and mathematics;…

  17. Manipulating the glial scar: chondroitinase ABC as a therapy for spinal cord injury.

    PubMed

    Bradbury, Elizabeth J; Carter, Lucy M

    2011-03-10

    Chondroitin sulphate proteoglycans (CSPGs) are potent inhibitors of growth in the adult CNS. Use of the enzyme chondroitinase ABC (ChABC) as a strategy to reduce CSPG inhibition in experimental models of spinal cord injury has led to observations of a remarkable capacity for repair. Here we review the evidence that treatment with ChABC, either as an individual therapy or in combination with other strategies, can have multiple beneficial effects on promoting repair following spinal cord injury. These include promoting regeneration of injured axons, plasticity of uninjured pathways and neuroprotection of injured projection neurons. More importantly, ChABC therapy has been demonstrated to promote significant recovery of function to spinal injured animals. Thus, there is robust pre-clinical evidence demonstrating beneficial effects of ChABC treatment following spinal cord injury. Furthermore, these effects have been replicated in a number of different injury models, with independent confirmation by different laboratories, providing an important validation of ChABC as a promising therapeutic strategy. We discuss putative mechanisms underlying ChABC-mediated repair as well as potential issues and considerations in translating ChABC treatment into a clinical therapy for spinal cord injury.

  18. Structural Validity of the Movement ABC-2 Test: Factor Structure Comparisons across Three Age Groups

    ERIC Educational Resources Information Center

    Schulz, Joerg; Henderson, Sheila E.; Sugden, David A.; Barnett, Anna L.

    2011-01-01

    Background: The Movement ABC test is one of the most widely used assessments in the field of Developmental Coordination Disorder (DCD). Improvements to the 2nd edition of the test (M-ABC-2) include an extension of the age range and reduction in the number of age bands as well as revision of tasks. The total test score provides a measure of motor…

  19. Creating an iPhone Application for Collecting Continuous ABC Data

    ERIC Educational Resources Information Center

    Whiting, Seth W.; Dixon, Mark R.

    2012-01-01

    This paper provides an overview and task analysis for creating a continuous ABC data- collection application using Xcode on a Mac computer. Behavior analysts can program an ABC data collection system, complete with a customized list of target clients, antecedents, behaviors, and consequences to be recorded, and have the data automatically sent to…

  20. Movement Assessment Battery for Children (M-ABC): Establishing Construct Validity for Israeli Children

    ERIC Educational Resources Information Center

    Engel-Yeger, Batya; Rosenblum, Sara; Josman, Naomi

    2010-01-01

    The Movement Assessment Battery for Children (M-ABC) is one of the most accepted tools, both in clinical practice and in research, for the diagnosis of Developmental Coordination Disorders (DCDs) in children. The present study aimed to: (1) establish the construct validity of M-ABC in Israel by comparing the motor performance of typically…

  1. Parents' Perspectives on Braille Literacy: Results from the ABC Braille Study

    ERIC Educational Resources Information Center

    Kamei-Hannan, Cheryl; Sacks, Sharon Zell

    2012-01-01

    Introduction: Parents who were the primary caretakers of children in the Alphabetic and Contracted Braille Study (ABC Braille Study) revealed their perspectives about braille literacy. Methods: A 30-item questionnaire was constructed by the ABC Braille research team, and researchers conducted telephone interviews with 31 parents who were the…

  2. The ABCs of Activity-Based Costing: A Cost Containment and Reallocation Tool.

    ERIC Educational Resources Information Center

    Turk, Frederick J.

    1992-01-01

    This article describes activity-based costing (ABC) and how this tool may help management understand the costs of major activities and identify possible alternatives. Also discussed are the traditional costing systems used by higher education and ways of applying ABC to higher education. (GLR)

  3. The Role of Activity Based Costing (ABC) in Educational Support Services: A White Paper.

    ERIC Educational Resources Information Center

    Edds, Daniel B.

    Many front-line managers who are assuming more financial responsibility for their organizations find traditional cost accounting inadequate for their needs and are turning to Activity Based Costing (ABC). ABC is not a financial reporting system to serve the needs of regulatory agencies, but a tool that tracks costs from the general ledger…

  4. Applying the Post-Modern Double ABC-X Model to Family Food Insecurity

    ERIC Educational Resources Information Center

    Hutson, Samantha; Anderson, Melinda; Swafford, Melinda

    2015-01-01

    This paper develops the argument that using the Double ABC-X model in family and consumer sciences (FCS) curricula is a way to educate nutrition and dietetics students regarding a family's perceptions of food insecurity. The Double ABC-X model incorporates ecological theory as a basis to explain family stress and the resulting adjustment and…

  5. A Comparison of the K-ABC and WISC-R: A Validity Study.

    ERIC Educational Resources Information Center

    Sapp, Gary L.; And Others

    The concurrent validity of the Kaufman Assessment Battery for Children (K-ABC) was examined by comparing K-ABC scores and Weschler Intelligence Scale for Children--Revised (WISC-R) scores for 58 school children in primary and intermediate grades. Thirty-seven of these children had either educable mental retardation, learning disabilities, or…

  6. Abrasive wear behavior of heat-treated ABC-silicon carbide

    SciTech Connect

    Zhang, Xiao Feng; Lee, Gun Y.; Chen, Da; Ritchie, Robert O.; De Jonghe, Lutgard C.

    2002-06-17

    Hot-pressed silicon carbide, containing aluminum, boron, and carbon additives (ABC-SiC), was subjected to three-body and two-body wear testing using diamond abrasives over a range of sizes. In general, the wear resistance of ABC-SiC, with suitable heat treatment, was superior to that of commercial SiC.

  7. Accuracy of the ABC/2 score for intracerebral hemorrhage: Systematic review and analysis of MISTIE, CLEAR-IVH, CLEAR III

    PubMed Central

    Webb, Alastair JS; Ullman, Natalie L; Morgan, Tim C; Muschelli, John; Kornbluth, Joshua; Awad, Issam A; Mayo, Stephen; Rosenblum, Michael; Ziai, Wendy; Zuccarrello, Mario; Aldrich, Francois; John, Sayona; Harnof, Sagi; Lopez, George; Broaddus, William C; Wijman, Christine; Vespa, Paul; Bullock, Ross; Haines, Stephen J; Cruz-Flores, Salvador; Tuhrim, Stan; Hill, Michael D; Narayan, Raj; Hanley, Daniel F

    2015-01-01

    Background and Purpose The ABC/2 score estimates intracerebral hemorrhage (ICH) volume, yet validations have been limited by small samples and inappropriate outcome measures. We determined accuracy of the ABC/2 score calculated at a specialized Reading Center (RC-ABC) or local site (site-ABC) versus the reference-standard CT-based planimetry (CTP). Methods In MISTIE-II, CLEAR-IVH and CLEAR-III trials, ICH volume was prospectively calculated by CTP, RC-ABC and site-ABC. Agreement between CTP and ABC/2 was defined as an absolute difference up to 5ml and relative difference within 20%. Determinants of ABC/2 accuracy were assessed by logistic regression. Results In 4369 scans from 507 patients, CTP was more strongly correlated with RC-ABC (r2=0.93) than site-ABC (r2=0.87). Although RC-ABC overestimated CTP-based volume on average (RC-ABC=15.2cm3, CTP=12.7cm3), agreement was reasonable when categorised into mild, moderate and severe ICH (kappa 0.75, p<0.001). This was consistent with overestimation of ICH volume in 6/8 previous studies. Agreement with CTP was greater for RC-ABC (84% within 5ml; 48% of scans within 20%) than for site-ABC (81% within 5ml; 41% within 20%). RC-ABC had moderate accuracy for detecting ≥ 5ml change in CTP volume between consecutive scans (sensitivity 0.76, specificity 0.86) and was more accurate with smaller ICH, thalamic haemorrhage and homogeneous clots. Conclusions ABC/2 scores at local or central sites are sufficiently accurate to categorise ICH volume and assess eligibility for the CLEAR III and MISTIE III studies, and moderately accurate for change in ICH volume. However, accuracy decreases with large, irregular or lobar clots. Clinical Trial Registration MISTIE-II NCT00224770; CLEAR-III NCT00784134; www.clinicaltrials.gov PMID:26243227

  8. Hijacking membrane transporters for arsenic phytoextraction

    PubMed Central

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

    2012-01-01

    Arsenic is a toxic metalloid and recognized carcinogen. Arsenate and arsenite are the most common arsenic species available for uptake by plants. As an inorganic phosphate (Pi) analog, arsenate is acquired by plant roots through endogenous Pi transport systems. Inside the cell, arsenate is reduced to the thiol-reactive form arsenite. Gluta