WorldWideScience

Sample records for antiporters

  1. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    We examined the expression and subcellular localization of antiporter regulating protein OsARP in a submergence tolerant rice (Oryza sativa L.) cultivar FR13A. In the public databases, this protein was designated as putative Os02g0465900 protein. The cDNA containing the full-length sequence of OsARP gene was ...

  2. Comparative analysis of cation/proton antiporter superfamily in plants

    Energy Technology Data Exchange (ETDEWEB)

    Ye, Chuyu [ORNL; Yang, Xiaohan [ORNL; Xia, Xinli [Beijing Forestry University, China; Yin, Weilun [Beijing Forestry University, China

    2013-01-01

    The cation/proton antiporter superfamily is associated with the transport of monovalent cations across membranes. This superfamily was annotated in the Arabidopsis genome and some members were functionally characterized. In the present study, a systematic analysis of the cation/proton antiporter genes in diverse plant specieswas reported.We identified 240 cation/proton antiporters in alga, moss, and angiosperm. A phylogenetic tree was constructed showing these 240members are separated into three families, i.e., Na+/H+ exchangers, K+ efflux antiporters, and cation/H+ exchangers. Our analysis revealed that tandem and/or segmental duplications contribute to the expansion of cation/H+ exchangers in the examined angiospermspecies. Sliding windowanalysis of the nonsynonymous/synonymous substitution ratios showed some differences in the evolutionary fate of cation/proton antiporter paralogs. Furthermore, we identified over-represented motifs among these 240 proteins and foundmostmotifs are family specific, demonstrating diverse evolution of the cation/proton antiporters among three families. In addition, we investigated the co-expressed genes of the cation/proton antiporters in Arabidopsis thaliana. The results showed some biological processes are enriched in the co-expressed genes, suggesting the cation/proton antiporters may be involved in these biological processes. Taken together, this study furthers our knowledge on cation/proton antiporters in plants.

  3. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    Md. Imtiaz Uddin

    2012-02-14

    Feb 14, 2012 ... We examined the expression and subcellular localization of antiporter regulating protein OsARP in a submergence .... Ten micro liters. PCR reaction consisted of template DNA 1 μL, primer (0.8 μM) 4. μL, Dye terminator V1.1- 8 μL and dH2O 7 μL. The PCR condition .... important role under those stresses.

  4. Isolation and preliminary function analysis of a Na + /H + antiporter ...

    African Journals Online (AJOL)

    A full-length cDNA Na+/H+ antiporter gene (MzNHX1) was isolated from Malus zumi according to the homologous Na+/H+ antiporter gene region in plants. Sequence analysis indicated that the cDNA was 2062 bp in length, including an open reading frame (ORF) of 1629 bp, which encoded a predicted polypeptide of 542 ...

  5. Mrp Antiporters Have Important Roles in Diverse Bacteria and Archaea.

    Science.gov (United States)

    Ito, Masahiro; Morino, Masato; Krulwich, Terry A

    2017-01-01

    Mrp (Multiple resistance and pH) antiporter was identified as a gene complementing an alkaline-sensitive mutant strain of alkaliphilic Bacillus halodurans C-125 in 1990. At that time, there was no example of a multi-subunit type Na + /H + antiporter comprising six or seven hydrophobic proteins, and it was newly designated as the monovalent cation: proton antiporter-3 (CPA3) family in the classification of transporters. The Mrp antiporter is broadly distributed among bacteria and archaea, not only in alkaliphiles. Generally, all Mrp subunits, mrpA-G , are required for enzymatic activity. Two exceptions are Mrp from the archaea Methanosarcina acetivorans and the eubacteria Natranaerobius thermophilus , which are reported to sustain Na + /H + antiport activity with the MrpA subunit alone. Two large subunits of the Mrp antiporter, MrpA and MrpD, are homologous to membrane-embedded subunits of the respiratory chain complex I, NuoL, NuoM, and NuoN, and the small subunit MrpC has homology with NuoK. The functions of the Mrp antiporter include sodium tolerance and pH homeostasis in an alkaline environment, nitrogen fixation in Schizolobium meliloti , bile salt tolerance in Bacillus subtilis and Vibrio cholerae , arsenic oxidation in Agrobacterium tumefaciens , pathogenesis in Pseudomonas aeruginosa and Staphylococcus aureus , and the conversion of energy involved in metabolism and hydrogen production in archaea. In addition, some Mrp antiporters transport K + and Ca 2+ instead of Na + , depending on the environmental conditions. Recently, the molecular structure of the respiratory chain complex I has been elucidated by others, and details of the mechanism by which it transports protons are being clarified. Based on this, several hypotheses concerning the substrate transport mechanism in the Mrp antiporter have been proposed. The MrpA and MrpD subunits, which are homologous to the proton transport subunit of complex I, are involved in the transport of protons and their

  6. Intracellular localization of Na + /H + antiporter from Malus zumi ...

    African Journals Online (AJOL)

    In this study, we examined the intracellular localization of the product of Na+/H+ antiporter gene (MzNHX1) cloned from Malus zumi. Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast.

  7. Isolation of Arachis hypogaea Na + /H + antiporter and its ...

    African Journals Online (AJOL)

    + antiporter (AhNHX1) gene was isolated from peanut (Arachis hypogaea) in the present work. The full-length cDNA of AhNHX1 was 2,331 bp, which contains a complete ORF of 1,620 bp. The deduced protein sequence contains 546 amino ...

  8. Functional comparison of Cnh1 antiporters from different Candida species

    Czech Academy of Sciences Publication Activity Database

    Krauke, Yannick; Zimmermannová, Olga; Sychrová, Hana

    2007-01-01

    Roč. 274, Suppl.1 (2007), s. 127-127 ISSN 1742-464X. [FEBS Congress Molecular Machines /32./. 07.07.2007-12.07.2007, Vienna] Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * Na/H antiporter * Candida * plasma membrane Subject RIV: EE - Microbiology, Virology

  9. Na+/H+ antiport is essential for Yersinia pestis virulence.

    Science.gov (United States)

    Minato, Yusuke; Ghosh, Amit; Faulkner, Wyatt J; Lind, Erin J; Schesser Bartra, Sara; Plano, Gregory V; Jarrett, Clayton O; Hinnebusch, B Joseph; Winogrodzki, Judith; Dibrov, Pavel; Häse, Claudia C

    2013-09-01

    Na(+)/H(+) antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na(+)/H(+) antiport in Yersinia pestis virulence and found that Y. pestis strains lacking the major Na(+)/H(+) antiporters, NhaA and NhaB, are completely attenuated in an in vivo model of plague. The Y. pestis derivative strain lacking the nhaA and nhaB genes showed markedly decreased survival in blood and blood serum ex vivo. Complementation of either nhaA or nhaB in trans restored the survival of the Y. pestis nhaA nhaB double deletion mutant in blood. The nhaA nhaB double deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na(+) levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na(+)/H(+) antiport is indispensable for the survival of Y. pestis in the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused by Y. pestis and possibly for those caused by other blood-borne bacterial pathogens.

  10. Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter.

    Science.gov (United States)

    Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro; Krulwich, Terry Ann

    2014-01-01

    Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).

  11. Intracellular localization of Na /H antiporter from Malus zumi (MzNHX1)

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... Analysis using yeast cells expressing a fusion protein of MzNHX1 and green fluorescent protein confirmed the localization of MzNHX1 on the tonoplast. Key words: Intracellular localization, eGFP, Malus zumi, Na+/H+ antiporter, yeast. INTRODUCTION. Na+/H+ antiporters are widespread membrane ...

  12. Complex Formation by the mrpABCDEFG Gene Products, Which Constitute a Principal Na+/H+ Antiporter in Bacillus subtilis▿

    OpenAIRE

    Kajiyama, Yusuke; Otagiri, Masato; Sekiguchi, Junichi; Kosono, Saori; Kudo, Toshiaki

    2007-01-01

    The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na+/H+ antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.

  13. Functional Role of MrpA in the MrpABCDEFG Na+/H+ Antiporter Complex from the Archaeon Methanosarcina acetivorans

    OpenAIRE

    Jasso-Ch?vez, Ricardo; Diaz-Perez, C?sar; Rodr?guez-Zavala, Jos? S.; Ferry, James G.

    2016-01-01

    The multisubunit cation/proton antiporter 3 family, also called Mrp, is widely distributed in all three phylogenetic domains (Eukarya, Bacteria, and Archaea). Investigations have focused on Mrp complexes from the domain Bacteria to the exclusion of Archaea, with a consensus emerging that all seven subunits are required for Na+/H+ antiport activity. The MrpA subunit from the MrpABCDEFG Na+/H+ antiporter complex of the archaeon Methanosarcina acetivorans was produced in antiporter-deficient Esc...

  14. Characterization of the Na⁺/H⁺ antiporter from Yersinia pestis.

    Science.gov (United States)

    Ganoth, Assaf; Alhadeff, Raphael; Kohen, Dovrat; Arkin, Isaiah T

    2011-01-01

    Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na⁺/H⁺ antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein's substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein's structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na⁺/H⁺ antiporter's structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na⁺/H⁺ antiporter to help in fighting Yersinia pestis -associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives.

  15. Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

    Science.gov (United States)

    Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2010-10-01

    Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

  16. Intracellular localization of Na /H antiporter from Malus zumi (MzNHX1)

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... 2 Yuan ming yuan xi road, Beijing 100193, China. 2Agronomy and ... show that the salt tolerance of plants and yeasts is highly related to the ... salt tolerance. To this day, Na+/H+ antiporter genes have been isolated from many different plants, including. OsNHX1 from rice (Fukuda et al., 1999), VvNHX1 from.

  17. Functional validation of a novel isoform of Na +/H+ antiporter from ...

    Indian Academy of Sciences (India)

    Madhu Sudhan

    2007-03-22

    Mar 22, 2007 ... We have cloned a novel isoform of a vacuolar Na+/H+ antiporter from Pennisetum glaucum (PgNHX1) that contains 5 transmembrane domains in contrast to AtNHX1 and OsNHX1 which have 9 transmembrane domains. Recently we have shown that PgNHX1 could confer high level of salinity tolerance ...

  18. pH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschii

    Science.gov (United States)

    Vinothkumar, Kutti R; Smits, Sander H J; Kühlbrandt, Werner

    2005-01-01

    Na+/H+ antiporters are pH-dependent membrane transport proteins that maintain the homeostasis of H+ and Na+ in living cells. MjNhaP1 from Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85°C, was cloned and expressed in Escherichia coli. Two-dimensional crystals were obtained from purified protein at pH 4. Electron cryomicroscopy yielded an 8 Å projection map. Like the related E. coli antiporter NhaA, MjNhaP1 is a dimer, but otherwise the structures of the two antiporters differ significantly. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4–6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1 was studied in situ. A major change in density distribution within the helix bundle, and a ∼2 Å shift in the position of the helix bundle relative to the dimer core occurred at pH 6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter. PMID:16015376

  19. An Na+/H+ antiporter gene from wheat plays an important role in ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    for salt tolerance but also for vacuolar pH regulation and the developmental processes of leaves. In the present study, we cloned a new Na+/H+ antiporter gene, TaNHX2, from wheat. The expression patterns of. TaNHX2 were analysed in response to treatment with salt, drought, cold and ABA. The function of TaNHX2 was.

  20. Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species

    Czech Academy of Sciences Publication Activity Database

    Krauke, Yannick; Sychrová, Hana

    2008-01-01

    Roč. 8, - (2008), s. 1-9 ISSN 1471-2180 R&D Projects: GA MŠk(CZ) LC531 Grant - others:EC(XE) MRTN-CT-2004-512481 Institutional research plan: CEZ:AV0Z50110509 Keywords : Na/H antiporters * Candida parapsilosis * Candida dubliniensis Subject RIV: EE - Microbiology, Virology Impact factor: 2.877, year: 2008 http://www.biomedcentral.com/1471-2180/8/80

  1. The Candida albicans Na(+)/H(+) antiporter exports potassium and rubidium

    Czech Academy of Sciences Publication Activity Database

    Kinclová, Olga; Potier, S.; Sychrová, Hana

    2001-01-01

    Roč. 504, 1-2 (2001), s. 11-15 ISSN 0014-5793 R&D Projects: GA AV ČR IAA5011005; GA ČR GA204/01/0272 Institutional research plan: CEZ:AV0Z5011922 Keywords : Na+/H+ antiporter * potassium efflux * salt tolerance Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.644, year: 2001

  2. The Na+,K+/H+-antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae

    Czech Academy of Sciences Publication Activity Database

    Zimmermannová, Olga; Gášková, D.; Sychrová, Hana

    2006-01-01

    Roč. 6, č. 5 (2006), s. 792-800 ISSN 1567-1356 R&D Projects: GA AV ČR(CZ) IAA5011407; GA ČR(CZ) GP204/02/D092 Institutional research plan: CEZ:AV0Z50110509 Keywords : membrane potential * Na+,K+/H+ antiporter * diS-C3(3) assay Subject RIV: BO - Biophysics Impact factor: 2.274, year: 2006

  3. An Na+/H+ antiporter gene from wheat plays an important role in ...

    Indian Academy of Sciences (India)

    A vacuole Na+/H+ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (nhx1), TaNHX2 ...

  4. Cloning and characterization of a novel vacuolar Na+/H+ antiporter gene (Dgnhx1 from chrysanthemum.

    Directory of Open Access Journals (Sweden)

    Qing-Lin Liu

    Full Text Available Plant vacuolar Na(+/H(+ antiporter genes play significant roles in salt tolerance. However, the roles of the chrysanthemum vacuolar Na(+/H(+ antiporter genes in salt stress response remain obscure. In this study, we isolated and characterized a novel vacuolar Na(+/H(+ antiporter gene DgNHX1 from chrysanthemum. The DgNHX1 sequence contained 1920 bp with a complete open reading frame of 1533 bp encoding a putative protein of 510 amino acids with a predicted protein molecular weight of 56.3 kDa. DgNHX1 was predicted containing nine transmembrane domains. Its expression in the chrysanthemum was up-regulated by salt stress, but not by abscisic acid (ABA. To assess roles of DgNHX1 in plant salt stress responses, we performed gain-of-function experiment. The DgNHX1-overexpression tobacco plants showed significant salt tolerance than the wild type (WT. The transgenic lines exhibited more accumulation of Na(+ and K(+ under salt stress. These findings suggest that DgNHX1 plays a positive regulatory role in salt stress response.

  5. The Vibrio cholerae Mrp system: cation/proton antiport properties and enhancement of bile salt resistance in a heterologous host.

    Science.gov (United States)

    Dzioba-Winogrodzki, Judith; Winogrodzki, Olga; Krulwich, Terry A; Boin, Markus A; Häse, Claudia C; Dibrov, Pavel

    2009-01-01

    The mrp operon from Vibrio cholerae encoding a putative multisubunit Na(+)/H(+) antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na(+) and Li(+) as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li(+), Na(+), and K(+) ions in pH-dependent manner with maximal activity at pH 9.0-9.5. Exchange was electrogenic (more than one H(+) translocated per cation moved in opposite direction). The apparent K(m) at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li(+), Na(+), and K(+), respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems. Copyright 2008 S. Karger AG, Basel.

  6. Co-overexpressing a plasma membrane and a vacuolar membrane sodium/proton antiporter significantly improves salt tolerance in transgenic Arabidopsis plants.

    Science.gov (United States)

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane bound sodium/proton (Sodium/Hydrogen) antiporter that transports sodium into the vacuole and exports hydrogen into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane bound sodium/hydrogen antiporter that exports sodium to the ex...

  7. A novel NhaD-type Na+/H+antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila.

    Science.gov (United States)

    Wang, Yanhong; Song, Na; Yang, Lina; Abdel-Motaal, Heba; Zhang, Rui; Zhang, Zhenglai; Meng, Fankui; Jiang, Juquan

    2017-07-01

    In this study, a NhaD-type Na + /H + antiporter gene designated Ha-nhaD was obtained by selection of genomic DNA from the moderate halophile and alkaliphile Halomonas alkaliphila in Escherichia coli KNabc lacking 3 major Na + /H + antiporters. The presence of Ha-NhaD conferred tolerance of E. coli KNabc to NaCl up to 0.6 mol·L -1 and to LiCl up to 0.2 mol·L -1 and to an alkaline pH. pH-dependent Na + (Li + )/H + antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/pUC-nhaD but not those of KNabc/pUC18. Ha-NhaD exhibited Na + (Li + )/H + antiport activity over a wide pH range from 7.0 to 9.5, with the highest activity at pH 9.0. Protein sequence alignment and phylogenetic analysis revealed that Ha-NhaD is significantly different from the 7 known NhaD-type Na + /H + antiporters, including Dw-NhaD, Dl-NhaD, Vp-NhaD, Vc-NhaD, Aa-NhaD, He-NhaD, and Ha-NhaD1. Although Ha-NhaD showed a closer phylogenetic relationship with Ha-NhaD2, a significant difference in pH-dependent activity profile exists between Ha-NhaD and Ha-NhaD2. Taken together, Ha-nhaD encodes a novel pH-dependent NhaD-type Na + /H + antiporter.

  8. Plasma-membrane Cnh1 Na+/H+ antiporter regulates potassium homeostasis in Candida albicans

    Czech Academy of Sciences Publication Activity Database

    Kinclová-Zimmermannová, Olga; Sychrová, Hana

    2007-01-01

    Roč. 153, č. 8 (2007), s. 2603-2612 ISSN 1350-0872 R&D Projects: GA MŠk(CZ) LC531; GA AV ČR KJB5011307 Grant - others:CANTRAIN(XE) MRTN-CT-2004-512481 Institutional research plan: CEZ:AV0Z50110509 Source of funding: R - rámcový projekt EK Keywords : C. albicans * Cnh1 antiporter * potassium homeostasis Subject RIV: EE - Microbiology, Virology Impact factor: 3.110, year: 2007

  9. Thermodynamic secrets of multidrug resistance: A new take on transport mechanisms of secondary active antiporters.

    Science.gov (United States)

    Zhang, Xuejun C; Liu, Min; Lu, Guangyuan; Heng, Jie

    2018-03-01

    Multidrug resistance (MDR) presents a growing challenge to global public health. Drug extrusion transporters play a critical part in MDR; thus, their mechanisms of substrate recognition are being studied in great detail. In this work, we review common structural features of key transporters involved in MDR. Based on our membrane potential-driving hypothesis, we propose a general energy-coupling mechanism for secondary-active antiporters. This putative mechanism provides a common framework for understanding poly-specificity of most-if not all-MDR transporters. © 2017 The Protein Society.

  10. Structural basis for dynamic mechanism of nitrate/nitrite antiport by NarK.

    Science.gov (United States)

    Fukuda, Masahiro; Takeda, Hironori; Kato, Hideaki E; Doki, Shintaro; Ito, Koichi; Maturana, Andrés D; Ishitani, Ryuichiro; Nureki, Osamu

    2015-05-11

    NarK belongs to the nitrate/nitrite porter (NNP) family in the major facilitator superfamily (MFS) and plays a central role in nitrate uptake across the membrane in diverse organisms, including archaea, bacteria, fungi and plants. Although previous studies provided insight into the overall structure and the substrate recognition of NarK, its molecular mechanism, including the driving force for nitrate transport, remained elusive. Here we demonstrate that NarK is a nitrate/nitrite antiporter, using an in vitro reconstituted system. Furthermore, we present the high-resolution crystal structures of NarK from Escherichia coli in the nitrate-bound occluded, nitrate-bound inward-open and apo inward-open states. The integrated structural, functional and computational analyses reveal the nitrate/nitrite antiport mechanism of NarK, in which substrate recognition is coupled to the transport cycle by the concomitant movement of the transmembrane helices and the key tyrosine and arginine residues in the substrate-binding site.

  11. Regulated release of Ca2+ from respiring mitochondria by Ca2+/2H+ antiport.

    Science.gov (United States)

    Fiskum, G; Lehninger, A L

    1979-07-25

    Simultaneous measurements of oxygen consumption and transmembrane transport of Ca2+, H+, and phosphate show that the efflux of Ca2+ from respiring tightly coupled rat liver mitochondria takes place by an electroneutral Ca2+/2H+ antiport process that is ruthenium red-insensitive and that is regulated by the oxidation-reduction state of the mitochondrial pyridine nucleotides. When mitochondrial pyridine nucleotides are kept in a reduced steady state, the efflux of Ca2+ is inhibited; when they are in an oxidized state, Ca2+ efflux is activated. These processes were demonstrated by allowing phosphate-depleted mitochondria respiring on succinate in the presence of rotenone to take up Ca2+ from the medium. Upon subsequent addition of ruthenium red to block Ca2+ transport via the electrophoretic influx pathway, and acetoacetate, to bring mitochondrial pyridine nucleotides into the oxidized state, Ca2+ efflux and H+ influx ensued. The observed H+ influx/Ca2+ efflux ratio was close to the value 2.0 predicted for the operation of an electrically neutral Ca2+/2H+ antiport process.

  12. Use of osmolytes during solubilization and reconstitution of phosphate: sugar phosphate antiport from bacteria

    International Nuclear Information System (INIS)

    Ambudkar, S.V.; Sonna, L.A.; Maloney, P.C.

    1986-01-01

    Phosphate:2-deoxyglucose 6-phosphate (Pi:2DG6P) antiport was extracted from Streptococcus lactis or Staphylococcus aureus with 1.1% octylglucoside in the presence of 0.37% E. coli lipid and reconstituted by detergent dilution. Because previous work suggested inactivation at an early stage, the authors introduced protein stabilants during solubilization. When 20% glycerol was used, proteoliposomes showed a 20-fold increase in 32 Pi transport. This enhanced recovery required phospholipid plus glycerol, and was found only when both were added together with the detergent. Glycerol protection yielded proteoliposomes in which antiporters retained their normal kinetic properties, and Pi exchange by the streptococcal example gave a maximal rate (200-400 nmol/min per mg protein) and a turnover number (30-50/s) which suggested that inactivation had been avoided. Further study showed that 20% glycerol could be replaced by equally high concentrations of compounds classified as osmolytes polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose) and certain amino acids (glycine, proline, but not valine). The authors suggest that osmolytes may be used to fully stabilize chemiosmotic transporters during reconstitution

  13. Potassium co-transport and antiport during the uptake of sucrose and glutamic acid from the xylem vessels

    NARCIS (Netherlands)

    Bel, A.J.E. van; Erven, A.J. van

    Perfusion experiments with excised internodes of tomato (Lycopersicon esculentum cv Moneymaker) showed that the uptake of glutamic acid and sucrose from the xylem vessels is accompanied with coupled proton co-transport and potassium antiport at low pH (<5.5). At high pH (5.5) both proton and

  14. Mechanistic model of sodium/proton antiport based on X-ray crystal structures and molecular dynamics simulations

    Science.gov (United States)

    Beckstein, Oliver; Dotson, David L.; Lee, Chiara; Yashiro, Shoko; Uzdavinys, Povilas; von Ballmoos, Christoph; Drew, David; Cameron, Alexander D.

    2015-03-01

    Na+/H+ antiporters are membrane proteins that are vital for cell homeostasis but the mechanistic details of their transport mechanism remain unclear, in particular, how Na+ and protons bind to the transporter. We recently solved X-ray crystal structures for two such antiporters (NhaA and NapA) in two different conformations of the transport cycle. All-atom molecular dynamics (MD) simulations (for a total simulated time > 10 μ s), indicate that sodium binding is dependent on the charge states of two conserved aspartate residues. A conserved lysine forms a previously unidentified salt bridge with one of the asparates. Under simulated physiological pH the presence of a Na+ ion disrupts and breaks the salt bridge in NhaA. To quantify proton binding, we then performed heuristic pKa calculations on our ensemble of simulations. The calculations support our novel hypothesis that the conserved lysine in these antiporter binds protons in a sodium-dependent manner and thus acts as part of the transport machinery. In conjunction with simulations of the conformational transition we propose a new mechanistic model of ion binding for the CPA2 class of antiporters within the larger framework of the alternating access mechanism of transmembrane transport.

  15. Conserved and Diversified Gene Families of Monovalent Cation/H+ Antiporters from Algae to Flowering Plants

    Directory of Open Access Journals (Sweden)

    Salil eChanroj

    2012-02-01

    Full Text Available All organisms have evolved strategies to regulate ion and pH homeostasis in response to developmental and environmental cues. One strategy is mediated by cation-proton antiporters (CPA. CPA1 genes found in bacteria, fungi, metazoa and plants have been functionally-characterized; though roles of plant CPA2 genes in KEA (K+-efflux antiporter and CHX (cation/H+ exchanger families are largely unknown. Phylogenetic analysis showed that three clades of the Na+-H+ exchanger (NHX family have been conserved from single-celled alga to Arabidopsis. These are i plasma membrane-bound SOS1/AtNHX7 that share ancestry with prokaryote NhaP, ii endosomal AtNHX5/6 that is part of the eukaryote Intracellular-NHE clade, and iii a vacuolar NHX clade (AtNHX1-4 specific to plants. Early diversification of KEA genes possibly from ancestral genes of a cyanobacterium is suggested for three K+-efflux antiporter clades (KEA/Kef seen in all plants. Intriguingly, the CHX gene family blossomed from a few members in early land plants to >40 genes in legumes. Homologs from spirogyra or moss share high similarity with guard cell-specific AtCHX20, suggesting that AtCHX20 and its relatives (AtCHX16-19 are founders of the family. Evolutionary analysis suggests pollen-expressed CHX genes appeared later in monocots and early eudicots. AtCHX proteins have been localized to intracellular and plasma membrane of plants, and shown to mediate K+ transport and pH homeostasis. Thus KEA genes are conserved from green algae to angiosperms, and their presence in red algae and secondary endosymbionts suggest a role in plastids. In contrast, AtNHX1-4 subtype evolved in ancestral plants to handle ion homeostasis of vacuoles in all cell types. The strong presence of CHX genes in land plants, but not in metazoa or fungi, would infer a role of ion and pH homeostasis at dynamic endomembranes to support vegetative and reproductive success of flowering plants.

  16. Atomic-level characterization of transport cycle thermodynamics in the glycerol-3-phosphate:phosphate antiporter.

    Science.gov (United States)

    Moradi, Mahmoud; Enkavi, Giray; Tajkhorshid, Emad

    2015-09-29

    Membrane transporters actively translocate their substrate by undergoing large-scale structural transitions between inward- (IF) and outward-facing (OF) states ('alternating-access' mechanism). Despite extensive structural studies, atomic-level mechanistic details of such structural transitions, and as importantly, their coupling to chemical events supplying the energy, remain amongst the most elusive aspects of the function of these proteins. Here we present a quantitative, atomic-level description of the functional thermodynamic cycle for the glycerol-3-phosphate:phosphate antiporter GlpT by using a novel approach in reconstructing the free energy landscape governing the IF↔OF transition along a cyclic transition pathway involving both apo and substrate-bound states. Our results provide a fully atomic description of the complete transport process, offering a structural model for the alternating-access mechanism and substantiating the close coupling between global structural transitions and local chemical events.

  17. The arginine-ornithine antiporter ArcD contributes to biological fitness of Streptococcus suis

    Directory of Open Access Journals (Sweden)

    Marcus eFulde

    2014-08-01

    Full Text Available The arginine-ornithine antiporter (ArcD is part of the Arginine Deiminase System (ADS, a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis. The ADS has recently been shown to play a role in the pathogenicity of S. suis, in particular in its survival in host cells. The contribution of arginine and arginine transport mediated by ArcD, however, has yet to be clarified. In the present study, we showed by experiments using [U-13C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine. To further study the role of ArcD in arginine metabolism, we generated an arcD-specific mutant strain and characterized its growth compared to the wild-type (WT strain, a virulent serotype 2 strain. The mutant strain showed a markedly reduced growth rate in chemically defined media supplemented with arginine when compared to the WT strain, indicating that ArcD promotes arginine uptake. To further evaluate the in vivo relevance of ArcD, we studied the intracellular bacterial survival of the arcD mutant strain in an epithelial cell culture infection model. The mutant strain was substantially attenuated, and its reduced intracellular survival rate correlated with a lower ability to neutralize the acidified environment. Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

  18. Molecular Basis of Substrate Polyspecificity of the Candida albicans Mdr1p Multidrug/H+Antiporter.

    Science.gov (United States)

    Redhu, Archana Kumari; Banerjee, Atanu; Shah, Abdul Haseeb; Moreno, Alexis; Rawal, Manpreet Kaur; Nair, Remya; Falson, Pierre; Prasad, Rajendra

    2018-03-02

    The molecular basis of polyspecificity of Mdr1p, a major drug/H + antiporter of Candida albicans, is not elucidated. We have probed the nature of the drug-binding pocket by performing systematic mutagenesis of the 12 transmembrane segments. Replacement of the 252 amino acid residues with alanine or glycine yielded 2/3 neutral mutations while 1/3 led to the complete or selective loss of resistance to drugs or substrates transported by the pump. Using the GlpT-based 3D-model of Mdr1p, we roughly categorized these critical residues depending on their type and localization, 1°/ main structural impact ("S" group), 2°/ exposure to the lipid interface ("L" group), 3°/ buried but not facing the main central pocket, inferred as critical for the overall H + /drug antiport mechanism ("M" group) and finally 4°/ buried and facing the main central pocket ("B" group). Among "B" category, 13 residues were essential for the large majority of drugs/substrates, while 5 residues were much substrate-specific, suggesting a role in governing polyspecificity (P group). 3D superposition of the substrate-specific MFS Glut1 and XylE with the MDR substrate-polyspecific MdfA and Mdr1p revealed that the B group forms a common substrate interaction core while the P group is only found in the 2 MDR MFS transporters, distributed into 3 areas around the B core. This specific pattern has let us to propose that the structural basis for polyspecificity of MDR MFS transporters is the extended capacity brought by residues located at the periphery of a binding core to accomodate compounds differing in size and type. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. Metagenome cloning and functional analysis of Na⁺/H⁺ antiporter genes from Keke Salt Lake in China.

    Science.gov (United States)

    Gao, Maio; Wang, Lei; Chen, San-Feng

    2012-02-01

    Na⁺/H⁺ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na⁺/Li⁺ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na⁺/H⁺ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na⁺/H⁺ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA⁻, nhaB⁻, chaA⁻) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na⁺/H⁺ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA⁻, nhaB⁻, chaA⁻) grow in the LBK medium containing 0.2-0.6 M Na⁺ or with 0.05-0.4 M Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na⁺/H⁺ and Li⁺/H⁺ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K⁺/H⁺ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.

  20. Physiology of the Vc-NhaP paralogous group of cation-proton antiporters in Vibrio cholerae.

    Science.gov (United States)

    Mourin, Muntahi; Schubiger, Carla B; Resch, Craig T; Häse, Claudia C; Dibrov, Pavel

    2017-04-01

    The genome of Vibrio cholerae encodes three cation-proton antiporters of NhaP-type, Vc-NhaP1, 2, and 3. To examine physiological roles of Vc-NhaP antiporters, triple ΔnhaP1ΔnhaP2ΔnhaP3 and single ΔnhaP3 deletion mutants of V. cholerae were constructed and characterized. Vc-NhaP3 was, for the first time, cloned and biochemically characterized. Activity measurements on the inside-out membrane vesicle experimental model defined Vc-NhaP3 as a potassium-specific cation-proton antiporter. While elimination of functional Vc-NhaP3 resulted in only minor growth defect in potassium-rich medium at pH 6.0, the triple Vc-NhaP mutant demonstrated severe growth defects at both low and high [K + ] at pH 6.0 and failed to grow at high [K + ] in mildly alkaline (pH 8.0 and 8.5) media, as well. Expressed from a plasmid, neither of the Vc-NhaP paralogues was able to complement the severe potassium-sensitive phenotype of the triple deletion mutant completely. Vc-NhaP1 provided much better complementation at acidic pH compared to Vc-NhaP2, despite the fact that Vc-NhaP2 showed much higher antiport activity in sub-bacterial vesicles. In mildly alkaline pH only Vc-NhaP2 complemented the potassium-sensitive phenotype of the triple deletion mutant. Taken together, these data suggest that in vivo all three isoforms operate in concert, contributing to K + resistance of V. cholerae. We suggest that the Vc-NhaP paralogue group might play a role in passing gastric acid barrier by ingested V. cholerae cells.

  1. Na+, K+/H+ antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development

    Czech Academy of Sciences Publication Activity Database

    Fan, B. H.; Zhao, L; Hu, X.; Li, W.; Novák, Ondřej; Strnad, Miroslav; Simon, S.; Friml, J.; Shen, J.; Jiang, L.; Qiu, Q. S.

    2018-01-01

    Roč. 41, č. 4 (2018), s. 850-864 ISSN 0140-7791 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis * AtNHX5 * AtNHX6 * endosomal Na ,K /H antiporters * pH * pin5 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 6.173, year: 2016

  2. Molecular characterization of a novel Na⁺/H⁺ antiporter cDNA from Eucalyptus globulus.

    Science.gov (United States)

    Baltierra, Fabiola; Castillo, Mabel; Gamboa, María Cecilia; Rothhammer, Matías; Krauskopf, Erwin

    2013-01-11

    Environmental stress factors such as salt, drought and heat are known to affect plant productivity. However, high salinity is spreading throughout the world, currently affecting more than 45 millionha. One of the mechanisms that allow plants to withstand salt stress consists on vacuolar sequestration of Na(+), through a Na(+)/H(+) antiporter. We isolated a new vacuolar Na(+)/H(+) antiporter from Eucalyptus globulus from a cDNA library. The cDNA had a 1626 bp open reading frame encoding a predicted protein of 542 amino acids with a deduced molecular weight of 59.1 KDa. Phylogenetic and bioinformatic analyses indicated that EgNHX1 localized in the vacuole. To assess its role in Na(+) exchange, we performed complementation studies using the Na(+) sensitive yeast mutant strain Δnhx1. The results showed that EgNHX1 partially restored the salt sensitive phenotype of the yeast Δnhx1 strain. However, its overexpression in transgenic Arabidopsis confers tolerance in the presence of increasing NaCl concentrations while the wild type plants exhibited growth retardation. Expression profiles of Eucalyptus seedlings subjected to salt, drought, heat and ABA treatment were established. The results revealed that Egnhx1 was induced significantly only by drought. Together, these results suggest that the product of Egnhx1 from E. globulus is a functional vacuolar Na(+)/H(+) antiporter. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Screening a novel Na+/H+ antiporter gene from a metagenomic library of halophiles colonizing in the Dagong Ancient Brine Well in China.

    Science.gov (United States)

    Xiang, Wenliang; Zhang, Jie; Li, Lin; Liang, Huazhong; Luo, Hai; Zhao, Jian; Yang, Zhirong; Sun, Qun

    2010-05-01

    Metagenomic DNA libraries constructed from the Dagong Ancient Brine Well were screened for genes with Na(+)/H(+) antiporter activity on the antiporter-deficient Escherichia coli KNabc strain. One clone with a stable Na(+)-resistant phenotype was obtained and its Na(+)/H(+) antiporter gene was sequenced and designated as m-nha. The deduced amino acid sequence of M-Nha protein consists of 523 residues with a calculated molecular weight of 58 147 Da and a pI of 5.50, which is homologous with NhaH from Halobacillus dabanensis D-8(T) (92%) and Halobacillus aidingensis AD-6(T) (86%), and with Nhe2 from Bacillus sp. NRRL B-14911 (64%). It had a hydropathy profile with 10 putative transmembrane domains and a long carboxyl terminal hydrophilic tail of 140 amino acid residues, similar to Nhap from Synechocystis sp. and Aphanothece halophytica, as well as NhaG from Bacillus subtilis. The m-nha gene in the antiporter-negative mutant E. coli KNabc conferred resistance to Na(+) and the ability to grow under alkaline conditions. The difference in amino acid sequence and the putative secondary structure suggested that the m-nha isolated from the Dagong Ancient Brine Well in this study was a novel Na(+)/H(+) antiporter gene.

  4. Lactose uptake driven by galactose efflux in Streptococcus thermophilus: Evidence for a galactose-lactose antiporter

    International Nuclear Information System (INIS)

    Hutkins, R.W.; Ponne, C.

    1991-01-01

    Galactose-nonfermenting (Gal - ) Streptococcus thermophilus TS2 releases galactose into the extracellular medium when grown in medium containing excess lactose. Starved and de-energized Gal - cells, however, could be loaded with galactose to levels approximately equal to the extracellular concentration (0 to 50 mM). When loaded cells were separated from the medium and resuspended in fresh broth containing 5 mM lactose, galactose efflux occurred. De-energized, galactose-loaded cells, resuspended in buffer or medium, accumulated [ 14 C]lactose at a greater rate and to significantly higher intracellular concentrations than unloaded cells. Uptake of lactose by loaded cells was inhibited more than that by unloaded cells in the presence of extracellular galactose, indicating that a galactose gradient was involved in the exchange system. When de-energized, galactose-loaded cells were resuspended in carbohydrate-free medium at pH 6.7, a proton motive force (Δp) of 86 to 90 mV was formed, whereas de-energized, nonloaded cells maintained a Δp of about 56 mV. However, uptake of lactose by loaded cells occurred when the proton motive force was abolished by the addition of an uncoupler or in the presence of a proton-translocating ATPase inhibitor. These results support the hypothesis that galactose efflux in Gal - S. thermophilus is electrogenic and that the exchange reaction (lactose uptake and galactose efflux) probably occurs via an antiporter system

  5. Expression of a mammalian Na+/H+ antiporter in Saccharomyces cerevisiae.

    Science.gov (United States)

    Montero-Lomelí, M; Okorokova Façanha, A L

    1999-01-01

    The basolateral Na+/H+ antiporter (NHE) from LLC-PK1 cells was expressed in Saccharomyces cerevisiae. Two different strategies were tested for expression. In the first, we used a yeast strain that contains a temperature-sensitive mutation in the SEC-6 gene, whose product is required for the fusion of secretory vesicles with the plasma membrane. This strain was transformed with a vector containing the coding region of the NHE1 isoform under control of a heat shock (HS) promoter (pYNHE1-HS). In the second strategy, we replaced the heat shock promoter from pYNHE1-HS with a galactose (GAL) promoter (pYNHEI-GAL) and transformed wild-type yeast. In both cases, Northern blots demonstrated a transcript that hybridized against a probe containing the membrane region of the exchanger. When an antibody against the last 40 amino acids of the carboxy-terminus of NHE1 was used for immunoblots, a protein with a Mr of 73000 was seen in total membranes from both yeast transformants. Subcellular fractionation revealed that NHE1 was expressed in the endoplasmic reticulum. In the case of the pYNHEI-GAL transformant, the 100000 x g membrane pellet was reconstituted in phosphatidylcholine liposomes, and ethylisopropylamiloride-sensitive Na+/H+ exchange was observed. These results have paved the way for expression of the Na+/H+ exchanger in a genetically well-known microorganism.

  6. Involvement of the Acr3 and DctA anti-porters in arsenite oxidation in Agrobacterium tumefaciens 5A.

    Science.gov (United States)

    Kang, Yoon-Suk; Shi, Zunji; Bothner, Brian; Wang, Gejiao; McDermott, Timothy R

    2015-06-01

    Microbial arsenite (AsIII) oxidation forms a critical piece of the arsenic cycle in nature, though our understanding of how and why microorganisms oxidize AsIII remains rudimentary. Our model organism Agrobacterium tumefaciens 5A contains two distinct ars operons (ars1 and ars2) that are similar in their coding region content. The ars1 operon is located nearby the aio operon that is essential for AsIII oxidation. The AsIII/H(+) anti-porters encoded by acr3-1 and acr3-2 are required for maximal AsIII and antimonite (SbIII) resistance, but acr3-1 (negatively regulated by ArsR-1) appears more active in this regard and also required for AsIII oxidation and expression of aioBA. A malate-phosphate anti-porter DctA is regulated by RpoN and AsIII, and is required for normal growth with malate as a sole carbon source. Qualitatively, a ΔdctA mutant was normal for AsIII oxidation and AsIII/SbIII resistance at metalloid concentrations inhibitory to the Δacr3-1 mutant; however, aioBA induction kinetics was significantly phase-shift delayed. Acr3 involvement in AsIII/SbIII resistance is reasonably well understood, but the role of Acr3 and DctA anti-porters in AsIII oxidation and its regulation is unexpected, and suggests that controlled AsIII trafficking across the cytoplasmic membrane is important to a process understood to occur in the periplasm. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Solving the Mechanism of Na+/H+ Antiporters Using Molecular Dynamics Simulations

    Science.gov (United States)

    Dotson, David L.

    Na+/H+ antiporters are vital membrane proteins for cell homeostasis, transporting Na+ ions in exchange for H+ across the lipid bilayer. In humans, dysfunction of these transporters are implicated in hypertension, heart failure, epilepsy, and autism, making them well-established drug targets. Although experimental structures for bacterial homologs of the human Na+/H+ have been obtained, the detailed mechanism for ion transport is still not well-understood. The most well-studied of these transporters, Escherichia coli NhaA, known to transport 2 H+ for every Na+ extruded, was recently shown to bind H+ and Na+ at the same binding site, for which the two ion species compete. Using molecular dynamics simulations, the work presented in this dissertation shows that Na+ binding disrupts a previously-unidentified salt bridge between two conserved residues, suggesting that one of these residues, Lys300, may participate directly in transport of H+. This work also demonstrates that the conformational change required for ion translocation in a homolog of NhaA, Thermus thermophilus NapA, thought by some to involve only small helical movements at the ion binding site, is a large-scale, rigid-body movement of the core domain relative to the dimerization domain. This elevator-like transport mechanism translates a bound Na+ up to 10 A across the membrane. These findings constitute a major shift in the prevailing thought on the mechanism of these transporters, and serve as an exciting launchpad for new developments toward understanding that mechanism in detail.

  8. Protein phylogenetic analysis of Ca2+/cation antiporters and insights into their evolution in plants

    Directory of Open Access Journals (Sweden)

    Laura eEmery

    2012-01-01

    Full Text Available Cation transport is a critical process in all organisms and is essential for mineral nutrition, ion stress tolerance, and signal transduction. Transporters that are members of the Ca2+/Cation Antiporter (CaCA superfamily are involved in the transport of Ca2+ and/or other cations using the counter exchange of another ion such as H+ or Na+. The CaCA superfamily has been previously divided into five transporter families: the YRBG, NCX, NCKX, CAX and CCX families, which include the well-characterized Na+/Ca2+ exchanger (NCX and H+/cation exchanger (CAX transporters. To examine the evolution of CaCA transporters within higher plants and the green plant lineage, CaCA genes were identified from the genomes of sequenced flowering plants, a bryophyte, lycophyte, and freshwater and marine algae, and compared with those from non-plant species. We found evidence of the expansion and increased diversity of flowering plant genes within the CAX and CCX families. Genes related to the NCX family are present in land plant though they encode distinct MHX homologs which probably have an altered transport function. In contrast, the NCX and NCKX genes which are absent in land plants have been retained in many species of algae, especially the marine algae, indicating that these organisms may share ‘animal-like’ characteristics of Ca2+ homeostasis and signaling. A group of genes encoding novel CAX-like proteins containing an EF hand domain were identified from plants and selected algae but appeared to be lacking in any other species. Lack of functional data for most of the CaCA proteins make it impossible to reliably predict substrate specificity and function for many of the groups or individual proteins. The abundance and diversity of CaCA genes throughout all branches of life indicates the importance of this class of cation transporter, and that many transporters with novel functions are waiting to be discovered.

  9. Differences in the phenotypic effects of mutations in homologous MrpA and MrpD subunits of the multi-subunit Mrp-type Na+/H+antiporter.

    Science.gov (United States)

    Morino, Masato; Ogoda, Shinichiro; Krulwich, Terry Ann; Ito, Masahiro

    2017-01-01

    Mrp antiporters are the sole antiporters in the Cation/Proton Antiporter 3 family of transporter databases because of their unusual structural complexity, 6-7 hydrophobic proteins that function as a hetero-oligomeric complex. The two largest and homologous subunits, MrpA and MrpD, are essential for antiport activity and have direct roles in ion transport. They also show striking homology with proton-conducting, membrane-embedded Nuo subunits of respiratory chain complex I of bacteria, e.g., Escherichia coli. MrpA has the closest homology to the complex I NuoL subunit and MrpD has the closest homology to the complex I NuoM and N subunits. Here, introduction of mutations in MrpD, in residues that are also present in MrpA, led to defects in antiport function and/or complex formation. No significant phenotypes were detected in strains with mutations in corresponding residues of MrpA, but site-directed changes in the C-terminal region of MrpA had profound effects, showing that the MrpA C-terminal region has indispensable roles in antiport function. The results are consistent with a divergence in adaptations that support the roles of MrpA and MrpD in secondary antiport, as compared to later adaptations supporting homologs in primary proton pumping by the respiratory chain complex I.

  10. Characterization of Bacillus subtilis YfkE (ChaA): a calcium-specific Ca2+/H+ antiporter of the CaCA family.

    Science.gov (United States)

    Fujisawa, Makoto; Wada, Yuko; Tsuchiya, Takahiro; Ito, Masahiro

    2009-08-01

    YfkE, a protein from Bacillus subtilis, exhibits homology to the Ca(2+):Cation Antiporter (CaCA) Family. In a fluorescence-based assay of everted membrane vesicles prepared from Na(+)(Ca(2+))/H(+) antiporter-defective mutant Escherichia coli KNabc, YfkE exhibited robust Ca(2+)/H(+) antiport activity, with a K (m) for Ca(2+) estimated at 12.5 muM at pH 8.5 and 113 muM at pH 7.5. Neither Na(+) nor K(+) served as a substrate. Mg(2+) also did not serve as a substrate, but inhibited the Ca(2+)/H(+) antiporter activity. The Ca(2+) transport capability of YfkE was also observed directly by transport assays in everted membrane vesicles using radiolabeled (45)Ca(2+). Transcriptional analysis from the putative yfkED operon using beta-garactosidase activity as a reporter revealed that both of the yfkE and yfkD genes are regulated by forespore-specific sigma factor, SigG, and the general stress response regulator, SigB. These results suggest that YfkE may be needed for Ca(2+) signaling in the sporulation or germination process in B. subtilis. ChaA is proposed as the designation for YfkE of B. subtilis.

  11. Protein Phylogenetic Analysis of Ca2+/cation Antiporters and Insights into their Evolution in Plants

    Science.gov (United States)

    Emery, Laura; Whelan, Simon; Hirschi, Kendal D.; Pittman, Jon K.

    2012-01-01

    Cation transport is a critical process in all organisms and is essential for mineral nutrition, ion stress tolerance, and signal transduction. Transporters that are members of the Ca2+/cation antiporter (CaCA) superfamily are involved in the transport of Ca2+ and/or other cations using the counter exchange of another ion such as H+ or Na+. The CaCA superfamily has been previously divided into five transporter families: the YRBG, Na+/Ca2+ exchanger (NCX), Na+/Ca2+, K+ exchanger (NCKX), H+/cation exchanger (CAX), and cation/Ca2+ exchanger (CCX) families, which include the well-characterized NCX and CAX transporters. To examine the evolution of CaCA transporters within higher plants and the green plant lineage, CaCA genes were identified from the genomes of sequenced flowering plants, a bryophyte, lycophyte, and freshwater and marine algae, and compared with those from non-plant species. We found evidence of the expansion and increased diversity of flowering plant genes within the CAX and CCX families. Genes related to the NCX family are present in land plant though they encode distinct MHX homologs which probably have an altered transport function. In contrast, the NCX and NCKX genes which are absent in land plants have been retained in many species of algae, especially the marine algae, indicating that these organisms may share “animal-like” characteristics of Ca2+ homeostasis and signaling. A group of genes encoding novel CAX-like proteins containing an EF-hand domain were identified from plants and selected algae but appeared to be lacking in any other species. Lack of functional data for most of the CaCA proteins make it impossible to reliably predict substrate specificity and function for many of the groups or individual proteins. The abundance and diversity of CaCA genes throughout all branches of life indicates the importance of this class of cation transporter, and that many transporters with novel functions are waiting to be discovered. PMID:22645563

  12. Bio-informatics Analysis of a Vacuolar Na+/H+ Antiporter (Alanhx) from the Salt Resistant Grass Aeluropus Lagopoides

    International Nuclear Information System (INIS)

    Ahmed, M. Z.; Khan, M. A.

    2016-01-01

    Sodium-hydrogen antiporter (NHX) protein regulates the trans-membrane transport of Na+ in higher plants. Vacuolar-NHX is a type of NHX protein located on tonoplast and minimizes the accumulation of Na+ in cytoplasm by compartmentalizing into vacuole especially in salt tolerant plants. In Aeluropus lagopoides, AlaNHX [NCBI: GU199336, Vacuolar-NHX] plays a vital role for efficient Na+ sequestration into the vacuole and helps in plant survival in saline areas. Therefore, sequence analysis, structural analysis and modeling of AlaNHX were performed through bioinformatics tools. Homology of AlaNHX was 99% similar with the Na+/H+ antiporter of Aeluropus littoralis. Sequence of AlaNHX consisted of 2353 bp including 337 bp of un-translated regions (UTR) at 5' and 393 at 3' end. In addition, AlaNHX have an open reading frame (ORF) of 1623 bp which translated into 59.4 KDa protein containing 540 amino acids (Leucine + Serine contributed in 22% of peptide chain). AlaNHX protein consists of 10 transmembrane domains (TMD; 3 primary and 7 secondary protein structural type) and a long (95 amino acids) carboxyl terminal end in cytoplasmic region. In addition, 3, 5, 7 and 8 TMD regions of AlaNHX were highly conserved. Different glycosylation, phosphorylation and myristoylation sites were also found in AlaNHX protein. Three-dimensional (3D) structure analysis revealed that this protein may be classified as stable and of hydrophobic nature containing a significant proportion of alpha helices. In this study, a three-dimensional structure of AlaNHX protein was predicted by using in-silico3D homology modeling technique. This study provides baseline information for understanding the importance of NHX protein structure. (author)

  13. Heterologous Expression of Pteris vittata Arsenite Antiporter PvACR3;1 Reduces Arsenic Accumulation in Plant Shoots.

    Science.gov (United States)

    Chen, Yanshan; Hua, Chen-Yu; Jia, Meng-Ru; Fu, Jing-Wei; Liu, Xue; Han, Yong-He; Liu, Yungen; Rathinasabapathi, Bala; Cao, Yue; Ma, Lena Q

    2017-09-19

    Arsenic (As) is a toxic carcinogen so it is crucial to decrease As accumulation in crops to reduce its risk to human health. Arsenite (AsIII) antiporter ACR3 protein is critical for As metabolism in organisms, but it is lost in flowering plants. Here, a novel ACR3 gene from As-hyperaccumulator Pteris vittata, PvACR3;1, was cloned and expressed in Saccharomyces cerevisiae (yeast), Arabidopsis thaliana (model plant), and Nicotiana tabacum (tobacco). Yeast experiments showed that PvACR3;1 functioned as an AsIII-antiporter to mediate AsIII efflux to an external medium. At 5 μM AsIII, PvACR3;1 transgenic Arabidopsis accumulated 14-29% higher As in the roots and 55-61% lower As in the shoots compared to WT control, showing lower As translocation. Besides, transgenic tobacco under 5 μM AsIII or AsV also showed similar results, indicating that expressing PvACR3;1 gene increased As retention in plant roots. Moreover, observation of PvACR3;1-green fluorescent protein fusions in transgenic Arabidopsis showed that PvACR3;1 protein localized to the vacuolar membrane, indicating that PvACR3;1 mediated AsIII sequestration into vacuoles, consistent with increased root As. In addition, soil experiments showed ∼22% lower As in the shoots of transgenic tobacco than control. Thus, our study provides a potential strategy to limit As accumulation in plant shoots, representing the first report to decrease As translocation by sequestrating AsIII into vacuoles, shedding light on engineering low-As crops to improve food safety.

  14. Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes*

    Science.gov (United States)

    Sasahara, Ayako; Nanatani, Kei; Enomoto, Masaru; Kuwahara, Shigefumi; Abe, Keietsu

    2011-01-01

    The aspartate:alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate:alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of l-aspartate1− with l-alanine0. Although physiological functions of AspT were well studied, l-aspartate1−:l-alanine0 antiport mechanisms are still unsolved. Here we report that the binding sites of l-aspartate and l-alanine are independently present in AspT by means of the kinetic studies. We purified His6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (Km = 0.35 ± 0.03 mm for l-aspartate, Km = 0.098 ± 0 mm for d-aspartate, Km = 26 ± 2 mm for l-alanine, Km = 3.3 ± 0.2 mm for d-alanine). Competitive inhibition by various amino acids of l-aspartate or l-alanine in self-exchange reactions revealed that l-cysteine selectively inhibited l-aspartate self-exchange but only weakly inhibited l-alanine self-exchange. Additionally, l-serine selectively inhibited l-alanine self-exchange but barely inhibited l-aspartate self-exchange. The aspartate analogs l-cysteine sulfinic acid, l-cysteic acid, and d-cysteic acid competitively and strongly inhibited l-aspartate self-exchange compared with l-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of l-aspartate and l-alanine are independently located in the substrate translocation pathway of AspT. PMID:21719707

  15. Multidrug resistance protein MdtM adds to the repertoire of antiporters involved in alkaline pH homeostasis in Escherichia coli

    Science.gov (United States)

    2013-01-01

    Background In neutralophilic bacteria, monovalent metal cation/H+ antiporters play a key role in pH homeostasis. In Escherichia coli, only four antiporters (NhaA, NhaB, MdfA and ChaA) are identified to function in maintenance of a stable cytoplasmic pH under conditions of alkaline stress. We hypothesised that the multidrug resistance protein MdtM, a recently characterised homologue of MdfA and a member of the major facilitator superfamily, also functions in alkaline pH homeostasis. Results Assays that compared the growth of an E. coli ΔmdtM deletion mutant transformed with a plasmid encoding wild-type MdtM or the dysfunctional MdtM D22A mutant at different external alkaline pH values (ranging from pH 8.5 to 10) revealed a potential contribution by MdtM to alkaline pH tolerance, but only when millimolar concentrations of sodium or potassium was present in the growth medium. Fluorescence-based activity assays using inverted vesicles generated from transformants of antiporter-deficient (ΔnhaA, ΔnhaB, ΔchaA) E. coli TO114 cells defined MdtM as a low-affinity antiporter that catalysed electrogenic exchange of Na+, K+, Rb+ or Li+ for H+. The K+/H+ antiport reaction had a pH optimum at 9.0, whereas the Na+/H+ exchange activity was optimum at pH 9.25. Measurement of internal cellular pH confirmed MdtM as contributing to maintenance of a stable cytoplasmic pH, acid relative to the external pH, under conditions of alkaline stress. Conclusions Taken together, the results support a role for MdtM in alkaline pH tolerance. MdtM can therefore be added to the currently limited list of antiporters known to function in pH homeostasis in the model organism E. coli. PMID:23701827

  16. Ligand-induced conformational dynamics of the Escherichia coli Na+/H+ antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Eisinger, Martin Lorenz; Dörrbaum, Aline Ricarda; Michel, Hartmut; Padan, Etana; Langer, Julian David

    2017-10-31

    Na + /H + antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li + binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases. Published under the PNAS license.

  17. Salt and cadmium stress tolerance caused by overexpression of the Glycine Max Na+/H+ Antiporter (GmNHX1) gene in duckweed (Lemna turionifera 5511).

    Science.gov (United States)

    Yang, Lin; Han, Yujie; Wu, Di; Yong, Wang; Liu, Miaomiao; Wang, Sutong; Liu, Wenxin; Lu, Meiyi; Wei, Ying; Sun, Jinsheng

    2017-11-01

    Cadmium (Cd) pollution has aroused increasing attention due to its toxicity. It has been proved that Na + /H + Antiporter (NHX1) encodes a well-documented protein in Na + /H + trafficking, which leads to salt tolerance. This study showed that Glycine max Na + /H + Antiporter (GmNHX1) improved short-term cadmium and salt resistance in Lemna turionifera 5511. Expression of GmNHX1 prevented root from abscission and cell membrane damage, which also can enhance antioxidant system, inhibited of reactive oxygen species (ROS) accumulation and cause a less absorption of Cd under cadmium and salt stress. The cadmium tolerance suggested that NHX1 was involved under the cadmium stress. Copyright © 2017. Published by Elsevier B.V.

  18. The Na+ transport in gram-positive bacteria defect in the Mrp antiporter complex measured with 23Na nuclear magnetic resonance.

    Science.gov (United States)

    Górecki, Kamil; Hägerhäll, Cecilia; Drakenberg, Torbjörn

    2014-01-15

    (23)Na nuclear magnetic resonance (NMR) has previously been used to monitor Na(+) translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na NMR method was adapted for measurements of internal Na(+) concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na(+) concentration than an external one. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Single gene deletions of mrpA to mrpG and mrpE point mutations affect activity of the Mrp Na+/H+ antiporter of alkaliphilic Bacillus and formation of hetero-oligomeric Mrp complexes.

    Science.gov (United States)

    Morino, Masato; Natsui, Shinsuke; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

    2008-06-01

    Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.

  20. Cnh1 Na+/H+ antiporter and Ena1 Na+-ATPase play different roles in cation homeostasis and cell physiology of Candida glabrata

    Czech Academy of Sciences Publication Activity Database

    Krauke, Yannick; Sychrová, Hana

    2011-01-01

    Roč. 11, č. 1 (2011), s. 29-41 ISSN 1567-1356 R&D Projects: GA MŠk(CZ) LC531 Grant - others:EC(XE) MRTN-CT-2004-512481 Institutional research plan: CEZ:AV0Z50110509 Keywords : Candida glabrata * potassium homeostasis * podium detoxification * Ena 1 ATPase * Cnh1 antiporter Subject RIV: EE - Microbiology, Virology Impact factor: 2.403, year: 2011

  1. Yeast 14-3-3 proteins participate in the regulation of cell cation homeostasis via interaction with Nha1 alkali-metal-cation/proton antiporter

    Czech Academy of Sciences Publication Activity Database

    Zahrádka, Jaromír; Van Heusden, G.P.H.; Sychrová, Hana

    2012-01-01

    Roč. 1820, č. 7 (2012), s. 849-858 ISSN 0304-4165 R&D Projects: GA MŠk(CZ) LC531; GA MŠk(CZ) OC10012; GA AV ČR(CZ) IAA500110801 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : yeast * 14-3-3 proteins * ion homeostasis * Nha1 antiporter Subject RIV: CE - Biochemistry Impact factor: 3.848, year: 2012

  2. Salicornia europaea L. Na⁺/H⁺ antiporter gene improves salt tolerance in transgenic alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Zhang, L Q; Niu, Y D; Huridu, H; Hao, J F; Qi, Z; Hasi, A

    2014-07-24

    In order to obtain a salt-tolerant perennial alfalfa (Medicago sativa L.), we transferred the halophyte Salicornia europaea L. Na(+)/H(+) antiporter gene, SeNHX1, to alfalfa by using the Agrobacterium-mediated transformation method. The transformants were confirmed by both PCR and RT-PCR analyses. Of 197 plants that were obtained after transformation, 36 were positive by PCR analysis using 2 primer pairs for the CaMV35S-SeNHX1 and SeNHX1-Nos fragments; 6 plants survived in a greenhouse. RT-PCR analysis revealed that SeNHX1 was expressed in 5 plants. The resultant transgenic alfalfa had better salt tolerance. After stress treatment for 21 days with 0.6% NaCl, the chlorophyll and MDA contents in transgenic plants were lower, but proline content and SOD, POD, and CAT activities were higher than those in wild-type plants. These results suggest that the salt tolerance of transgenic alfalfa was improved by the overexpression of the SeNHX1 gene.

  3. Cystine uptake through the cystine/glutamate antiporter xCT triggers glioblastoma cell death under glucose deprivation.

    Science.gov (United States)

    Goji, Takeo; Takahara, Kazuhiko; Negishi, Manabu; Katoh, Hironori

    2017-12-01

    Oncogenic signaling in cancer cells alters glucose uptake and utilization to supply sufficient energy and biosynthetic intermediates for survival and sustained proliferation. Oncogenic signaling also prevents oxidative stress and cell death caused by increased production of reactive oxygen species. However, elevated glucose metabolism in cancer cells, especially in glioblastoma, results in the cells becoming sensitive to glucose deprivation ( i.e. in high glucose dependence), which rapidly induces cell death. However, the precise mechanism of this type of cell death remains unknown. Here, we report that glucose deprivation alone does not trigger glioblastoma cell death. We found that, for cell death to occur in glucose-deprived glioblastoma cells, cystine and glutamine also need to be present in culture media. We observed that cystine uptake through the cystine/glutamate antiporter xCT under glucose deprivation rapidly induces NADPH depletion, reactive oxygen species accumulation, and cell death. We conclude that although cystine uptake is crucial for production of antioxidant glutathione in cancer cells its transport through xCT also induces oxidative stress and cell death in glucose-deprived glioblastoma cells. Combining inhibitors targeting cancer-specific glucose metabolism with cystine and glutamine treatment may offer a therapeutic approach for glioblastoma tumors exhibiting high xCT expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Functional analysis of the Na+,K+/H+ antiporter PeNHX3 from the tree halophyte Populus euphratica in yeast by model-guided mutagenesis.

    Directory of Open Access Journals (Sweden)

    Liguang Wang

    Full Text Available Na+,K+/H+ antiporters are H+-coupled cotransporters that are crucial for cellular homeostasis. Populus euphratica, a well-known tree halophyte, contains six Na+/H+ antiporter genes (PeNHX1-6 that have been shown to function in salt tolerance. However, the catalytic mechanisms governing their ion transport remain largely unknown. Using the crystal structure of the Na+/H+ antiporter from the Escherichia coli (EcNhaA as a template, we built the three-dimensional structure of PeNHX3 from P. euphratica. The PeNHX3 model displays the typical TM4-TM11 assembly that is critical for ion binding and translocation. The PeNHX3 structure follows the 'positive-inside' rule and exhibits a typical physicochemical property of the transporter proteins. Four conserved residues, including Tyr149, Asn187, Asp188, and Arg356, are indentified in the TM4-TM11 assembly region of PeNHX3. Mutagenesis analysis showed that these reserved residues were essential for the function of PeNHX3: Asn187 and Asp188 (forming a ND motif controlled ion binding and translocation, and Tyr149 and Arg356 compensated helix dipoles in the TM4-TM11 assembly. PeNHX3 mediated Na+, K+ and Li+ transport in a yeast growth assay. Domain-switch analysis shows that TM11 is crucial to Li+ transport. The novel features of PeNHX3 in ion binding and translocation are discussed.

  5. Topology of AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, determined by site-directed fluorescence labeling.

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C; Abe, Keietsu

    2007-10-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of L-aspartate (Asp) with release of L-alanine (Ala) and CO(2). The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an L-aspartate-beta-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

  6. Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants

    Science.gov (United States)

    Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

    2016-01-01

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na+/H+) antiporter that transports Na+ into the vacuole and exports H+ into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na+/H+ antiporter that exports Na+ to the extracellular space and imports H+ into the plant cell. Plants rely on these enzymes either to keep Na+ out of the cell or to sequester Na+ into vacuoles to avoid the toxic level of Na+ in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. PMID:26985021

  7. Flavone Glucoside Uptake into Barley Mesophyll and Arabidopsis Cell Culture Vacuoles. Energization Occurs by H+-Antiport and ATP-Binding Cassette-Type Mechanisms1

    Science.gov (United States)

    Frangne, Nathalie; Eggmann, Thomas; Koblischke, Carsten; Weissenböck, Gottfried; Martinoia, Enrico; Klein, Markus

    2002-01-01

    In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a Km of 50 to 100 μm. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H+ antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta. PMID:11842175

  8. Detecting molecular interactions that stabilize, activate and guide ligand-binding of the sodium/proton antiporter MjNhaP1 from Methanococcus jannaschii.

    Science.gov (United States)

    Kedrov, Alexej; Wegmann, Susanne; Smits, Sander H J; Goswami, Panchali; Baumann, Hella; Muller, Daniel J

    2007-08-01

    Integral membrane proteins are involved in virtually every cellular process. Precisely regulating these machineries would allow controlling many human and vertebrate diseases. Embedded into cellular membranes, membrane proteins establish molecular interactions that sensitively react to environmental changes and to molecular compounds, such as ligands or inhibitors. We applied atomic force microscopy (AFM) to image the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii, and single-molecule force spectroscopy (SMFS) to probe molecular interactions that drive the protein structure-function relationship. High-resolution AFM topographs showed the dimeric assembly of MjNhaP1 being reconstituted into a lipid bilayer. SMFS of MjNhaP1 unraveled molecular interactions stabilizing individual structural domains. Transmembrane domains exhibited certain probabilities to unfold individually or cooperatively with other domains resulting in different unfolding pathways. Helices VIII and X established pH sensitive interactions altering significantly upon MjNhaP1 activation, while removal of the ligand (Na(+)) destabilized the entire antiporter except helix VIII. It is assumed that Asp234/235 of helix VIII are involved in the ligand-binding site and that helix X plays a functional role in the activation of the transporter.

  9. Co-overexpressing a Plasma Membrane and a Vacuolar Membrane Sodium/Proton Antiporter Significantly Improves Salt Tolerance in Transgenic Arabidopsis Plants.

    Science.gov (United States)

    Pehlivan, Necla; Sun, Li; Jarrett, Philip; Yang, Xiaojie; Mishra, Neelam; Chen, Lin; Kadioglu, Asim; Shen, Guoxin; Zhang, Hong

    2016-05-01

    The Arabidopsis gene AtNHX1 encodes a vacuolar membrane-bound sodium/proton (Na(+)/H(+)) antiporter that transports Na(+) into the vacuole and exports H(+) into the cytoplasm. The Arabidopsis gene SOS1 encodes a plasma membrane-bound Na(+)/H(+) antiporter that exports Na(+) to the extracellular space and imports H(+) into the plant cell. Plants rely on these enzymes either to keep Na(+) out of the cell or to sequester Na(+) into vacuoles to avoid the toxic level of Na(+) in the cytoplasm. Overexpression of AtNHX1 or SOS1 could improve salt tolerance in transgenic plants, but the improved salt tolerance is limited. NaCl at concentration >200 mM would kill AtNHX1-overexpressing or SOS1-overexpressing plants. Here it is shown that co-overexpressing AtNHX1 and SOS1 could further improve salt tolerance in transgenic Arabidopsis plants, making transgenic Arabidopsis able to tolerate up to 250 mM NaCl treatment. Furthermore, co-overexpression of AtNHX1 and SOS1 could significantly reduce yield loss caused by the combined stresses of heat and salt, confirming the hypothesis that stacked overexpression of two genes could substantially improve tolerance against multiple stresses. This research serves as a proof of concept for improving salt tolerance in other plants including crops. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  10. Efficient gamma-aminobutyric acid bioconversion by employing synthetic complex between glutamate decarboxylase and glutamate/GABA antiporter in engineered Escherichia coli.

    Science.gov (United States)

    Le Vo, Tam Dinh; Ko, Ji-seun; Park, Si Jae; Lee, Seung Hwan; Hong, Soon Ho

    2013-08-01

    Gamma-aminobutyric acid (GABA) is a precursor of one of the most promising heat-resistant biopolymers, Nylon-4, and can be produced by the decarboxylation of monosodium glutamate (MSG). In this study, a synthetic protein complex was applied to improve the GABA conversion in engineered Escherichia coli. Complexes were constructed by assembling a single protein-protein interaction domain SH3 to the glutamate decarboxylase (GadA and GadB) and attaching a cognate peptide ligand to the glutamate/GABA antiporter (GadC) at the N-terminus, C-terminus, and the 233rd amino acid residue. When GadA and GadC were co-overexpressed via the C-terminus complex, a GABA concentration of 5.65 g/l was obtained from 10 g/l MSG, which corresponds to a GABA yield of 93 %. A significant increase of the GABA productivity was also observed where the GABA productivity increased 2.5-fold in the early culture period due to the introduction of the synthetic protein complex. The GABA pathway efficiency and GABA productivity were enhanced by the introduction of the complex between Gad and glutamate/GABA antiporter.

  11. Cooperation of Antiporter LAT2/CD98hc with Uniporter TAT1 for Renal Reabsorption of Neutral Amino Acids.

    Science.gov (United States)

    Vilches, Clara; Boiadjieva-Knöpfel, Emilia; Bodoy, Susanna; Camargo, Simone; López de Heredia, Miguel; Prat, Esther; Ormazabal, Aida; Artuch, Rafael; Zorzano, Antonio; Verrey, François; Nunes, Virginia; Palacín, Manuel

    2018-04-02

    Background Reabsorption of amino acids (AAs) across the renal proximal tubule is crucial for intracellular and whole organism AA homeostasis. Although the luminal transport step is well understood, with several diseases caused by dysregulation of this process, the basolateral transport step is not understood. In humans, only cationic aminoaciduria due to malfunction of the basolateral transporter y + LAT1/CD98hc (SLC7A7/SLC3A2), which mediates the export of cationic AAs, has been described. Thus, the physiologic roles of basolateral transporters of neutral AAs, such as the antiporter LAT2/CD98hc (SLC7A8/SLC3A2), a heterodimer that exports most neutral AAs, and the uniporter TAT1 (SLC16A10), which exports only aromatic AAs, remain unclear. Functional cooperation between TAT1 and LAT2/CD98hc has been suggested by in vitro studies but has not been evaluated in vivo Methods To study the functional relationship of TAT1 and LAT2/CD98hc in vivo , we generated a double-knockout mouse model lacking TAT1 and LAT2, the catalytic subunit of LAT2/CD98hc (dKO LAT2-TAT1 mice). Results Compared with mice lacking only TAT1 or LAT2, dKO LAT2-TAT1 mice lost larger amounts of aromatic and other neutral AAs in their urine due to a tubular reabsorption defect. Notably, dKO mice also displayed decreased tubular reabsorption of cationic AAs and increased expression of y + LAT1/CD98hc. Conclusions The LAT2/CD98hc and TAT1 transporters functionally cooperate in vivo , and y + LAT1/CD98hc may compensate for the loss of LAT2/CD98hc and TAT1, functioning as a neutral AA exporter at the expense of some urinary loss of cationic AAs. Cooperative and compensatory mechanisms of AA transporters may explain the lack of basolateral neutral aminoacidurias in humans. Copyright © 2018 by the American Society of Nephrology.

  12. Ca2+/cation antiporters (CaCA: Identification, characterization and expression profiling in bread wheat (Triticum aestivum L.

    Directory of Open Access Journals (Sweden)

    Mehak Taneja

    2016-11-01

    Full Text Available The Ca2+/cation antiporters (CaCA superfamily proteins play vital function in Ca2+ ion homeostasis, which is an important event during development and defense response. Molecular characterization of these proteins has been performed in certain plants, but they are still not characterized in Triticum aestivum (bread wheat. Herein, we identified thirty four TaCaCA superfamily proteins, which were classified into TaCAX, TaCCX, TaNCL and TaMHX protein families based on their structural organization and evolutionary relation with earlier reported proteins. Since the T. aestivum comprises an allohexaploid genome, TaCaCA genes were derived from each A, B and D subgenome and homeologous chromosome (HC, except chromosome-group 1. Majority of genes were derived from more than one HCs in each family that were considered as homeologous genes (HGs due to their high similarity with each other. These HGs showed comparable gene and protein structures in terms of exon/intron organization and domain architecture. Majority of TaCaCA proteins comprised two Na_Ca_ex domains. However, TaNCLs consisted of an additional EF-hand domain with calcium binding motifs. Each TaCaCA protein family consisted of about ten transmembrane and two α-repeat regions with specifically conserved signature motifs except TaNCL, which had single α-repeat. Variable expression of most of the TaCaCA genes during various developmental stages suggested their specified role in development. However, constitutively high expression of a few genes like TaCAX1-A and TaNCL1-B indicated their role throughout the plant growth and development. The modulated expression of certain genes during biotic (fungal infections and abiotic stresses (heat, drought, salt suggested their role in stress response. Majority of TaCCX and TaNCL family genes were found highly affected during various abiotic stresses. However the role of individual gene needs to be established. The present study unfolded the opportunity

  13. Ca2+/Cation Antiporters (CaCA): Identification, Characterization and Expression Profiling in Bread Wheat (Triticum aestivum L.).

    Science.gov (United States)

    Taneja, Mehak; Tyagi, Shivi; Sharma, Shailesh; Upadhyay, Santosh Kumar

    2016-01-01

    The Ca 2+ /cation antiporters (CaCA) superfamily proteins play vital function in Ca 2+ ion homeostasis, which is an important event during development and defense response. Molecular characterization of these proteins has been performed in certain plants, but they are still not characterized in Triticum aestivum (bread wheat). Herein, we identified 34 TaCaCA superfamily proteins, which were classified into TaCAX, TaCCX, TaNCL, and TaMHX protein families based on their structural organization and evolutionary relation with earlier reported proteins. Since the T. aestivum comprises an allohexaploid genome, TaCaCA genes were derived from each A, B, and D subgenome and homeologous chromosome (HC), except chromosome-group 1. Majority of genes were derived from more than one HCs in each family that were considered as homeologous genes (HGs) due to their high similarity with each other. These HGs showed comparable gene and protein structures in terms of exon/intron organization and domain architecture. Majority of TaCaCA proteins comprised two Na_Ca_ex domains. However, TaNCLs consisted of an additional EF-hand domain with calcium binding motifs. Each TaCaCA protein family consisted of about 10 transmembrane and two α-repeat regions with specifically conserved signature motifs except TaNCL, which had single α-repeat. Variable expression of most of the TaCaCA genes during various developmental stages suggested their specified role in development. However, constitutively high expression of a few genes like TaCAX1-A and TaNCL1-B indicated their role throughout the plant growth and development. The modulated expression of certain genes during biotic (fungal infections) and abiotic stresses (heat, drought, salt) suggested their role in stress response. Majority of TaCCX and TaNCL family genes were found highly affected during various abiotic stresses. However, the role of individual gene needs to be established. The present study unfolded the opportunity for detail

  14. Role of the Group 2 Mrp sodium/proton antiporter in rapid response to high alkaline shock in the alkaline- and salt-tolerant Dietzia sp. DQ12-45-1b.

    Science.gov (United States)

    Fang, Hui; Qin, Xiao-Yu; Zhang, Kai-Duan; Nie, Yong; Wu, Xiao-Lei

    2018-04-01

    The six- and seven-subunit Na + /H + antiporters (Mrp) are widely distributed in bacteria. They are reported to be integral for pH homeostasis in alkaliphilic bacteria when adapting to high pH environments. In this study, operons encoding for the six-subunit Na + /H + antiporters were found in the genomes of all studied Dietzia strains, which have different alkaline-resistant abilities. Disruption of the operon in the strain Dietzia sp. DQ12-45-1b which leads to declined growth in presence of hypersaline and alkaline conditions suggested that the six-subunit Na + /H + antiporter played an important role in hypersaline and alkaline resistance. Although the complexes DqMrp from DQ12-45-1b (strain with high alkaline resistance) and DaMrp from D. alimentaria 72 T (strain with low alkaline resistance) displayed Na + (Li + )/H + antiport activities, they functioned optimally at different pH levels (9.0 for DQ12-45-1b and 8.0 for 72 T ). While both antiporters functioned properly to protect Escherichia coli cells from salt shock, only the DqMrp-containing strain survived the high alkaline shock. Furthermore, real-time PCR results showed that the expression of mrpA and mrpD induced only immediately after DQ12-45-1b cells were subjected to the alkaline shock. These results suggested that the expression of DqMrp might be induced by a pH gradient across the cell membrane, and DqMrp mainly functioned at an early stage to respond to the alkaline shock.

  15. Parallel adaptation of the rabbit renal cortical sodium/proton antiporter and sodium/bicarbonate cotransporter in metabolic acidosis and alkalosis.

    Science.gov (United States)

    Akiba, T; Rocco, V K; Warnock, D G

    1987-01-01

    Recent studies have shown that the bicarbonate reabsorptive capacity of the proximal tubule is increased in metabolic acidosis. For net bicarbonate reabsorption to be regulated, there may be changes in the rate of apical H+ secretion as well as in the basolateral base exit step. The present studies examined the rate of Na+/H+ exchange (acridine orange method) and Na+/HCO3 cotransport (22Na uptake) in apical and basolateral membranes prepared from the rabbit renal cortex by sucrose density gradient centrifugation. NH4Cl loading was used to produce acidosis (arterial pH, 7.27 +/- 0.03), and Cl-deficient diet with furosemide was used to produce alkalosis (arterial pH, 7.51 +/- 0.02). Maximal transport rate (Vmax) of Na+/H+ antiporter and Na+/HCO3 cotransporter were inversely related with plasma bicarbonate concentration from 6 to 39 mM. Furthermore, the maximal transport rates of both systems varied in parallel; when Vmax for the Na+/HCO3 cotransporter was plotted against Vmax for the Na+/H+ antiporter for each of the 24 groups of rabbits, the regression coefficient (r) was 0.648 (P less than 0.001). There was no effect of acidosis or alkalosis on affinity for Na+ of either transporter. We conclude that both apical and basolateral H+/HCO3 transporters adapt during acid-base disturbances, and that the maximal transport rates of both systems vary in parallel during such acid-base perturbations. PMID:3038953

  16. Lysine 300 is essential for stability but not for electrogenic transport of theEscherichia coliNhaA Na+/H+antiporter.

    Science.gov (United States)

    Călinescu, Octavian; Dwivedi, Manish; Patiño-Ruiz, Miyer; Padan, Etana; Fendler, Klaus

    2017-05-12

    Na + /H + antiporters are located in the cytoplasmic and intracellular membranes and play crucial roles in regulating intracellular pH, Na + , and volume. The NhaA antiporter of Escherichia coli is the best studied member of the Na + /H + exchanger family and a model system for all related Na + /H + exchangers, including eukaryotic representatives. Several amino acid residues are important for the transport activity of NhaA, including Lys-300, a residue that has recently been proposed to carry one of the two H + ions that NhaA exchanges for one Na + ion during one transport cycle. Here, we sought to characterize the effects of mutating Lys-300 of NhaA to amino acid residues containing side chains of different polarity and length ( i.e. Ala, Arg, Cys, His, Glu, and Leu) on transporter stability and function. Salt resistance assays, acridine-orange fluorescence dequenching, solid supported membrane-based electrophysiology, and differential scanning fluorometry were used to characterize Na + and H + transport, charge translocation, and thermal stability of the different variants. These studies revealed that NhaA could still perform electrogenic Na + /H + exchange even in the absence of a protonatable residue at the Lys-300 position. However, all mutants displayed lower thermal stability and reduced ion transport activity compared with the wild-type enzyme, indicating the critical importance of Lys-300 for optimal NhaA structural stability and function. On the basis of these experimental data, we propose a tentative mechanism integrating the functional and structural role of Lys-300. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Infrared spectroscopic study of the structural and functional properties of the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii.

    Science.gov (United States)

    Dzafić, E; Klein, O; Goswami, P; Kühlbrandt, W; Mäntele, W

    2009-06-01

    In this study, structural, functional, and mechanistic properties of the Na(+)/H(+) antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na(+)/H(+) antiporters are generally responsible for the regulation of cytoplasmic pH and Na(+) concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive. The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to alpha-helical segments. In contrast, the temperature-induced structural changes for beta-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.

  18. Identification of a Proton-Chloride Antiporter (EriC) by Himar1 Transposon Mutagenesis in Lactobacillus reuteri and Its Role in Histamine Production

    Science.gov (United States)

    Hemarajata, P; Spinler, JK; Balderas, MA; Versalovic, J

    2014-01-01

    The gut microbiome may modulate intestinal immunity by luminal conversion of dietary amino acids to biologically active signals. The model probiotic organism Lactobacillus reuteri ATCC PTA 6475 is indigenous to the human microbiome, and converts the amino acid L-histidine to the biogenic amine, histamine. Histamine suppresses TNF production by human myeloid cells and is a product of L-histidine decarboxylation, which is a proton-facilitated reaction. A transposon mutagenesis strategy was developed based on a single-plasmid nisin-inducible Himar1 transposase/transposon delivery system for L. reuteri. A highly conserved proton-chloride antiporter gene (eriC), a gene widely present in the gut microbiome was discovered by Himar1 transposon (Tn)-mutagenesis presented in this study. Genetic inactivation of eriC by transposon insertion and genetic recombineering resulted in reduced ability of L. reuteri to inhibit TNF production by activated human myeloid cells, diminished histamine production by the bacteria and downregulated expression of histidine decarboxylase (hdc) cluster genes compared to those of WT 6475. EriC belongs to a large family of ion transporters that includes chloride channels and proton-chloride antiporters and may facilitate the availability of protons for the decarboxylation reaction, resulting in histamine production by L. reuteri. This report leverages the tools of bacterial genetics for probiotic gene discovery. The findings highlight the widely conserved nature of ion transporters in bacteria and how ion transporters are coupled with amino acid decarboxylation and contributed to microbiome-mediated immunomodulation. PMID:24488273

  19. Impact of AtNHX1, a vacuolar Na+/H+ antiporter, upon gene expression during short- and long-term salt stress in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Blumwald Eduardo

    2007-04-01

    Full Text Available Abstract Background AtNHX1, the most abundant vacuolar Na+/H+ antiporter in Arabidopsis thaliana, mediates the transport of Na+ and K+ into the vacuole, influencing plant development and contributing to salt tolerance. In this report, microarray expression profiles of wild type plants, a T-DNA insertion knockout mutant of AtNHX1 (nhx1, and a 'rescued' line (NHX1::nhx1 were exposed to both short (12 h and 48 h and long (one and two weeks durations of a non-lethal salt stress to identify key gene transcripts associated with the salt response that are influenced by AtNHX1. Results 147 transcripts showed both salt responsiveness and a significant influence of AtNHX1. Fifty-seven of these genes showed an influence of the antiporter across all salt treatments, while the remaining genes were influenced as a result of a particular duration of salt stress. Most (69% of the genes were up-regulated in the absence of AtNHX1, with the exception of transcripts encoding proteins involved with metabolic and energy processes that were mostly down-regulated. Conclusion While part of the AtNHX1-influenced transcripts were unclassified, other transcripts with known or putative roles showed the importance of AtNHX1 to key cellular processes that were not necessarily limited to the salt stress response; namely calcium signaling, sulfur metabolism, cell structure and cell growth, as well as vesicular trafficking and protein processing. Only a small number of other salt-responsive membrane transporter transcripts appeared significantly influenced by AtNHX1.

  20. Cloning and identification of Group 1 mrp operon encoding a novel monovalent cation/proton antiporter system from the moderate halophile Halomonas zhaodongensis.

    Science.gov (United States)

    Meng, Lin; Hong, Shan; Liu, Henan; Huang, Haipeng; Sun, Hao; Xu, Tong; Jiang, Juquan

    2014-11-01

    The novel species Halomonas zhaodongensis NEAU-ST10-25(T) recently identified by our group is a moderate halophile which can grow at the range of 0-2.5 M NaCl (optimum 0.5 M) and pH 6-12 (optimum pH 9). To explore its halo-alkaline tolerant mechanism, genomic DNA was screened from NEAU-ST10-25(T) in this study for Na(+)(Li(+))/H(+) antiporter genes by selection in Escherichia coli KNabc lacking three major Na(+)(Li(+))/H(+) antiporters. One mrp operon could confer tolerance of E. coli KNabc to 0.8 M NaCl and 100 mM LiCl, and an alkaline pH. This operon was previously mainly designated mrp (also mnh, pha or sha) due to its multiple resistance and pH-related activity. Here, we will also use mrp to designate the homolog from H. zhaodongensis (Hz_mrp). Sequence analysis and protein alignment showed that Hz_mrp should belong to Group 1 mrp operons. Further phylogenetic analysis reveals that Hz_Mrp system should represent a novel sub-class of Group 1 Mrp systems. This was confirmed by a significant difference in pH-dependent activity profile or the specificity and affinity for the transported monovalent cations between Hz_Mrp system and all the known Mrp systems. Therefore, we propose that Hz_Mrp should be categorized as a novel Group 1 Mrp system.

  1. Functional Differentiation of Antiporter-Like Polypeptides in Complex I; a Site-Directed Mutagenesis Study of Residues Conserved in MrpA and NuoL but Not in MrpD, NuoM, and NuoN.

    Directory of Open Access Journals (Sweden)

    Eva Sperling

    Full Text Available It has long been known that the three largest subunits in the membrane domain (NuoL, NuoM and NuoN of complex I are homologous to each other, as well as to two subunits (MrpA and MrpD from a Na+/H+ antiporter, Mrp. MrpA and NuoL are more similar to each other and the same is true for MrpD and NuoN. This suggests a functional differentiation which was proven experimentally in a deletion strain model system, where NuoL could restore the loss of MrpA, but not that of MrpD and vice versa. The simplest explanation for these observations was that the MrpA and MrpD proteins are not antiporters, but rather single subunit ion channels that together form an antiporter. In this work our focus was on a set of amino acid residues in helix VIII, which are only conserved in NuoL and MrpA (but not in any of the other antiporter-like subunits. and to compare their effect on the function of these two proteins. By combining complementation studies in B. subtilis and 23Na-NMR, response of mutants to high sodium levels were tested. All of the mutants were able to cope with high salt levels; however, all but one mutation (M258I/M225I showed differences in the efficiency of cell growth and sodium efflux. Our findings showed that, although very similar in sequence, NuoL and MrpA seem to differ on the functional level. Nonetheless the studied mutations gave rise to interesting phenotypes which are of interest in complex I research.

  2. Transgenic salt-tolerant sugar beet (Beta vulgaris L.) constitutively expressing an Arabidopsis thaliana vacuolar Na/H antiporter gene, AtNHX3, accumulates more soluble sugar but less salt in storage roots.

    Science.gov (United States)

    Liu, Hua; Wang, Qiuqing; Yu, Mengmeng; Zhang, Yanyan; Wu, Yingbao; Zhang, Hongxia

    2008-09-01

    In Arabidopsis thaliana, six vacuolar Na(+)/H(+) antiporters (AtNHX1-6) were identified. Among them, AtNHX1, 2 and 5 are functional Na(+)/H(+) antiporters with the most abundant expression levels in seedling shoots and roots. However, the expression of AtNHX3 in Arabidopsis can only be detected by RT-PCR, and its physiological function still remains unclear. In this work, we demonstrate that constitutive expression of AtNHX3 in sugar beet (Beta vulgaris L.) conferred augmented resistance to high salinity on transgenic plants. In the presence of 300 or 500 mm NaCl, transgenic plants showed very high potassium accumulation in the roots and storage roots. Furthermore, the transcripts of sucrose phosphate synthase (SPS), sucrose synthase (SS) and cell wall sucrose invertase (SI) genes were maintained in transgenic plants. The accumulation of soluble sugar in the storage roots of transgenic plants grown under high salt stress condition was also higher. Our results implicate that AtNHX3 is also a functional antiporter responsible for salt tolerance by mediating K(+)/H(+) exchange in higher plants. The salt accumulation in leaves but not in the storage roots, and the increased yield of storage roots with enhanced constituent soluble sugar contents under salt stress condition demonstrate a great potential use of this gene in improving the quality and yield of crop plants.

  3. Cloning and characterization of the Salicornia brachiata Na(+)/H(+) antiporter gene SbNHX1 and its expression by abiotic stress.

    Science.gov (United States)

    Jha, Anupama; Joshi, Mukul; Yadav, Narendra Singh; Agarwal, Pradeep K; Jha, Bhavanath

    2011-03-01

    Salinity causes multifarious adverse effects to plants. Plants response to salt stress involves numerous processes that function in coordination to alleviate both cellular hyperosmolarity and ion disequilibrium. A Na(+)/H(+) antiporter NHX1 gene has been isolated from a halophytic plant Salicornia brachiata in this study. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in SbNHX1 classify it as a plant vacuolar NHX gene. The SbNHX1 cDNA has an open reading frame of 1,683 bp, encoding a polypeptide of 560 amino acid residues with an estimated molecular mass 62.44 kDa. The SbNHX1 shows high amino acid similarity with other halophytic NHX gene and belongs to Class-I type NHXs. TMpred suggests that SbNHX1 contains 11 strong transmembrane (TM). Real time PCR analysis revealed that SbNHX1 transcript expresses maximum at 0.5 M. Transcript increases gradually by increasing the treatment duration at 0.5 M NaCl, however, maximum expression was observed at 48 h. The overexpression of SbNHX1 gene in tobacco plant showed NaCl tolerance. This study shows that SbNHX1 is a potential gene for salt tolerance, and can be used in future for developing salt tolerant crops.

  4. Enhanced salt resistance in apple plants overexpressing a Malus vacuolar Na+/H+ antiporter gene is associated with differences in stomatal behavior and photosynthesis.

    Science.gov (United States)

    Li, Chao; Wei, Zhiwei; Liang, Dong; Zhou, Shasha; Li, Yonghong; Liu, Changhai; Ma, Fengwang

    2013-09-01

    High salinity is a major abiotic factor that limits crop production. The dwarfing apple rootstock M.26 is sensitive to such stress. To obtain an apple that is adaptable to saline soils, we transformed this rootstock with a vacuolar Na(+)/H(+) antiporter, MdNHX1. Differences in salt tolerance between transgenic and wild-type (WT) rootstocks were examined under field conditions. We also compared differences when 'Naganofuji No. 2' apple was grafted onto these transgenic or WT rootstocks. Plants on the transgenic rootstocks grew well during 60 d of mild stress (100 mM NaCl) while the WT exhibited chlorosis, inhibited growth and even death. Compared with the untreated control, the stomatal density was greater in both non-grafted and grafted WT plants exposed to 200 mM NaCl. In contrast, that density was significantly decreased in leaves from grafted transgenic plants. At 200 mM NaCl, net photosynthesis, stomatal conductance, intercellular CO2 concentration, and chlorophyll contents were markedly reduced in the WT, whereas the declines in those values were only minor in similarly stressed transgenic plants. Therefore, we conclude that overexpressing plants utilize a better protective mechanism for retaining higher photosynthetic capacity. Furthermore, this contrast in tolerance and adaptability to stress is linked to differences in stomatal behavior and photosynthetic rates. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  5. Nitric oxide enhances salt secretion and Na(+) sequestration in a mangrove plant, Avicennia marina, through increasing the expression of H(+)-ATPase and Na(+)/H(+) antiporter under high salinity.

    Science.gov (United States)

    Chen, Juan; Xiao, Qiang; Wu, Feihua; Dong, Xuejun; He, Junxian; Pei, Zhenming; Zheng, Hailei

    2010-12-01

    Modulation of nitric oxide (NO) on ion homeostasis, by enhancing salt secretion in the salt glands and Na(+) sequestration into the vacuoles, was investigated in a salt-secreting mangrove tree, Avicennia marina (Forsk.) Vierh. The major results are as follows: (i) under 400 mM NaCl treatment, the application of 100 µM sodium nitroprusside (SNP), an NO donor, significantly increased the density of salt crystals and salt secretion rate of the leaves, along with maintaining a low Na(+) to K(+) ratio in the leaves. (ii) The measurement of element contents by X-ray microanalysis in the epidermis and transversal sections of A. marina leaves revealed that SNP (100 µM) significantly increased the accumulation of Na(+) in the epidermis and hypodermal cells, particularly the Na(+) to K(+) ratio in the salt glands, but no such effects were observed in the mesophyll cells. (iii) Using non-invasive micro-test technology (NMT), both long-term SNP (100 µM) and transient SNP (30 µM) treatments significantly increased net Na(+) efflux in the salt glands. On the contrary, NO synthesis inhibitors and scavenger reversed the effects of NO on Na(+) flux. These results indicate that NO enhanced salt secretion by increasing net Na(+) efflux in the salt glands. (iv) Western blot analysis demonstrated that 100 µM SNP stimulated protein expressions of plasma membrane (PM) H(+)-ATPase and vacuolar membrane Na(+)/H(+) antiporter. (v) To further clarify the molecular mechanism of the effects of NO on enhancing salt secretion and Na(+) sequestration, partial cDNA fragments of PM H(+)-ATPase (HA1), PM Na(+)/H(+) antiporter (SOS1) and vacuolar Na(+)/H(+) antiporter (NHX1) were isolated and transcriptional expression of HA1, SOS1, NHX1 and vacuolar H(+)-ATPase subunit c (VHA-c1) genes were analyzed using real-time quantitative polymerase chain reaction. The relative transcript abundance of the four genes were markedly increased in 100 µM SNP-treated A. marina. Moreover, the increase

  6. Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling▿ †

    Science.gov (United States)

    Nanatani, Kei; Fujiki, Takashi; Kanou, Kazuhiko; Takeda-Shitaka, Mayuko; Umeyama, Hideaki; Ye, Liwen; Wang, Xicheng; Nakajima, Tasuku; Uchida, Takafumi; Maloney, Peter C.; Abe, Keietsu

    2007-01-01

    The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an l-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity. PMID:17660287

  7. Co-expression of Pennisetum glaucum vacuolar Na⁺/H⁺ antiporter and Arabidopsis H⁺-pyrophosphatase enhances salt tolerance in transgenic tomato.

    Science.gov (United States)

    Bhaskaran, Shimna; Savithramma, D L

    2011-11-01

    Salinity is one of the major abiotic stresses affecting plant productivity. Tomato (Solanum lycopersicum L.), an important and widespread crop in the world, is sensitive to moderate levels of salt in the soil. To generate tomato plants that can adapt to saline soil, AVP1, a vacuolar H(+)-pyrophosphatase gene from Arabidopsis thaliana, and PgNHX1, a vacuolar Na(+)/H(+) antiporter gene from Pennisetum glaucum, were co-expressed by Agrobacterium tumefaciens-mediated transformation. A sample of transformants was self-pollinated, and progeny were evaluated for salt tolerance in vitro and in vivo. It is reported here that co-expression of AVP1 and PgNHX1 confers enhanced salt tolerance to the transformed tomato compared with the AVP1 and PgNHX1 single gene transgenic plants and the wild-type. These transgenic plants grew well in the presence of 200 mM NaCl while wild-type plants exhibited chlorosis and died within 3 weeks. The transgenic line co-expressing AVP1 and PgNHX1 retained more chlorophyll and accumulated 1.4 times more proline as a response to stress than single gene transformants. Moreover, these transgenic plants accumulated a 1.5 times higher Na(+) content in their leaf tissue than the single gene transformants. The toxic effect of Na(+) accumulation in the cytosol is reduced by its sequestration into the vacuole. The physiological analysis of the transgenic lines clearly demonstrates that co-expression of AVP1 and PgNHX1 improved the osmoregulatory capacity of double transgenic lines by enhanced sequestration of ions into the vacuole by increasing the availability of protons and thus alleviating the toxic effect of Na(+).

  8. Phylogenetic analysis and protein structure modelling identifies distinct Ca(2+)/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species.

    Science.gov (United States)

    Pittman, Jon K; Hirschi, Kendal D

    2016-12-01

    The Ca(2+)/Cation Antiporter (CaCA) superfamily is an ancient and widespread family of ion-coupled cation transporters found in nearly all kingdoms of life. In animals, K(+)-dependent and K(+)-indendent Na(+)/Ca(2+) exchangers (NCKX and NCX) are important CaCA members. Recently it was proposed that all rice and Arabidopsis CaCA proteins should be classified as NCX proteins. Here we performed phylogenetic analysis of CaCA genes and protein structure homology modelling to further characterise members of this transporter superfamily. Phylogenetic analysis of rice and Arabidopsis CaCAs in comparison with selected CaCA members from non-plant species demonstrated that these genes form clearly distinct families, with the H(+)/Cation exchanger (CAX) and cation/Ca(2+) exchanger (CCX) families dominant in higher plants but the NCKX and NCX families absent. NCX-related Mg(2+)/H(+) exchanger (MHX) and CAX-related Na(+)/Ca(2+) exchanger-like (NCL) proteins are instead present. Analysis of genomes of ten closely-related rice species and four Arabidopsis-related species found that CaCA gene family structures are highly conserved within related plants, apart from minor variation. Protein structures were modelled for OsCAX1a and OsMHX1. Despite exhibiting broad structural conservation, there are clear structural differences observed between the different CaCA types. Members of the CaCA superfamily form clearly distinct families with different phylogenetic, structural and functional characteristics, and therefore should not be simply classified as NCX proteins, which should remain as a separate gene family.

  9. Chloride Ion Transport by the E. coli CLC Cl−/H+ Antiporter: A Combined Quantum-Mechanical and Molecular-Mechanical Study

    Directory of Open Access Journals (Sweden)

    Chun-Hung Wang

    2018-03-01

    Full Text Available We performed steered molecular dynamics (SMD and umbrella sampling simulations of Cl− ion migration through the transmembrane domain of a prototypical E. coli CLC Cl−/H+ antiporter by employing combined quantum-mechanical (QM and molecular-mechanical (MM calculations. The SMD simulations revealed interesting conformational changes of the protein. While no large-amplitude motions of the protein were observed during pore opening, the side chain rotation of the protonated external gating residue Glu148 was found to be critical for full access of the channel entrance by Cl−. Moving the anion into the external binding site (Sext induced small-amplitude shifting of the protein backbone at the N-terminal end of helix F. As Cl− traveled through the pore, rigid-body swinging motions of helix R separated it from helix D. Helix R returned to its original position once Cl− exited the channel. Population analysis based on polarized wavefunction from QM/MM calculations discovered significant (up to 20% charge loss for Cl− along the ion translocation pathway inside the pore. The delocalized charge was redistributed onto the pore residues, especially the functional groups containing π bonds (e.g., the Tyr445 side chain, while the charges of the H atoms coordinating Cl− changed almost negligibly. Potentials of mean force computed from umbrella sampling at the QM/MM and MM levels both displayed barriers at the same locations near the pore entrance and exit. However, the QM/MM PMF showed higher barriers (~10 kcal/mol than the MM PMF (~2 kcal/mol. Binding energy calculations indicated that the interactions between Cl− and certain pore residues were overestimated by the semi-empirical PM3 Hamiltonian and underestimated by the CHARMM36 force fields, both of which were employed in the umbrella sampling simulations. In particular, CHARMM36 underestimated binding interactions for the functional groups containing π bonds, missing the stabilizations of

  10. The dual role of candida glabrata drug:H+ antiporter CgAqr1 (ORF CAGL0J09944g) in antifungal drug and acetic acid resistance

    Science.gov (United States)

    Costa, Catarina; Henriques, André; Pires, Carla; Nunes, Joana; Ohno, Michiyo; Chibana, Hiroji; Sá-Correia, Isabel; Teixeira, Miguel C.

    2013-01-01

    Opportunistic Candida species often have to cope with inhibitory concentrations of acetic acid, in the acidic environment of the vaginal mucosa. Given that the ability of these yeast species to tolerate stress induced by weak acids and antifungal drugs appears to be a key factor in their persistence and virulence, it is crucial to understand the underlying mechanisms. In this study, the drug:H+ antiporter CgAqr1 (ORF CAGL0J09944g), from Candida glabrata, was identified as a determinant of resistance to acetic acid, and also to the antifungal agents flucytosine and, less significantly, clotrimazole. These antifungals were found to act synergistically with acetic acid against this pathogen. The action of CgAqr1 in this phenomenon was analyzed. Using a green fluorescent protein fusion, CgAqr1 was found to localize to the plasma membrane and to membrane vesicles when expressed in C. glabrata or, heterologously, in Saccharomyces cerevisiae. Given its ability to complement the susceptibility phenotype of its S. cerevisiae homolog, ScAqr1, CgAqr1 was proposed to play a similar role in mediating the extrusion of chemical compounds. Significantly, the expression of this gene was found to reduce the intracellular accumulation of 3H-flucytosine and, to a moderate extent, of 3H-clotrimazole, consistent with a direct role in antifungal drug efflux. Interestingly, no effect of CgAQR1 deletion could be found on the intracellular accumulation of 14C-acetic acid, suggesting that its role in acetic acid resistance may be indirect, presumably through the transport of a still unidentified physiological substrate. Although neither of the tested chemicals induces changes in CgAQR1 expression, pre-exposure to flucytosine or clotrimazole was found to make C. glabrata cells more sensitive to acetic acid stress. Results from this study show that CgAqr1 is an antifungal drug resistance determinant and raise the hypothesis that it may play a role in C. glabrata persistent colonization and

  11. Clotrimazole Drug Resistance in Candida glabrata Clinical Isolates Correlates with Increased Expression of the Drug:H(+) Antiporters CgAqr1, CgTpo1_1, CgTpo3, and CgQdr2.

    Science.gov (United States)

    Costa, Catarina; Ribeiro, Jonathan; Miranda, Isabel M; Silva-Dias, Ana; Cavalheiro, Mafalda; Costa-de-Oliveira, Sofia; Rodrigues, Acácio G; Teixeira, Miguel C

    2016-01-01

    For years, antifungal drug resistance in Candida species has been associated to the expression of ATP-Binding Cassette (ABC) multidrug transporters. More recently, a few drug efflux pumps from the Drug:H(+) Antiporter (DHA) family have also been shown to play a role in this process, although to date only the Candida albicans Mdr1 transporter has been demonstrated to be relevant in the clinical acquisition of antifungal drug resistance. This work provides evidence to suggest the involvement of the C. glabrata DHA transporters CgAqr1, CgQdr2, CgTpo1_1, and CgTpo3 in the clinical acquisition of clotrimazole drug resistance. A screening for azole drug resistance in 138 C. glabrata clinical isolates, from patients attending two major Hospitals in Portugal, was performed. Based on this screening, 10 clotrimazole susceptible and 10 clotrimazole resistant isolates were selected for further analysis. The transcript levels of CgAQR1, CgQDR2, CgTPO1_1, and CgTPO3 were found to be significantly up-regulated in resistant isolates when compared to the susceptible ones, with a level of correlation that was found to be similar to that of CgCDR2, an ABC gene known to be involved in the clinical acquisition of resistance. As a proof-of-concept experiment, the CgTPO3 gene was deleted in an azole resistant C. glabrata isolate, exhibiting high levels of expression of this gene. The deletion of CgTPO3 in this isolate was found to lead to decreased resistance to clotrimazole and fluconazole, and increased accumulation of azole drugs, thus suggesting the involvement of this transporter in the manifestation of azole resistance.

  12. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT.

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C; Abe, Keietsu

    2009-04-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  13. Structural and Functional Importance of Transmembrane Domain 3 (TM3) in the Aspartate:Alanine Antiporter AspT: Topology and Function of the Residues of TM3 and Oligomerization of AspT▿

    Science.gov (United States)

    Nanatani, Kei; Maloney, Peter C.; Abe, Keietsu

    2009-01-01

    AspT, the aspartate:alanine antiporter of Tetragenococcus halophilus, a membrane protein of 543 amino acids with 10 putative transmembrane (TM) helices, is the prototype of the aspartate:alanine exchanger (AAE) family of transporters. Because TM3 (isoleucine 64 to methionine 85) has many amino acid residues that are conserved among members of the AAE family and because TM3 contains two charged residues and four polar residues, it is thought to be located near (or to form part of) the substrate translocation pathway that includes the binding site for the substrates. To elucidate the role of TM3 in the transport process, we carried out cysteine-scanning mutagenesis. The substitutions of tyrosine 75 and serine 84 had the strongest inhibitory effects on transport (initial rates of l-aspartate transport were below 15% of the rate for cysteine-less AspT). Considerable but less-marked effects were observed upon the replacement of methionine 70, phenylalanine 71, glycine 74, arginine 76, serine 83, and methionine 85 (initial rates between 15% and 30% of the rate for cysteine-less AspT). Introduced cysteine residues at the cytoplasmic half of TM3 could be labeled with Oregon green maleimide (OGM), whereas cysteines close to the periplasmic half (residues 64 to 75) were not labeled. These results suggest that TM3 has a hydrophobic core on the periplasmic half and that hydrophilic residues on the cytoplasmic half of TM3 participate in the formation of an aqueous cavity in membranes. Furthermore, the presence of l-aspartate protected the cysteine introduced at glycine 62 against a reaction with OGM. In contrast, l-aspartate stimulated the reactivity of the cysteine introduced at proline 79 with OGM. These results demonstrate that TM3 undergoes l-aspartate-induced conformational alterations. In addition, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses and a glutaraldehyde cross-linking assay suggest that functional AspT forms homo-oligomers as a

  14. Harmaline-resistant mutant of Methanothermobacter thermautotrophicus with a lesion in Na+/H +antiport

    Czech Academy of Sciences Publication Activity Database

    Vidová, M.; Bobálová, Janette; Šmigáň, P.

    2011-01-01

    Roč. 30, SI (2011), S54-S60 ISSN 0231-5882 Institutional research plan: CEZ:AV0Z40310501 Keywords : methanogens * harmaline resistance * sodium/proton exchange Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.192, year: 2011

  15. An Na+/H+ antiporter gene from wheat plays an important role in ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    tissue (Blumwald et al1985). A novel Na+/H+ ... might play an important role in salt and osmotic stress tolerance in plant cells. [Yu J N, Huang J .... This culture was further diluted to an OD600 of 0.002 and four aliquots of 100 µl were spread onto YPGAL medium. Colony- forming units were counted after 2 days. Another four.

  16. Functional validation of a novel isoform of Na +/H+ antiporter from ...

    Indian Academy of Sciences (India)

    Madhu Sudhan

    2007-03-22

    Mar 22, 2007 ... large areas of the world (Boyer 1982; Zhu 2001). Mechanisms that confer salt tolerance vary with the plant species, however the basic strategy works towards the maintenance of Na+ homeostasis in the cytosol (Blumwald. 2000). Active exclusion of Na+ mediated by the plasma membrane localized Na+/H+ ...

  17. Functional validation of a novel isoform of Na/H antiporter from ...

    Indian Academy of Sciences (India)

    2007-03-22

    Mar 22, 2007 ... Transgenic rice plants overexpressing PgNHX1 developed more extensive root system and completed their life cycle by setting flowers and seeds in the presence of 150 mM NaCl. Our data demonstrate the potential of PgNHX1 for imparting enhanced salt tolerance capabilities to salt-sensitive crop plants ...

  18. Transport of Diamines by Enterococcus faecalis Is Mediated by an Agmatine-Putrescine Antiporter

    NARCIS (Netherlands)

    Driessen, Arnold J.M.; Smid, Eddy J.; Konings, Wilhelmus

    1988-01-01

    Enterococcusfaecalis ATCC 11700 is able to use arginine and the diamine agmatine as a sole energy source. Via the highly homologous deiminase pathways, arginine and agmatine are converted into CO2, NH3, and the end products ornithine and putrescine, respectively. In the arginine deiminase pathway,

  19. Dicyclohexylcarbodiimide (DCCD) sensitive organic cation/H/sup +/ antiporter in renal brush border membrane vesicles (BBMV)

    Energy Technology Data Exchange (ETDEWEB)

    Sokol, P.P.; Holohan, P.D.; Ross, C.R.

    1986-03-01

    We tested the hypothesis that organic cation transport in BBMV is coupled to the countertransport of a H/sup +/ by employing a prototypic organic cation, N/sup 1/-methylnicotinamide (NMN), and a rapid filtration assay. Two H/sup +/ gradient uncouplers, carbonyl cyanide m-chlorophenylhydrazone (CCCP)/sub 3/ and gramicidin D were effective in dissipating H/sup +/ driven (/sup 3/H)NMN transport. Nigericin, a K/sup +//H/sup +/ exchanger, generated a H/sup +/ gradient in situ which drove the net accumulation of NMN. The molecular mechanism of H/sup +/ coupling was examined employing DCCD, a hydrophobic carbodiimide, which inactivates essential carboxylate groups, the putative H/sup +/ receptor. DCCD inactivated NMN transport irreversibly with an IC/sub 50/ of 2.6..mu..M whereas the hydrophilic carbodiimide, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, did not. DCCD inactivation followed pseudo-first-order kinetics and was not affected by NMN. A double logarithmic plot of the apparent rate constants vs. (DCCD) gave a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of DCCD inactivates one carboxylate group per active transport unit. The results show that (1) the transport of organic cations is coupled to the countermovement of a H/sup +/ and (2) a carboxylate group is essential for H/sup +/ binding and translocation.

  20. The oxidative stress-inducible cystine/glutamate antiporter, system x (c) (-) : cystine supplier and beyond.

    Science.gov (United States)

    Conrad, Marcus; Sato, Hideyo

    2012-01-01

    The oxidative stress-inducible cystine/glutamate exchange system, system x (c) (-) , transports one molecule of cystine, the oxidized form of cysteine, into cells and thereby releases one molecule of glutamate into the extracellular space. It consists of two protein components, the 4F2 heavy chain, necessary for membrane location of the heterodimer, and the xCT protein, responsible for transport activity. Previously, system x (c) (-) has been regarded to be a mere supplier of cysteine to cells for the synthesis of proteins and the antioxidant glutathione (GSH). In that sense, oxygen, electrophilic agents, and bacterial lipopolysaccharide trigger xCT expression to accommodate with increased oxidative stress by stimulating GSH biosynthesis. However, emerging evidence established that system x (c) (-) may act on its own as a GSH-independent redox system by sustaining a redox cycle over the plasma membrane. Hallmarks of this cycle are cystine uptake, intracellular reduction to cysteine and secretion of the surplus of cysteine into the extracellular space. Consequently, increased levels of extracellular cysteine provide a reducing microenvironment required for proper cell signaling and communication, e.g. as already shown for the mechanism of T cell activation. By contrast, the enhanced release of glutamate in exchange with cystine may trigger neurodegeneration due to glutamate-induced cytotoxic processes. This review aims to provide a comprehensive picture from the early days of system x (c) (-) research up to now.

  1. A Hydrophobic Filter Confers the Cation Selectivity of Zygosaccharomyces rouxii Plasma-Membrane Na (+)/H (+) Antiporter

    Czech Academy of Sciences Publication Activity Database

    Kinclová-Zimmermannová, Olga; Falson, P.; Cmunt, Denis; Sychrová, Hana

    2015-01-01

    Roč. 427, č. 8 (2015), s. 1681-1697 ISSN 0022-2836 R&D Projects: GA ČR(CZ) GAP503/10/0307; GA MŠk(CZ) LD13037 Institutional support: RVO:67985823 Keywords : yeast * plasma membrane * sodium proton exchanger * substrate specificity * potassium transport Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.517, year: 2015

  2. Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions

    Czech Academy of Sciences Publication Activity Database

    Zahrádka, Jaromír; Sychrová, Hana

    2012-01-01

    Roč. 12, č. 4 (2012), s. 439-446 ISSN 1567-1356 R&D Projects: GA MŠk(CZ) LC531; GA AV ČR(CZ) IAA500110801 Grant - others:Univerzita Karlova(CZ) 33779266 Institutional research plan: CEZ:AV0Z50110509 Keywords : potassium uptake * potassium efflux * yeast * plasma-membrane potential Subject RIV: EE - Microbiology, Virology Impact factor: 2.462, year: 2012

  3. GENERATION OF A PROTON MOTIVE FORCE BY HISTIDINE DECARBOXYLATION AND ELECTROGENIC HISTIDINE HISTAMINE ANTIPORT IN LACTOBACILLUS-BUCHNERI

    NARCIS (Netherlands)

    MOLENAAR, D; BOSSCHER, JS; TENBRINK, B; DRIESSEN, AJM; KONINGS, WN

    Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (DELTApsi), inside negative, upon

  4. Excretion of putrescine by the putrescine-ornithine antiporter encoded by the potE gene of Escherichia coli.

    OpenAIRE

    Kashiwagi, K; Miyamoto, S; Suzuki, F; Kobayashi, H; Igarashi, K

    1992-01-01

    Excretion of putrescine from Escherichia coli was assessed by measuring its uptake into inside-out membrane vesicles. The vesicles were prepared from wild-type E. coli or E. coli transformed with plasmids containing one of the three polyamine transport systems. The results indicate that excretion of putrescine is catalyzed by the putrescine transport protein, encoded by the potE gene located at 16 min on the E. coli chromosome. Loading of ornithine (or lysine) inside the vesicles was essentia...

  5. Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH

    International Nuclear Information System (INIS)

    Olsnes, S.; Ludt, J.; Tonnessen, T.I.; Sandvig, K.

    1987-01-01

    The rates of bicarbonate-dependent uptake and efflux of 22 Na + in Vero cells were studied and compared with the uptake and efflux of 36 Cl - . Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na + was much less affected by changes in pH. The bicarbonate-linked uptake of 22 Na + was dependent on internal Cl- but not on internal Na + . At a constant external concentration of HCO 3 -, the amount of 22 Na + associated with the cells increased when the internal concentration of HCO 3 - decreased and vice versa, which is compatible with the possibility that the ion pair NaCO 3 - is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36 Cl - both in the absence and presence of Na + . At alkaline internal pH, HCO 3 - stimulated the efflux of 36 Cl - from preloaded cells, while at acidic internal pH both Na + and HCO 3 - were required to induce 36 Cl - efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane

  6. Photorespiration maintains carbon recycling efficiency at low irradiance despite impaired glycolate/glycerate antiport or hydroxypyruvate reduction

    Science.gov (United States)

    Photorespiration partially recycles fixed carbon that would otherwise be lost following the oxygenation reaction of Ribulose, 1-5, carboxylase oxygenase (Rubisco) and significantly reduces net photosynthesis in C3 plants. The recycling of photorespiratory C2 to C3 intermediates is not perfectly effi...

  7. Expression of an "Arabidopsis" Ca(2+)/H(+) antiporter CAX1 variant in petunia enhances cadmium tolerance and accumulation

    Science.gov (United States)

    Phytoremediation is a cost-effective and minimally invasive technology to cleanse soils contaminated with heavy metals. However, few plant species are suitable for phytoremediation of metals such as cadmium (Cd). Genetic engineering offers a powerful tool to generate plants that can hyperaccumulate ...

  8. The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato.

    Science.gov (United States)

    Huertas, Raúl; Rubio, Lourdes; Cagnac, Olivier; García-Sánchez, María Jesús; Alché, Juan De Dios; Venema, Kees; Fernández, José Antonio; Rodríguez-Rosales, María Pilar

    2013-12-01

    The endosomal LeNHX2 ion transporter exchanges H(+) with K(+) and, to lesser extent, Na(+) . Here, we investigated the response to NaCl supply and K(+) deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K(+) the opposite was found. Analysis of mineral composition showed a higher K(+) content in roots, shoots and xylem sap of transgenic plants and no differences in Na(+) content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na(+)/H(+) and, above all, K(+)/H(+) transport activity in root intracellular membrane vesicles. Under K(+) limiting conditions, transgenic plants enhanced root expression of the high-affinity K(+) uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K(+) depletion rates and half cytosolic K(+) activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K(+) and lower cytosolic K(+) activity than untransformed plants. These results indicate the fundamental role of K(+) homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress. © 2013 John Wiley & Sons Ltd.

  9. The Ca2+/H+ antiporter TMEM165 expression, localization in the developing, lactating and involuting mammary gland parallels the secretory pathway Ca2+ATPase (SPCA1)

    Science.gov (United States)

    Plasma membrane Ca2+-ATPase 2 (PMCA2) knockout mice showed that ~ 60 % of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca2+-ATPase’s 1 and/or 2 (SP...

  10. CA2+ AND ZN2+ ARE TRANSPORTED BY THE ELECTROGENIC 2NA+/1H+ ANTIPORTER IN ECHINODERM GASTROINTESTINAL EPITHELIUM. (R823068)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  11. High expression of cystine-glutamate antiporter xCT (SLC7A11) is an independent biomarker for epileptic seizures at diagnosis in glioma

    DEFF Research Database (Denmark)

    Sørensen, Mai Froberg; Heimisdóttir, Sólborg Berglind; Sørensen, Mia Dahl

    2018-01-01

    Epileptic seizures are an important cause of morbidity in glioma patients. Substantial lines of evidence support the concept of the excitatory neurotransmitter glutamate being a crucial mediator of glioma-associated seizures. In gliomas, non-vesicular secretion of glutamate via the cystine-glutamate...... exchanger (SLC7A11, xCT) constitutes the main mechanism contributing to high extracellular glutamate concentrations. However, a convincing "proof-of-relevance" of this mechanism in patient material is lacking. A cohort of 229 consecutive patients with newly diagnosed glioma was analyzed with respect...... tumor using tissue microarrays. In addition to histological grading of the tumors, isocitrate dehydrogenase 1 (IDH1) R132H mutational status was determined by immunohistochemistry. 215 consecutive glioma patients were included in the study (7.4% grade II, 7.0% grade III, 85.6% grade IV). High x...

  12. Erv14 cargo receptor participates in yeast salt tolerance via its interaction with the plasma-membrane Nha1 cation/proton antiporter

    Czech Academy of Sciences Publication Activity Database

    Rosas-Santiago, P.; Zimmermannová, Olga; Vera-Estrella, R.; Sychrová, Hana; Pantoja, O.

    2016-01-01

    Roč. 1858, č. 1 (2016), s. 67-74 ISSN 0005-2736 Institutional support: RVO:67985823 Keywords : Erv14p * Nha1p * protein–protein interaction * mislocalization * salt-tolerance * yeast Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.498, year: 2016

  13. Activation of the plasma membrane Na/H antiporter salt-overly-sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

    KAUST Repository

    Quintero, Francisco J.

    2011-01-24

    The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex upregulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

  14. Co-expression of tonoplast Cation/H(+) antiporter and H(+)-pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity, drought and field conditions.

    Science.gov (United States)

    Bao, Ai-Ke; Du, Bao-Qiang; Touil, Leila; Kang, Peng; Wang, Qiang-Long; Wang, Suo-Min

    2016-03-01

    Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H(+)-PPase genes, ZxNHX and ZxVP1-1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens-mediated transformation. Compared with wild-type (WT) plants, transgenic alfalfa plants co-expressing ZxNHX and ZxVP1-1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water-deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na(+), K(+) and Ca(2+) accumulation in leaves and roots, as a result of co-expression of ZxNHX and ZxVP1-1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water-deficit stress. Furthermore, the transgenic alfalfa co-expressing ZxNHX and ZxVP1-1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water-use efficiency than WT plants. Our results indicate that co-expression of tonoplast NHX and H(+)-PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  15. Genetic interactions among the Arl1 GTPase and intracellular Naplus/Hplus antiporters in pH homeostasis and cation detoxification

    Czech Academy of Sciences Publication Activity Database

    Marešová, Lydie; Sychrová, Hana

    2010-01-01

    Roč. 10, č. 7 (2010), s. 802-811 ISSN 1567-1356 R&D Projects: GA MŠk(CZ) LC531; GA AV ČR(CZ) KJB500110701; GA AV ČR(CZ) IAA500110801 Institutional research plan: CEZ:AV0Z50110509 Keywords : Saccharomyces cerevisiae * cation transport Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.279, year: 2010

  16. Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex--the evolutionary progenitor of the long horizontal helix in complex I.

    Science.gov (United States)

    Virzintiene, Egle; Moparthi, Vamsi K; Al-Eryani, Yusra; Shumbe, Leonard; Górecki, Kamil; Hägerhäll, Cecilia

    2013-10-11

    MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. NCBI nr-aa BLAST: CBRC-AGAM-07-0021 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-07-0021 ref|YP_001402419.1| Na+/H+ antiporter NhaA [Yersinia pseudotuberculosis... IP 31758] gb|ABS48792.1| Na+/H+ antiporter NhaA [Yersinia pseudotuberculosis IP 31758] YP_001402419.1 3e-83 69% ...

  18. NCBI nr-aa BLAST: CBRC-DNOV-01-2526 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DNOV-01-2526 ref|ZP_00371235.1| NA+/H+ antiporter (napA) [Campylobacter upsaliens...is RM3195] gb|EAL53227.1| NA+/H+ antiporter (napA) [Campylobacter upsaliensis RM3195] ZP_00371235.1 0.025 22% ...

  19. NCBI nr-aa BLAST: CBRC-PABE-13-0007 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PABE-13-0007 ref|YP_572835.1| Na+/H+ antiporter NhaC [Chromohalobacter salexig...ens DSM 3043] gb|ABE58136.1| Na+/H+ antiporter NhaC [Chromohalobacter salexigens DSM 3043] YP_572835.1 8.2 35% ...

  20. NCBI nr-aa BLAST: CBRC-DYAK-06-0027 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-06-0027 ref|YP_001273600.1| Ca2+/Na+ antiporter (K+-dependent) [Methanobrevibacter smith...ii ATCC 35061] gb|ABQ87232.1| Ca2+/Na+ antiporter (K+-dependent) [Methanobrevibacter smithii ATCC 35061] YP_001273600.1 0.007 23% ...

  1. NCBI nr-aa BLAST: CBRC-CREM-01-1346 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1346 ref|NP_939160.1| Na(+)/H(+) antiporter homolog [Corynebacterium diphtheria...e NCTC 13129] emb|CAE49312.1| Na(+)/H(+) antiporter homolog [Corynebacterium diphtheriae] NP_939160.1 2e-34 34% ...

  2. NCBI nr-aa BLAST: CBRC-CREM-01-1301 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1301 ref|ZP_01168030.1| Na+/H+ antiporter, putative [Oceanospirillum s...p. MED92] gb|EAR59909.1| Na+/H+ antiporter, putative [Oceanospirillum sp. MED92] ZP_01168030.1 3e-81 52% ...

  3. NCBI nr-aa BLAST: CBRC-ACAR-01-0808 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0808 ref|YP_001335185.1| putative arginine/ornithine antiporter [Klebsiella pneumonia...e subsp. pneumoniae MGH 78578] gb|ABR76955.1| putative arginine/ornithine antiporter [Klebsiella pneumoniae subsp. pneumoniae MGH 78578] YP_001335185.1 0.32 27% ...

  4. AtCHX13 is a plasma membrane K(+) transporter

    Science.gov (United States)

    Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K(+) required for growth and development is poorly understood. The Arabidopsis ("Arabidopsis thaliana") genome contains numerous cation:proton antiporte...

  5. AtCHX13 is a plasma membrane K+ transporter

    Science.gov (United States)

    Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (...

  6. Substrate-bound structure of the E. coli multidrug resistance transporter MdfA.

    Science.gov (United States)

    Heng, Jie; Zhao, Yan; Liu, Ming; Liu, Yue; Fan, Junping; Wang, Xianping; Zhao, Yongfang; Zhang, Xuejun C

    2015-09-01

    Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.

  7. Induction of ferroptotic cell death for overcoming cisplatin resistance of head and neck cancer.

    Science.gov (United States)

    Roh, Jong-Lyel; Kim, Eun Hye; Jang, Hye Jin; Park, Jin Young; Shin, Daiha

    2016-10-10

    Inhibition of key molecules related to ferroptosis, cystine/glutamate antiporter and glutathione peroxidase, may induce eradication of chemotherapy/radiotherapy-resistant cancer cells. The present study investigated whether ferroptosis could overcome head and neck cancer (HNC) resistance to cisplatin treatment. Three cisplatin-resistant HNC cell lines (AMC-HN3R, -HN4R, and -HN9R) and their parental lines were used. The effects of cystine and glutamate alteration and pharmacological and genetic inhibition of cystine/glutamate antiporter were assessed by measuring viability, death, reactive oxygen species production, protein expression, and preclinical mouse tumor xenograft models. Conditioned media with no cystine or glutamine excess induced ferroptosis of both cisplatin-sensitive and -resistant HNC cells without any apparent changes to necrosis and apoptosis markers. The cystine/glutamate antiporter inhibitors erastin and sulfasalazine inhibited HNC cell growth and accumulated lipid reactive oxygen species, thereby inducing ferroptosis. Genetic silencing of cystine/glutamate antiporter with siRNA or shRNA treatment also induced effective ferroptotic cell death of resistant HNC cells and enhanced the cisplatin cytotoxicity of resistant HNC cells. Pharmacological and genetic inhibition of cystine/glutamate antiporter significantly sensitized resistant HNC cells to cisplatin in vitro and in vivo. Pharmacological and genetic inhibition of cystine/glutamate antiporter overcomes the cisplatin resistance of HNC cells by inducing ferroptosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. Metagenomic cloning and characterization of Na⁺ transporters from Huamachi Salt Lake in China.

    Science.gov (United States)

    Gao, Miao; Tao, Li; Chen, Sanfeng

    2013-02-22

    Moderately halophilic bacteria are a kind of extreme environment microorganism that can tolerate moderate salt concentrations ranging from 0.5M to 2.5M. Here, via a metagenomic library screen, we identified four putative Na(+) transporters, designated H7-Nha, H16-Mppe, H19-Cap and H35-Mrp, from moderately halophilic community in the hypersaline soil of Huamachi Salt Lake, China. Functional complementation observed in a Na(+)(Ca(2+))/H(+) antiporter-defective Escherichia coli mutant (KNabc) suggests that the four putative Na(+) transporters could confer cells a capacity of Na(+) resistance probably by enhancing Na(+) or Ca(2+) efflux, but not Li(+) or K(+) exchange. Blastp analysis of the deduced amino-acid sequences indicates that H7-Nha has 71% identity to the NhaG Na(+)/H(+) antiporter of Bacillus subtilis, while H19-Cap shows 99% identity to Enterobacter cloacae Ca(2+) antiporter. Interestingly, H16-Mppe shares 59% identity to the metallophosphoesterase of Bacillus cellulosilyticus and H35-Mrp shows 68% identity to multidrug resistance protein of Lysinibacillus sphaericus. This is the first report that predicts a potential role of metallophosphoesterase in Na(+) resistance in halophilic bacteria. Furthermore, everted membrane vesicles prepared from E. coli cells harboring H7-Nha exhibit Na(+)/H(+) antiporter activity, but not Li(+) (K(+))/H(+) antiporter activity, confirming that H7-Nha supports Na(+) resistance mainly via Na(+)/H(+) antiport. Our report also demonstrates that metagenomic library screen is a convenient and effective way to explore more novel types of Na(+) transporters. Copyright © 2012 Elsevier GmbH. All rights reserved.

  9. Alkaline pH homeostasis in bacteria: new insights.

    Science.gov (United States)

    Padan, Etana; Bibi, Eitan; Ito, Masahiro; Krulwich, Terry A

    2005-11-30

    The capacity of bacteria to survive and grow at alkaline pH values is of widespread importance in the epidemiology of pathogenic bacteria, in remediation and industrial settings, as well as in marine, plant-associated and extremely alkaline ecological niches. Alkali-tolerance and alkaliphily, in turn, strongly depend upon mechanisms for alkaline pH homeostasis, as shown in pH shift experiments and growth experiments in chemostats at different external pH values. Transcriptome and proteome analyses have recently complemented physiological and genetic studies, revealing numerous adaptations that contribute to alkaline pH homeostasis. These include elevated levels of transporters and enzymes that promote proton capture and retention (e.g., the ATP synthase and monovalent cation/proton antiporters), metabolic changes that lead to increased acid production, and changes in the cell surface layers that contribute to cytoplasmic proton retention. Targeted studies over the past decade have followed up the long-recognized importance of monovalent cations in active pH homeostasis. These studies show the centrality of monovalent cation/proton antiporters in this process while microbial genomics provides information about the constellation of such antiporters in individual strains. A comprehensive phylogenetic analysis of both eukaryotic and prokaryotic genome databases has identified orthologs from bacteria to humans that allow better understanding of the specific functions and physiological roles of the antiporters. Detailed information about the properties of multiple antiporters in individual strains is starting to explain how specific monovalent cation/proton antiporters play dominant roles in alkaline pH homeostasis in cells that have several additional antiporters catalyzing ostensibly similar reactions. New insights into the pH-dependent Na(+)/H(+) antiporter NhaA that plays an important role in Escherichia coli have recently emerged from the determination of the

  10. H+ V-ATPase-Energized Transporters in Brush Border Membrane Vesicles from Whole Larvae of Aedes Aegypti

    Science.gov (United States)

    Brush Border Membrane vesicles (BBMVs) from Whole larvae of Aedes aegypti (AeBBMVWs ) contain an H+ V-ATPase (V), a Na+/H+ antiporter, NHA1 (A) and a Na+-coupled, nutrient amino acid transporter, NAT8 (N), VAN for short. All V-ATPase subunits are present in the Ae. aegypti genome and in the vesicles...

  11. The "Arabidopsis cax3" mutants display altered salt tolerance, pH sensitivity and reduced plasma membrane H(+)-ATPase activity

    Science.gov (United States)

    Perturbing CAX1, an "Arabidopsis" vacuolar H(+)/Ca(2+) antiporter, and the related vacuolar transporter CAX3, has been previously shown to cause severe growth defects; however, the specific function of CAX3 has remained elusive. Here, we describe plant phenotypes that are shared among "cax1" and "ca...

  12. Reference: 55 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available /H+ antiporter in leaf vacuoles in Arabidopsis and also suggest that its contribu...nce of NaCl, nhx1 seedling establishment was impaired. These results place AtNHX1 as the dominant K+ and Na+

  13. NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli

    NARCIS (Netherlands)

    Rowe, John J.; Ubbink-Kok, Trees; Molenaar, Douwe; Konings, Wilhelmus; Driessen, Arnold J.M.

    Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest-lived radioactive isotope of

  14. The secondary multidrug transporter LmrP contains multiple drug interaction sites

    NARCIS (Netherlands)

    Putman, M; Koole, LA; van Veen, HW; Konings, WN

    1999-01-01

    The secondary multidrug transporter LmrP of Lactococcus lactis mediates the efflux of Hoechst 33342 from the cytoplasmic leaflet of the membrane. Kinetic analysis of Hoechst 33342 transport in inside-out membrane vesicles of L. lactis showed that the LmrP-mediated H+/Hoechst 33342 antiport reaction

  15. Over-expressing Salicornia europaea (SeNHX1) gene in tobacco ...

    African Journals Online (AJOL)

    use

    2011-11-21

    Nov 21, 2011 ... These results suggested that the impor- tance of vacuolar Na+/H+ antiporters to improve plants salinity tolerance is regulating ion homeostasis. This capacity of vacuolar compartmentalization can be taken as an adaptation mechanism to high salt environment of halophytes and glycophytes (Blumwald et al.

  16. Fulltext PDF

    Indian Academy of Sciences (India)

    2014-08-08

    Aug 8, 2014 ... antiporters play important roles in cellular ion homeostasis,vacuolar pH regulation and sequestration of Na. + ions into the vacuole. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na. +. /H. + transporting activity. In this study, we truncated the ...

  17. Journal of Genetics | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Plant vacuolar Na+/H+ antiporters play important roles in cellular ion homeostasis,vacuolar pH regulation and sequestration of Na+ ions into the vacuole. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na+/H+ transporting activity. In this study, we truncated the ...

  18. Screening of protein kinase inhibitors identifies PKC inhibitors as inhibitors of osteoclastic acid secretion and bone resorption

    DEFF Research Database (Denmark)

    Sørensen, Mette G; Karsdal, Morten A; Dziegiel, Morten H

    2010-01-01

    Bone resorption is initiated by osteoclastic acidification of the resorption lacunae. This process is mediated by secretion of protons through the V-ATPase and chloride through the chloride antiporter ClC-7. To shed light on the intracellular signalling controlling extracellular acidification, we...... screened a protein kinase inhibitor library in human osteoclasts....

  19. Seizure-like afterdischarges simulated in a model neuron.

    NARCIS (Netherlands)

    Kager, J.; Wadman, W.J.; Somjen, G.G.

    2006-01-01

    To explore non-synaptic mechanisms in paroxysmal discharges, we used a computer model of a simplified hippocampal pyramidal cell, surrounded by interstitial space and a "glial-endothelial" buffer system. Ion channels for Na(+), K(+), Ca(2+) and Cl(-) (,) ion antiport 3Na/Ca, and "active" ion pumps

  20. Heterodimerization of Arabidopsis calcium/proton exchangers contributes to regulation of guard cell dynamics and plant defense responses

    Science.gov (United States)

    "Arabidopsis thaliana" cation exchangers (CAX1 and CAX3) are closely related tonoplast-localized calcium/proton (Ca(2+)/H+) antiporters that contribute to cellular Ca(2+) homeostasis. CAX1 and CAX3 were previously shown to interact in yeast; however, the function of this complex in plants has remain...

  1. Fulltext PDF

    Indian Academy of Sciences (India)

    2014-08-08

    Aug 8, 2014 ... Jing Ji, jijing@tju.edu.cn. (a calcium-binding protein). Previous research has shown that AtSOS1 is essential to maintain low intracellular levels of toxic Na. + under salt stress (Qiu et al. 2002). Vac- uolar Na. +. /H. + antiporters have been investigated as the key to salt tolerance in plants (Blumwald et al.

  2. Hydrophilic C terminus of Salicornia europaea vacuolar Na/H ...

    Indian Academy of Sciences (India)

    Plant vacuolar Na+/H+ antiporters play important roles in cellular ion homeostasis,vacuolar pH regulation and sequestration of Na+ ions into the vacuole. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na+/H+ transporting activity. In this study, we truncated the ...

  3. Effects of glutamate decarboxylase and gamma-aminobutyric acid (GABA) transporter on the bioconversion of GABA in engineered Escherichia coli.

    Science.gov (United States)

    Le Vo, Tam Dinh; Kim, Tae Wan; Hong, Soon Ho

    2012-05-01

    Gamma-aminobutyric acid (GABA) is a non-essential amino acid and a precursor of pyrrolidone, a monomer of nylon 4. GABA can be biosynthesized through the decarboxylation of L: -glutamate by glutamate decarboxylase. In this study, the effects of glutamate decarboxylase (gadA, gadB), glutamate/GABA antiporter (gadC) and GABA aminotransferase (gabT) on GABA production were investigated in Escherichia coli. Glutamate decarboxylase was overexpressed alone or with the glutamate/GABA antiporter to enhance GABA synthesis. GABA aminotransferase, which redirects GABA into the TCA cycle, was knock-out mutated. When gadB and gadC were co-overexpressed in the gabT mutant strain, a final GABA concentration of 5.46 g/l was obtained from 10 g/l of monosodium glutamate (MSG), which corresponded to a GABA yield of 89.5%.

  4. Citrin deficiency: A treatable cause of acute psychosis in adults.

    OpenAIRE

    Bijarnia-Mahay Sunita; Häberle Johannes; Rüfenacht Véronique; Shigematsu Yosuke; Saxena Renu; Verma Ishwar C

    2015-01-01

    Citrin deficiency is an autosomal recessive genetic disorder caused by a defect in the mitochondrial aspartate/glutamate antiporter, citrin. The disorder manifests either as neonatal intra-hepatic cholestasis or occurs in adulthood with recurrent hyperammonemia and neuropsychiatric disturbances. It has a high prevalence in the East Asian population, but is actually pan-ethnic. We report the case of a 26-year-old male patient presenting with episodes of abnormal neuro-psychiatric behavior asso...

  5. Energetics of sodium efflux from Escherichia coli

    International Nuclear Information System (INIS)

    Borbolla, M.G.; Rosen, B.P.

    1984-01-01

    When energy-starved cells of Escherichia coli were passively loaded with 22 Na+, efflux of sodium could be initiated by addition of a source of metabolic energy. Conditions were established where the source of energy was phosphate bond energy, an electrochemical proton gradient, or both. Only an electrochemical proton gradient was required for efflux from intact cells. These results are consistent with secondary exchange of Na+ for H+ catalyzed by a sodium/proton antiporter

  6. Curcumin-Protected PC12 Cells Against Glutamate-Induced Oxidative Toxicity

    OpenAIRE

    Chang, Chi-Huang; Chen, Hua-Xin; Yü, George; Peng, Chiung-Chi; Peng, Robert Y.

    2014-01-01

    Glutamate is a major excitatory neurotransmitter present in the central nervous system. The glutamate/cystine antiporter system xc– connects the antioxidant defense with neurotransmission and behaviour. Overactivation of ionotropic glutamate receptors induces neuronal death, a pathway called excitotoxicity. Glutamate-induced oxidative stress is a major contributor to neurodegenerative diseases including cerebral ischemia, Alzheimer’s and Huntington’s disease. Curcuma has a wide spectrum of bi...

  7. A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli--expression and characterization of cytochrome-tagged Complex I subunits.

    Science.gov (United States)

    Gustavsson, Tobias; Trane, Maria; Moparthi, Vamsi K; Miklovyte, Egle; Moparthi, Lavanya; Górecki, Kamil; Leiding, Thom; Arsköld, Sindra Peterson; Hägerhäll, Cecilia

    2010-08-01

    Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane-spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C-terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c(550). Compared with other available fusion-protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter-like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo-cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c(550) domain in all the fusion proteins exhibited normal spectra and redox properties, with an E(m) of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c-tag. Finally, a his-tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.

  8. THE ENERGETIC FUNCTIONS OF PLANT MITOCHONDRIA UNDER STRESS

    Directory of Open Access Journals (Sweden)

    Grabelnych O.I.

    2005-09-01

    Full Text Available This article reviews the involvement of the mitochondrial systems, which maintain the balance of cell energy at different stress conditions. It is shown the functioning of the alternative oxidase, free fatty acids, uncoupling proteins, the rotenone-insensitive NAD(PH dehydrogenases, the ADP/ATP-antiporter, the permeability transition pore and ATP-sensitive potassium channel (К+ATP. It is discussed data about physiological role of these systems in plant cell.

  9. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    A vacuole Na+/H+ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (nhx1), TaNHX2 ...

  10. Expression of heterologous transporters in Saccharomyces kudriavzevii: A strategy for improving yeast salt tolerance and fermentation performance

    Czech Academy of Sciences Publication Activity Database

    Dibalová-Čuláková, Hana; Alonso-del-Real, J.; Querol, A.; Sychrová, Hana

    2018-01-01

    Roč. 268, Mar 2 (2018), s. 27-34 ISSN 0168-1605 R&D Projects: GA ČR(CZ) GA15-03708S Institutional support: RVO:67985823 Keywords : fermentation * salt tolerance * alkali-metal-cation exporter * non-conventional yeasts * Nhal antiporter * Ena ATPase Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.339, year: 2016

  11. Monosodium glutamate-induced oxidative kidney damage and possible mechanisms: a mini-review.

    Science.gov (United States)

    Sharma, Amod

    2015-10-22

    Animal studies suggest that chronic monosodium glutamate (MSG) intake induces kidney damage by oxidative stress. However, the underlying mechanisms are still unclear, despite the growing evidence and consensus that α-ketoglutarate dehydrogenase, glutamate receptors and cystine-glutamate antiporter play an important role in up-regulation of oxidative stress in MSG-induced renal toxicity. This review summaries evidence from studies into MSG-induced renal oxidative damage, possible mechanisms and their importance from a toxicological viewpoint.

  12. Cystine uptake by cultured cells originating from dog proximal tubule segments

    International Nuclear Information System (INIS)

    States, B.; Reynolds, R.; Lee, J.; Segal, S.

    1990-01-01

    Large numbers of kidney epithelial cells were cultured successfully from isolated dog proximal tubule segments. Cells in primary culture and in first passage retained the cystine-dibasic amino acid co-transporter system which is found in vivo and in freshly isolated proximal tubule segments. In contrast to other cultured cells, the cystine-glutamate anti-porter was absent in primary cultures. However, this anti-porter system seemed to be developing in cells in first passage. The intracellular ratio of cysteine:reduced glutathione (CSH:GSH) was maintained at 1:36 in both primary cultures and in low passage cells. Incubation of cells in primary culture for 5 min at 37 degrees C with 0.025 mM [ 35 S]L-cystine resulted in incorporation of approximately 36 and 8.5% of the label into intracellular CSH and GSH, respectively. These cultured cells, therefore, seem to be an excellent model system for the eventual elucidation of (a) the inticacies of cystine metabolism and (b) regulation of (1) the cystine-dibasic amino acid co-transporter system and (2) the development of the cysteine-glutamate anti-porter system

  13. Elevated compartmentalization of Na+ into vacuoles improves salt and cold stress tolerance in sweet potato (Ipomoea batatas).

    Science.gov (United States)

    Fan, Weijuan; Deng, Gaifang; Wang, Hongxia; Zhang, Hongxia; Zhang, Peng

    2015-08-01

    Salinity and low temperature are the main limiting factors for sweet potato (Ipomoea batatas) growth and agricultural productivity. Various studies have shown that plant NHX-type antiporter plays a crucial role in regulating plant tolerance to salt stress by intracellular Na(+) compartmentalization. The Arabidopsis thaliana AtNHX1 gene that encodes a vacuolar Na(+) /H(+) antiporter was introduced into the sweet potato cultivar Xushu-22 by Agrobacterium-mediated transformation to confer abiotic stress tolerance. Stable insertion of AtNHX1 into the sweet potato genome and its expression was confirmed by Southern blot and reverse transcription-polymerase chain reaction (RT-PCR). A remarkably higher Na(+) /H(+) exchange activity of tonoplast membrane from transgenic sweet potato lines (NOE) in comparison with wild-type (WT) plants confirmed the vacuolar antiporter function in mediating Na(+) /H(+) exchange. Under salt stress, NOE plants accumulated higher Na(+) and K(+) levels in their tissues compared with WT plants, maintaining high K(+) /Na(+) ratios. Consequently, NOE plants showed enhanced protection against cell damage due to the increased proline accumulation, preserved cell membrane integrity, enhanced reactive oxygen species (ROS) scavenging (e.g. increased superoxide dismutase activity), and reduced H2 O2 and malondialdehyde (MDA) production. Moreover, the transgenic plants showed improved cold tolerance through multiple mechanisms of action, revealing the first molecular evidence for NHX1 function in cold response. The transgenic plants showed better biomass production and root yield under stressful conditions. These findings demonstrate that overexpressing AtNHX1 in sweet potato renders the crop tolerant to both salt and cold stresses, providing a greater capacity for the use of AtNHX1 in improving crop performance under combined abiotic stress conditions. © 2014 Scandinavian Plant Physiology Society.

  14. Scanning ion-selective electrode technique and X-ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt-sensitive cucumber and salt-tolerant pumpkin under NaCl stress.

    Science.gov (United States)

    Lei, Bo; Huang, Yuan; Sun, Jingyu; Xie, Junjun; Niu, Mengliang; Liu, Zhixiong; Fan, Molin; Bie, Zhilong

    2014-12-01

    Grafting onto salt-tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na(+) in salt-tolerant pumpkin and salt-sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion-selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na(+), and a correspondingly increased H(+) influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na(+)/H(+) exchange in the root was inhibited by amiloride (a Na(+)/H(+) antiporter inhibitor) or vanadate [a plasma membrane (PM) H(+) -ATPase inhibitor], indicating that Na(+) exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na(+)/H(+) antiporter across the PM, and the Na(+)/H(+) antiporter system in salt stressed pumpkin roots was sufficient to exclude Na(+) X-ray microanalysis showed higher Na(+) in the cortex, but lower Na(+) in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na(+), limit the radial transport of Na(+) to the stele and thus restrict the transport of Na(+) to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots. © 2014 Scandinavian Plant Physiology Society.

  15. Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity

    International Nuclear Information System (INIS)

    Oppedisano, Francesca; Catto, Marco; Koutentis, Panayiotis A.; Nicolotti, Orazio; Pochini, Lorena; Koyioni, Maria; Introcaso, Antonellina; Michaelidou, Sophia S.; Carotti, Angelo; Indiveri, Cesare

    2012-01-01

    The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20 μM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the protein's Cys residue(s). A dose–response analysis of the most active compounds gave IC 50 values in the range of 3–30 μM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein. -- Highlights: ► Non‐competitive inhibition of ASCT2 by 1,2,3-dithiazoles was studied in proteoliposomes. ► Different 1,2,3-dithiazoles were synthesized and evaluated as transporter inhibitors. ► Many compounds potently inhibited the glutamine/glutamine antiport catalyzed by ASCT2. ► The inhibition was reversed by DTE indicating reaction with protein Cys. ► The most active compounds gave IC 50 in the range of 3–30 μM.

  16. In vivo imaging of system xc- as a novel approach to monitor multiple sclerosis

    International Nuclear Information System (INIS)

    Martin, Abraham; Szczupak, Boguslaw; Arrieta, Ander; Vazquez-Villoldo, Nuria; Soria, Federico N.; Domercq, Maria; Matute, Carlos; Gomez-Vallejo, Vanessa; Llop, Jordi; Padro, Daniel; Plaza-Garcia, Sandra; Reese, Torsten

    2016-01-01

    Glutamate excitotoxicity contributes to oligodendroglial and axonal damage in multiple sclerosis pathology. Extracellular glutamate concentration in the brain is controlled by cystine/glutamate antiporter (system xc-), a membrane antiporter that imports cystine and releases glutamate. Despite this, the system xc - activity and its connection to the inflammatory reaction in multiple sclerosis (MS) is largely unknown. Longitudinal in vivo magnetic resonance (MRI) and positron emission tomography (PET) imaging studies with 2-[ 18 F]Fluoro-2-deoxy-D-glucose ([ 18 F]FDG), [ 11 C]-(R)-1-(2-chlorophenyl)-N-methyl-N-1(1-methylpropyl) -3-isoquinolinecarbox amide ([ 11 C]PK11195) and (4S)-4-(3- 18 F-fluoropropyl)-L-glutamate ([ 18 F]FSPG) were carried out during the course of experimental autoimmune encephalomyelitis (EAE) induction in rats. [ 18 F]FSPG showed a significant increase of system xc - function in the lumbar section of the spinal cord at 14 days post immunization (dpi) that stands in agreement with the neurological symptoms and ventricle edema formation at this time point. Likewise, [ 18 F]FDG did not show significant changes in glucose metabolism throughout central nervous system and [ 11 C]PK11195 evidenced a significant increase of microglial/macrophage activation in spinal cord and cerebellum 2 weeks after EAE induction. Therefore, [ 18 F]FSPG showed a major capacity to discriminate regions of the central nervous system affected by the MS in comparison to [ 18 F]FDG and [ 11 C]PK11195. Additionally, clodronate-treated rats showed a depletion in microglial population and [ 18 F]FSPG PET signal in spinal cord confirming a link between neuroinflammatory reaction and cystine/glutamate antiporter activity in EAE rats. Altogether, these results suggest that in vivo PET imaging of system xc - could become a valuable tool for the diagnosis and treatment evaluation of MS. (orig.)

  17. Inactivation of the glutamine/amino acid transporter ASCT2 by 1,2,3-dithiazoles: proteoliposomes as a tool to gain insights in the molecular mechanism of action and of antitumor activity

    Energy Technology Data Exchange (ETDEWEB)

    Oppedisano, Francesca [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy); Catto, Marco [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Koutentis, Panayiotis A. [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Nicolotti, Orazio [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Pochini, Lorena [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy); Koyioni, Maria [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Introcaso, Antonellina [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Michaelidou, Sophia S. [Department of Chemistry, University of Cyprus, P.O. Box 20537, 1678 Nicosia (Cyprus); Carotti, Angelo, E-mail: carotti@farmchim.uniba.it [Dipartimento Farmaco-Chimico, Università degli Studi “Aldo Moro,”, via Orabona 4, 70125 Bari (Italy); Indiveri, Cesare, E-mail: indiveri@unical.it [Dipartimento di Biologia Cellulare Università della Calabria, via P. Bucci 4 c, 87036 Arcavacata di Rende (CS) (Italy)

    2012-11-15

    The ASCT2 transport system catalyses a sodium-dependent antiport of glutamine and other neutral amino acids which is involved in amino acid metabolism. A library of 1,2,3-dithiazoles was designed, synthesized and evaluated as inhibitors of the glutamine/amino acid ASCT2 transporter in the model system of proteoliposomes reconstituted with the rat liver transporter. Fifteen of the tested compounds at concentration of 20 μM or below, inhibited more than 50% the glutamine/glutamine antiport catalysed by the reconstituted transporter. These good inhibitors bear a phenyl ring with electron withdrawing substituents. The inhibition was reversed by 1,4-dithioerythritol indicating that the effect was likely owed to the formation of mixed sulfides with the protein's Cys residue(s). A dose–response analysis of the most active compounds gave IC{sub 50} values in the range of 3–30 μM. Kinetic inhibition studies indicated a non-competitive inhibition, presumably because of a potential covalent interaction of the dithiazoles with cysteine thiol groups that are not located at the substrate binding site. Indeed, computational studies using a homology structural model of ASCT2 transporter, suggested as possible binding targets, Cys-207 or Cys-210, that belong to the CXXC motif of the protein. -- Highlights: ► Non‐competitive inhibition of ASCT2 by 1,2,3-dithiazoles was studied in proteoliposomes. ► Different 1,2,3-dithiazoles were synthesized and evaluated as transporter inhibitors. ► Many compounds potently inhibited the glutamine/glutamine antiport catalyzed by ASCT2. ► The inhibition was reversed by DTE indicating reaction with protein Cys. ► The most active compounds gave IC{sub 50} in the range of 3–30 μM.

  18. Structural and functional aspects of the multidrug efflux pump AcrB.

    Science.gov (United States)

    Eicher, Thomas; Brandstätter, Lorenz; Pos, Klaas M

    2009-08-01

    The tripartite efflux system AcrA/AcrB/TolC is the main pump in Escherichia coli for the efflux of multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB is central to substrate recognition and energy transduction and acts as a proton/drug antiporter. Recent structural studies show that homotrimeric AcrB can adopt different monomer conformations representing consecutive states in an allosteric functional rotation transport cycle. The conformational changes create an alternate access drug transport tunnel including a hydrophobic substrate binding pocket in one of the cycle intermediates.

  19. One-Pot Synthesis and Transmembrane Chloride Transport Properties of C3-Symmetric Benzoxazine Urea.

    Science.gov (United States)

    Roy, Arundhati; Saha, Debasis; Mukherjee, Arnab; Talukdar, Pinaki

    2016-11-18

    One-pot synthesis of a C 3 -symmetric benzoxazine-based tris-urea compound is discussed. 1 H NMR titrations indicate a stronger Cl - binding compared that of Br - and I - by the receptor. Effective Cl - transport across liposomal membranes via a Cl - /X - antiport mechanism is confirmed. Theoretical calculation suggests that a few water molecules with N-H, C═O, and the aromatic ring of the receptor create a H-bonded polar cavity where a Cl - is recognized by O-H···Cl - interactions from five bridged water molecules.

  20. The anionic basis of fluid secretion by the rabbit mandibular salivary gland

    DEFF Research Database (Denmark)

    Case, R M; Hunter, M; Novak, I

    1984-01-01

    . In control glands (i.e. perfused with both HCO3- and Cl-), administration of furosemide, an inhibitor of Na+/Cl- co-transport, reduced the secretion rate and increased salivary HCO3- in a manner indistinguishable from that seen when perfusate Cl- was replaced with ise -. In control perfused glands...... on two independent transport systems. One is a Cl- -dependent, furosemide-sensitive system, probably a Na+/Cl- symport. The other is an HCO3- -dependent, methazolamide-sensitive system, and is probably an Na+/H+ antiport....

  1. SpAHA1 and SpSOS1 Coordinate in Transgenic Yeast to Improve Salt Tolerance.

    Directory of Open Access Journals (Sweden)

    Yang Zhou

    Full Text Available In plant cells, the plasma membrane Na+/H+ antiporter SOS1 (salt overly sensitive 1 mediates Na+ extrusion using the proton gradient generated by plasma membrane H+-ATPases, and these two proteins are key plant halotolerance factors. In the present study, two genes from Sesuvium portulacastrum, encoding plasma membrane Na+/H+ antiporter (SpSOS1 and H+-ATPase (SpAHA1, were cloned. Localization of each protein was studied in tobacco cells, and their functions were analyzed in yeast cells. Both SpSOS1 and SpAHA1 are plasma membrane-bound proteins. Real-time polymerase chain reaction (PCR analyses showed that SpSOS1 and SpAHA1 were induced by salinity, and their expression patterns in roots under salinity were similar. Compared with untransformed yeast cells, SpSOS1 increased the salt tolerance of transgenic yeast by decreasing the Na+ content. The Na+/H+ exchange activity at plasma membrane vesicles was higher in SpSOS1-transgenic yeast than in the untransformed strain. No change was observed in the salt tolerance of yeast cells expressing SpAHA1 alone; however, in yeast transformed with both SpSOS1 and SpAHA1, SpAHA1 generated an increased proton gradient that stimulated the Na+/H+ exchange activity of SpSOS1. In this scenario, more Na+ ions were transported out of cells, and the yeast cells co-expressing SpSOS1 and SpAHA1 grew better than the cells transformed with only SpSOS1 or SpAHA1. These findings demonstrate that the plasma membrane Na+/H+ antiporter SpSOS1 and H+-ATPase SpAHA1 can function in coordination. These results provide a reference for developing more salt-tolerant crops via co-transformation with the plasma membrane Na+/H+ antiporter and H+-ATPase.

  2. A quantitative assay for lysosomal acidification rates in human osteoclasts

    DEFF Research Database (Denmark)

    Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld

    2011-01-01

    lacunae. The electroneutrality of the lacunae is maintained by chloride transport through the chloride-proton antiporter chloride channel 7. Inhibition of either proton or chloride transport prevents bone resorption. The aims of this study were to validate the human osteoclastic microsome- based influx......M valinomycin increased the acid influx by 129%. Total abrogation of acid influx was observed using both H(+) and Cl(-) ionophores. Finally, investigation of the anion profile demonstrated that Cl(-) and Br(-) are the preferred anions for the transporter. In conclusion, the acid influx assay based on microsomes...

  3. Propionate induces cell swelling and K+ accumulation in shark rectal gland

    Energy Technology Data Exchange (ETDEWEB)

    Feldman, G.M.; Ziyadeh, F.N.; Mills, J.W.; Booz, G.W.; Kleinzeller, A. (Mount Desert Island Biological Laboratory, Salsbury Cove, ME (USA))

    1989-08-01

    Small organic anions have been reported to induce cell solute accumulation and swelling. To investigate the mechanism of swelling, we utilized preparations of rectal gland cells from Squalus acanthias incubated in medium containing propionate. Propionate causes cells to swell by diffusing across membranes in its nonionic form, acidifying cell contents, and activating the Na+-H+ antiporter. The Na+-H+ exchange process tends to correct intracellular pH (pHi), and thus it maintains a favorable gradient for propionic acid diffusion and allows propionate to accumulate. Activation of the Na+-H+ antiport also facilitates Na+ entry into the cell and Nai accumulation. At the same time Na+-K+-ATPase activity, unaffected by propionate, replaces Nai with Ki, whereas the K+ leak rate, decreased by propionate, allows Ki to accumulate. As judged by {sup 86}Rb+ efflux, the reduction in K+ leak was not due to propionate-induced cell acidification or reduction in Cli concentration. Despite inducing cell swelling, propionate did not disrupt cell structural elements and F actin distribution along cell membranes.

  4. The yeast CLC protein counteracts vesicular acidification during iron starvation.

    Science.gov (United States)

    Braun, Nikolai A; Morgan, Bruce; Dick, Tobias P; Schwappach, Blanche

    2010-07-01

    Ion gradients across intracellular membranes contribute to the physicochemical environment inside compartments. CLC anion transport proteins that localise to intracellular organelles are anion-proton exchangers involved in anion sequestration or vesicular acidification. By homology, the only CLC protein of Saccharomyces cerevisiae, Gef1, belongs to this family of intracellular exchangers. Gef1 localises to the late Golgi and prevacuole and is essential in conditions of iron limitation. In the absence of Gef1, a multicopper oxidase involved in iron uptake, Fet3, fails to acquire copper ion cofactors. The precise role of the exchanger in this physiological context is unknown. Here, we show that the Gef1-containing compartment is adjusted to a more alkaline pH under iron limitation. This depends on the antiport function of Gef1, because an uncoupled mutant of Gef1 (E230A) results in the acidification of the lumen and fails to support Fet3 maturation. Furthermore, we found that Gef1 antiport activity correlates with marked effects on cellular glutathione homeostasis, raising the possibility that the effect of Gef1 on Fet3 copper loading is related to the control of compartmental glutathione concentration or redox status. Mutational inactivation of a conserved ATP-binding site in the cytosolic cystathione beta-synthetase domain of Gef1 (D732A) suggests that Gef1 activity is regulated by energy metabolism.

  5. CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gamma -butyrobetaine exchange.

    Science.gov (United States)

    Jung, Heinrich; Buchholz, Marion; Clausen, Jurgen; Nietschke, Monika; Revermann, Anne; Schmid, Roland; Jung, Kirsten

    2002-10-18

    l-Carnitine is essential for beta-oxidation of fatty acids in mitochondria. Bacterial metabolic pathways are used for the production of this medically important compound. Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose function is required for l-carnitine conversion in Escherichia coli. The caiT gene was overexpressed in E. coli, and the gene product was purified by affinity chromatography and reconstituted into proteoliposomes. Functional analyses with intact cells and proteoliposomes demonstrated that CaiT is able to catalyze the exchange of l-carnitine for gamma-butyrobetaine, the excreted end product of l-carnitine conversion in E. coli, and related betaines. Electrochemical ion gradients did not significantly stimulate l-carnitine uptake. Analysis of l-carnitine counterflow yielded an apparent external K(m) of 105 microm and a turnover number of 5.5 s(-1). Contrary to related proteins, CaiT activity was not modulated by osmotic stress. l-Carnitine binding to CaiT increased the protein fluorescence and caused a red shift in the emission maximum, an observation explained by ligand-induced conformational alterations. The fluorescence effect was specific for betaine structures, for which the distance between trimethylammonium and carboxyl groups proved to be crucial for affinity. Taken together, the results suggest that CaiT functions as an exchanger (antiporter) for l-carnitine and gamma-butyrobetaine according to the substrate/product antiport principle.

  6. Functional expression of a human GDP-L-fucose transporter in Escherichia coli.

    Science.gov (United States)

    Förster-Fromme, Karin; Schneider, Sarah; Sprenger, Georg A; Albermann, Christoph

    2017-02-01

    To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes. The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-L-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of 3 H-GDP-L-fucose with a V max of 8 pmol/min mg with a K m of 4 µM. The functional expression of SLC35C1 in GDP-L-fucose overproducing E. coli led to the export of GDP-L-fucose to the culture supernatant. The export of GDP-L-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

  7. Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis[W][OA

    Science.gov (United States)

    Barragán, Verónica; Leidi, Eduardo O.; Andrés, Zaida; Rubio, Lourdes; De Luca, Anna; Fernández, José A.; Cubero, Beatriz; Pardo, José M.

    2012-01-01

    Intracellular NHX proteins are Na+,K+/H+ antiporters involved in K+ homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na+,K+/H+ antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K+. By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na+ sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K+ pool, which in turn provoked greater K+ retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K+ uptake at the tonoplast, for turgor regulation, and for stomatal function. PMID:22438021

  8. Intracellular pH in increased after transformation of Chinese hamster embryo fibroblasts

    International Nuclear Information System (INIS)

    Ober, S.S.; Pardee, A.B.

    1987-01-01

    These studies reveal that a series of tumorigenic Chinese hamster embryo fibroblast (CHEF) cell lines maintain an internal pH (pH/sub i/) that is 0.12 +/- 0.04 pH unit above that of the nontumorigenic CHEF/18 parental line. pH measurements were made with [ 14 C]-benzoic acid. This increase of pH/sub i/ in the tumorigenic CHEF cells is not due to autocrine growth factor production or to the persistent activation of pathways previously shown to modulate Na + /H + -antiporter activity present in the CHEF/18 line. These findings suggest that the defect in pH/sub i/ regulation in the tumorigenic CHEF/18 derivatives lies in the Na + /H + antiporter itself. Further studies to determine the biological significance of an increased pH/sub i/ show that the external pH (pH 0 )-dependence curve for initiation of DNA synthesis in the tumorigenic CHEF lines is shifted by approximately 0.2 pH unit toward acidic values relative to that of the nontumorigenic CHEF/18 parent. These data show a critical role for pH/sub i/ in the regulation of DNA synthesis in Chinese hamster embryo fibroblasts and demonstrate that aberrations in pH/sub i/ can contribute to the acquisition of altered growth properties

  9. Propionate induces cell swelling and K+ accumulation in shark rectal gland

    International Nuclear Information System (INIS)

    Feldman, G.M.; Ziyadeh, F.N.; Mills, J.W.; Booz, G.W.; Kleinzeller, A.

    1989-01-01

    Small organic anions have been reported to induce cell solute accumulation and swelling. To investigate the mechanism of swelling, we utilized preparations of rectal gland cells from Squalus acanthias incubated in medium containing propionate. Propionate causes cells to swell by diffusing across membranes in its nonionic form, acidifying cell contents, and activating the Na+-H+ antiporter. The Na+-H+ exchange process tends to correct intracellular pH (pHi), and thus it maintains a favorable gradient for propionic acid diffusion and allows propionate to accumulate. Activation of the Na+-H+ antiport also facilitates Na+ entry into the cell and Nai accumulation. At the same time Na+-K+-ATPase activity, unaffected by propionate, replaces Nai with Ki, whereas the K+ leak rate, decreased by propionate, allows Ki to accumulate. As judged by 86 Rb+ efflux, the reduction in K+ leak was not due to propionate-induced cell acidification or reduction in Cli concentration. Despite inducing cell swelling, propionate did not disrupt cell structural elements and F actin distribution along cell membranes

  10. Cell potentials, cell resistance, and proton fluxes in corn root tissue. Effects of dithioerythritol

    Energy Technology Data Exchange (ETDEWEB)

    Lin, W.; Hanson, J.B.

    1976-09-01

    Studies were made of the effect of dithioerythritol on net proton flux, potassium influx and efflux, cell potential, and cell resistance in fresh and washed corn (Zea mays L. WF9XM14) root tissue. Dithioerythritol induces equal proton influx and potassium efflux rates, decreases membrane resistance, and hyperpolarizes the cell potential. Greater effects on H/sup +/ and K/sup +/ fluxes are secured at pH 7 than at pH 5. Other sulfhydryl-protecting reagents produced the same responses. No evidence could be found that dithioerythritol affected energy metabolism or membrane ATPase, and proton influx was induced in the presence of uncoupling agents. We deduce that dithioerythritol activates a passive H/sup +//K/sup +/ antiport, driven in these experiments by the outwardly directed electrochemical gradient of K/sup +/. The net effect on H/sup +/ and K/sup +/ fluxes is believed to reside with the combined activity of a polarized H/sup +//K/sup +/ exchanging ATPase and the passive H/sup +//K/sup +/ antiport. A model is presented to show how the combined system might produce stable potential differences and K/sup +/ content.

  11. Energy conservation by oxidation of formate to carbon dioxide and hydrogen via a sodium ion current in a hyperthermophilic archaeon.

    Science.gov (United States)

    Lim, Jae Kyu; Mayer, Florian; Kang, Sung Gyun; Müller, Volker

    2014-08-05

    Thermococcus onnurineus NA1 is known to grow by the anaerobic oxidation of formate to CO2 and H2, a reaction that operates near thermodynamic equilibrium. Here we demonstrate that this reaction is coupled to ATP synthesis by a transmembrane ion current. Formate oxidation leads to H(+) translocation across the cytoplasmic membrane that then drives Na(+) translocation. The ion-translocating electron transfer system is rather simple, consisting of only a formate dehydrogenase module, a membrane-bound hydrogenase module, and a multisubunit Na(+)/H(+) antiporter module. The electrochemical Na(+) gradient established then drives ATP synthesis. These data give a mechanistic explanation for chemiosmotic energy conservation coupled to formate oxidation to CO2 and H2. Because it is discussed that the membrane-bound hydrogenase with the Na(+)/H(+) antiporter module are ancestors of complex I of mitochondrial and bacterial electron transport these data also shed light on the evolution of ion transport in complex I-like electron transport chains.

  12. In vivo imaging of system xc- as a novel approach to monitor multiple sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Abraham; Szczupak, Boguslaw; Arrieta, Ander [CIC biomaGUNE, Molecular Imaging Unit, San Sebastian (Spain); Vazquez-Villoldo, Nuria; Soria, Federico N.; Domercq, Maria; Matute, Carlos [University of the Basque Country, Department of Neurosciences, Leioa (Spain); UPV/EHU, Achucarro Basque Center for Neuroscience, Zamudio (Spain); Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Leioa (Spain); Gomez-Vallejo, Vanessa; Llop, Jordi [CIC biomaGUNE, Molecular Imaging Unit, San Sebastian (Spain); CIC biomaGUNE, Radiochemistry and Nuclear Imaging, San Sebastian (Spain); Padro, Daniel; Plaza-Garcia, Sandra; Reese, Torsten [CIC biomaGUNE, Molecular Imaging Unit, San Sebastian (Spain); CIC biomaGUNE, Magnetic Resonance Imaging, San Sebastian (Spain)

    2016-06-15

    Glutamate excitotoxicity contributes to oligodendroglial and axonal damage in multiple sclerosis pathology. Extracellular glutamate concentration in the brain is controlled by cystine/glutamate antiporter (system xc-), a membrane antiporter that imports cystine and releases glutamate. Despite this, the system xc{sup -} activity and its connection to the inflammatory reaction in multiple sclerosis (MS) is largely unknown. Longitudinal in vivo magnetic resonance (MRI) and positron emission tomography (PET) imaging studies with 2-[{sup 18}F]Fluoro-2-deoxy-D-glucose ([{sup 18}F]FDG), [{sup 11}C]-(R)-1-(2-chlorophenyl)-N-methyl-N-1(1-methylpropyl) -3-isoquinolinecarbox amide ([{sup 11}C]PK11195) and (4S)-4-(3-{sup 18}F-fluoropropyl)-L-glutamate ([{sup 18}F]FSPG) were carried out during the course of experimental autoimmune encephalomyelitis (EAE) induction in rats. [{sup 18}F]FSPG showed a significant increase of system xc{sup -} function in the lumbar section of the spinal cord at 14 days post immunization (dpi) that stands in agreement with the neurological symptoms and ventricle edema formation at this time point. Likewise, [{sup 18}F]FDG did not show significant changes in glucose metabolism throughout central nervous system and [{sup 11}C]PK11195 evidenced a significant increase of microglial/macrophage activation in spinal cord and cerebellum 2 weeks after EAE induction. Therefore, [{sup 18}F]FSPG showed a major capacity to discriminate regions of the central nervous system affected by the MS in comparison to [{sup 18}F]FDG and [{sup 11}C]PK11195. Additionally, clodronate-treated rats showed a depletion in microglial population and [{sup 18}F]FSPG PET signal in spinal cord confirming a link between neuroinflammatory reaction and cystine/glutamate antiporter activity in EAE rats. Altogether, these results suggest that in vivo PET imaging of system xc{sup -} could become a valuable tool for the diagnosis and treatment evaluation of MS. (orig.)

  13. Mitochondrial complex I deactivation is related to superoxide production in acute hypoxia

    Directory of Open Access Journals (Sweden)

    Pablo Hernansanz-Agustín

    2017-08-01

    Full Text Available Mitochondria use oxygen as the final acceptor of the respiratory chain, but its incomplete reduction can also produce reactive oxygen species (ROS, especially superoxide. Acute hypoxia produces a superoxide burst in different cell types, but the triggering mechanism is still unknown. Herein, we show that complex I is involved in this superoxide burst under acute hypoxia in endothelial cells. We have also studied the possible mechanisms by which complex I could be involved in this burst, discarding reverse electron transport in complex I and the implication of PTEN-induced putative kinase 1 (PINK1. We show that complex I transition from the active to ‘deactive’ form is enhanced by acute hypoxia in endothelial cells and brain tissue, and we suggest that it can trigger ROS production through its Na+/H+ antiporter activity. These results highlight the role of complex I as a key actor in redox signalling in acute hypoxia.

  14. A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization

    DEFF Research Database (Denmark)

    Schiøtt, Morten; Romanowsky, Shawn M; Bækgaard, Lone

    2004-01-01

    Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome...... encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor......-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential...

  15. Structural basis for the counter-transport mechanism of a H+/Ca2+ exchanger.

    Science.gov (United States)

    Nishizawa, Tomohiro; Kita, Satomi; Maturana, Andrés D; Furuya, Noritaka; Hirata, Kunio; Kasuya, Go; Ogasawara, Satoshi; Dohmae, Naoshi; Iwamoto, Takahiro; Ishitani, Ryuichiro; Nureki, Osamu

    2013-07-12

    Ca(2+)/cation antiporters catalyze the exchange of Ca(2+) with various cations across biological membranes to regulate cytosolic calcium levels. The recently reported structure of a prokaryotic Na(+)/Ca(2+) exchanger (NCX_Mj) revealed its overall architecture in an outward-facing state. Here, we report the crystal structure of a H(+)/Ca(2+) exchanger from Archaeoglobus fulgidus (CAX_Af) in the two representatives of the inward-facing conformation at 2.3 Å resolution. The structures suggested Ca(2+) or H(+) binds to the cation-binding site mutually exclusively. Structural comparison of CAX_Af with NCX_Mj revealed that the first and sixth transmembrane helices alternately create hydrophilic cavities on the intra- and extracellular sides. The structures and functional analyses provide insight into the mechanism of how the inward- to outward-facing state transition is triggered by the Ca(2+) and H(+) binding.

  16. pH-Gated Chloride Transport by a Triazine-Based Tripodal Semicage.

    Science.gov (United States)

    Roy, Arundhati; Saha, Debasis; Mandal, Prashant Sahebrao; Mukherjee, Arnab; Talukdar, Pinaki

    2017-01-26

    Triazine-based preorganized tripodal receptors are reported as efficient transmembrane Cl - carriers. These receptors were designed based on triazine core and 3,7-diazabicyclo[3.3.1]nonane arms to facilitate preorganized cavity formation. Each bicyclic arm was further functionalized to control protonation and lipophilicity, which are crucial for their efficient anion binding and effective transport through liposomal membranes. The benzyl-substituted receptor was the most effective ion transporter followed by the pentafluorobenzyl-substituted derivative. The nonsubstituted receptor was least active owing to its high polarity. Two active transporters were found to function as mobile carriers for Cl - via an antiport exchange mechanism. Molecular dynamic simulations with the most active receptor show a strong Cl - binding within the cavity by direct and water-mediated H-bonds with its N-H groups. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Ion transporters involved in acidification of the resorption lacuna in osteoclasts

    DEFF Research Database (Denmark)

    Henriksen, K.; Sorensen, M.G.; Jensen, V.K.

    2008-01-01

    Osteoclasts possess a large amount of ion transporters, which participate in bone resorption; of these, the vacuolar-adenosine trisphosphatase (V-ATPase) and the chloride-proton antiporter ClC-7 acidify the resorption lacuna. However, whether other ion transporters participate in this process is ......, including carbonic anhydrase II, the NHEs, and potassium-chloride cotransporters, are all involved in resorption but do not seem to directly be involved in acidification of the lysosomes Udgivelsesdato: 2008/9...... experiments, or (4) lysed in trizol for mRNA isolation for Affymetrix array analysis. Inhibitors targeted toward most of the ion transporters showed low potency in the acidification-based assays, although some inhibitors, such as carbonic anhydrase II and the sodium-hydrogen exchanger (NHE) inhibitors...

  18. Structure and operation of bacterial tripartite pumps.

    Science.gov (United States)

    Hinchliffe, Philip; Symmons, Martyn F; Hughes, Colin; Koronakis, Vassilis

    2013-01-01

    In bacteria such as Pseudomonas aeruginosa and Escherichia coli, tripartite membrane machineries, or pumps, determine the efflux of small noxious molecules, such as detergents, heavy metals, and antibiotics, and the export of large proteins including toxins. They are therefore influential in bacterial survival, particularly during infections caused by multidrug-resistant pathogens. In these tripartite pumps an inner membrane transporter, typically an ATPase or proton antiporter, binds and translocates export or efflux substrates. In cooperation with a periplasmic adaptor protein it recruits and opens a TolC family cell exit duct, which is anchored in the outer membrane and projects across the periplasmic space between inner and outer membranes. Assembled tripartite pumps thus span the entire bacterial cell envelope. We review the atomic structures of each of the three pump components and discuss how these have allowed high-resolution views of tripartite pump assembly, operation, and possible inhibition.

  19. Na(+)/K(+) pump interacts with the h-current to control bursting activity in central pattern generator neurons of leeches.

    Science.gov (United States)

    Kueh, Daniel; Barnett, William H; Cymbalyuk, Gennady S; Calabrese, Ronald L

    2016-09-02

    The dynamics of different ionic currents shape the bursting activity of neurons and networks that control motor output. Despite being ubiquitous in all animal cells, the contribution of the Na(+)/K(+) pump current to such bursting activity has not been well studied. We used monensin, a Na(+)/H(+) antiporter, to examine the role of the pump on the bursting activity of oscillator heart interneurons in leeches. When we stimulated the pump with monensin, the period of these neurons decreased significantly, an effect that was prevented or reversed when the h-current was blocked by Cs(+). The decreased period could also occur if the pump was inhibited with strophanthidin or K(+)-free saline. Our monensin results were reproduced in model, which explains the pump's contributions to bursting activity based on Na(+) dynamics. Our results indicate that a dynamically oscillating pump current that interacts with the h-current can regulate the bursting activity of neurons and networks.

  20. Influence of bicarbonate on the sensitivity of renin release to sodium chloride

    DEFF Research Database (Denmark)

    Skøtt, O; Jensen, B L

    1989-01-01

    glomeruli treated with bicarbonate/chloride exchange inhibitor (DNDS), NaCl/KCl cotransport inhibitor (bumetanide), or Na+/H+ antiport inhibitor (amiloride) in the presence or absence of bicarbonate. In addition, the sensitivity to increases in osmolality by addition of sucrose was tested in the presence...... or absence of bicarbonate. Renin release from time controls superfused with a bicarbonate-free Ringer was identical to release from glomeruli superfused with a bicarbonate Ringer. DNDS (0.11 or 1.1 mM) had no effect on renin release in a bicarbonate Ringer. 30 mM sucrose inhibited renin release independently...... of bicarbonate. 15 mM NaCl stimulated renin release when bicarbonate was absent, while it caused an inhibition in the presence of bicarbonate. When bicarbonate/chloride exchange was inhibited, addition of NaCl stimulated renin release even when bicarbonate was present. The effect of NaCl on renin release...

  1. Consequences of SOS1 deficiency: Intracellular physiology and transcription

    KAUST Repository

    Ha, OhDong

    2010-06-01

    As much as there is known about the function of the sodium/proton antiporter SOS1 in plants, recent studies point towards a more general role for this protein. The crucial involvement in salt stress protection is clearly one of its functions –confined to the N-terminus, but the modular structure of the protein includes a segment with several domains that are functionally not studied but comprise more than half of the protein’s length. Additional functions of the protein appear to be an influence on vesicle trafficking, vacuolar pH and general ion homeostasis during salt stress. Eliminating SOS1 leads to the expression of genes that are not strictly salinity stress related. Functions that are regulated in sos1 mutants included pathogen responses, and effects on circadian rhythm.

  2. The effect of lithium chloride on the biooxidation of aqueous methanol/acetone mixtures.

    Science.gov (United States)

    O'Brien, M; Hamer, G

    2001-08-01

    Lithium chloride, more specifically the lithium cation, has been implicated in interference in biological systems. In the case of Escherichia coli, interference involves the Na+(Li+)/H+ antiporter transport system. The study reported here concerns the effects of LiCl on a mixed enrichment culture that is able to biodegrade both methanol and acetone under aerobic conditions. The results obtained using unsteady state continuous flow culture techniques demonstrate a significant disruptive effect of LiCl on culture performance. In addition, a reduction in the substrate-based biomass yield coefficient, which is a clear advantage as far as biotreatment process performance is concerned, also occurs. The ultimate fate of the LiCl was not determined.

  3. Partial purification of the ATP-driven calcium pump of Streptococcus sanguis

    International Nuclear Information System (INIS)

    Lynn, A.R.; Rosen, B.P.

    1986-01-01

    ATP-dependent transport of calcium has been observed in several species of streptococci as uptake of 45 Ca 2+ into everted membrane vesicles. Membranes from Streptococcus sanguis and Streptococcus faecalis were solubilized with octyl-β-D-glucoside or Triton X-100, and the extracts reconstituted into proteoliposomes containing Escherichia coli or soybean phospholipid. Calcium transport in reconstituted proteoliposomes was insensitive to the ionophores nigericin and valinomycin and was unaffected by the F 0 F 1 inhibitor N,N'-dicyclohexylcarbodiimide. Uptake was inhibited by ortho-vanadate with a K/sub i/ in the micromolar range. These results demonstrate that the reconstituted transport activities are not the result of ATP-driven proton pumping via the F 0 F 1 coupled to a calcium/proton antiporter and suggest that existence of a calcium translocating ATPase. Partial purification of the transport activity from Streptococcus sanguis has been achieved using density gradient centrifugation and FPLC

  4. Variation in tissue Na(+) content and the activity of SOS1 genes among two species and two related genera of Chrysanthemum.

    Science.gov (United States)

    Gao, Jiaojiao; Sun, Jing; Cao, Peipei; Ren, Liping; Liu, Chen; Chen, Sumei; Chen, Fadi; Jiang, Jiafu

    2016-04-21

    Chrysanthemum, a leading ornamental species, does not tolerate salinity stress, although some of its related species do. The current level of understanding regarding the mechanisms underlying salinity tolerance in this botanical group is still limited. A comparison of the physiological responses to salinity stress was made between Chrysanthemum morifolium 'Jinba' and its more tolerant relatives Crossostephium chinense, Artemisia japonica and Chrysanthemum crassum. The stress induced a higher accumulation of Na(+) and more reduction of K(+) in C. morifolium than in C. chinense, C. crassum and A. japonica, which also showed higher K(+)/Na(+) ratio. Homologs of an Na(+)/H(+) antiporter (SOS1) were isolated from each species. The gene carried by the tolerant plants were more strongly induced by salt stress than those carried by the non-tolerant ones. When expressed heterologously, they also conferred a greater degree of tolerance to a yeast mutant lacking Na(+)-pumping ATPase and plasma membrane Na(+)/H(+) antiporter activity. The data suggested that the products of AjSOS1, CrcSOS1 and CcSOS1 functioned more effectively as Na (+) excluders than those of CmSOS1. Over expression of four SOS1s improves the salinity tolerance of transgenic plants and the overexpressing plants of SOS1s from salt tolerant plants were more tolerant than that from salt sensitive plants. In addition, the importance of certain AjSOS1 residues for effective ion transport activity and salinity tolerance was established by site-directed mutagenesis and heterologous expression in yeast. AjSOS1, CrcSOS1 and CcSOS1 have potential as transgenes for enhancing salinity tolerance. Some of the mutations identified here may offer opportunities to better understand the mechanistic basis of salinity tolerance in the chrysanthemum complex.

  5. Overexpression of PeHA1 enhances hydrogen peroxide signaling in salt-stressed Arabidopsis.

    Science.gov (United States)

    Wang, Meijuan; Wang, Yang; Sun, Jian; Ding, Mingquan; Deng, Shurong; Hou, Peichen; Ma, Xujun; Zhang, Yuhong; Wang, Feifei; Sa, Gang; Tan, Yeqing; Lang, Tao; Li, Jinke; Shen, Xin; Chen, Shaoliang

    2013-10-01

    The plant plasma membrane (PM) H(+)-ATPase plays a crucial role in controlling K(+)/Na(+) homeostasis under salt stress. Our previous microarray analysis indicated that Populus euphratica retained a higher abundance of PM H(+)-ATPase transcript versus a salt-sensitive poplar. To clarify the roles of the PM H(+)-ATPase in salt sensing and adaptation, we isolated the PM H(+)-ATPase gene PeHA1 from P. euphratica and introduced it into Arabidopsis thaliana. Compared to wild-type, PeHA1-transgenic Arabidopsis had a greater germination rate, root length, and biomass under NaCl stress (50-150 mM). Ectopic expression of PeHA1 remarkably enhanced the capacity to control the homeostasis of ions and reactive oxygen species in salinized Arabidopsis. Flux data from salinized roots showed that transgenic plants exhibited a more pronounced Na(+)/H(+) antiport and less reduction of K(+) influx versus wild-type. Enhanced PM ATP hydrolytic activity, proton pumping, and Na(+)/H(+) antiport in PeHA1-transgenic plants, were consistent to those observed in vivo, i.e., H(+) extrusion, external acidification, and Na(+) efflux. Activities of the antioxidant enzymes ascorbate peroxidase and catalase were typically higher in transgenic seedlings irrespective of salt concentration. In transgenic Arabidopsis roots, H2O2 production was higher under control conditions and increased more rapidly than wild-type when plants were subjected to NaCl treatment. Interestingly, transgenic plants were unable to control K(+)/Na(+) homeostasis when salt-induced H2O2 production was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase. These observations suggest that PeHA1 accelerates salt tolerance partially through rapid H2O2 production upon salt treatment, which triggers adjustments in K(+)/Na(+) homeostasis and antioxidant defense in Arabidopsis. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  6. Decreased capacity for sodium export out of Arabidopsis chloroplasts impairs salt tolerance, photosynthesis and plant performance.

    Science.gov (United States)

    Müller, Maria; Kunz, Hans-Henning; Schroeder, Julian I; Kemp, Grant; Young, Howard S; Neuhaus, H Ekkehard

    2014-05-01

    Salt stress is a widespread phenomenon, limiting plant performance in large areas around the world. Although various types of plant sodium/proton antiporters have been characterized, the physiological function of NHD1 from Arabidopsis thaliana has not been elucidated in detail so far. Here we report that the NHD1-GFP fusion protein localizes to the chloroplast envelope. Heterologous expression of AtNHD1 was sufficient to complement a salt-sensitive Escherichia coli mutant lacking its endogenous sodium/proton exchangers. Transport competence of NHD1 was confirmed using recombinant, highly purified carrier protein reconstituted into proteoliposomes, proving Na(+) /H(+) antiport. In planta NHD1 expression was found to be highest in mature and senescent leaves but was not induced by sodium chloride application. When compared to wild-type controls, nhd1 T-DNA insertion mutants showed decreased biomasses and lower chlorophyll levels after sodium feeding. Interestingly, if grown on sand and supplemented with high sodium chloride, nhd1 mutants exhibited leaf tissue Na(+) levels similar to those of wild-type plants, but the Na(+) content of chloroplasts increased significantly. These high sodium levels in mutant chloroplasts resulted in markedly impaired photosynthetic performance as revealed by a lower quantum yield of photosystem II and increased non-photochemical quenching. Moreover, high Na(+) levels might hamper activity of the plastidic bile acid/sodium symporter family protein 2 (BASS2). The resulting pyruvate deficiency might cause the observed decreased phenylalanine levels in the nhd1 mutants due to lack of precursors. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  7. The role of protons in fast and slow gating of the Torpedo chloride channel ClC-0.

    Science.gov (United States)

    Zifarelli, Giovanni; Pusch, Michael

    2010-05-01

    Transmembrane proton transport is of fundamental importance for life. The list of H(+) transporting proteins has been recently expanded with the discovery that some members of the CLC gene family are stoichiometrically coupled Cl(-)/H(+) antiporters. Other CLC proteins are instead passive Cl(-) selective anion channels. The gating of these CLC channels is, however, strongly regulated by pH, likely reflecting the evolutionary relationship with CLC Cl(-)/H(+) antiporters. The role of protons in the gating of the model Torpedo channel ClC-0 is best understood. ClC-0 is a homodimer with separate pores in each subunit. Each protopore can be opened and closed independently from the other pore by a "fast gate". A common, slow gate acts on both pores simultaneously. The opening of the fast gate is controlled by a critical glutamate (E166), whose protonation state determines the fast gate's pH dependence. Extracellular protons likely can arrive directly at E166. In contrast, protonation of E166 from the inside has been proposed to be mediated by the dissociation of an intrapore water molecule. The OH(-) anion resulting from the water dissociation is stabilized in one of the anion binding sites of the channel, competing with intracellular Cl(-) ions. The pH dependence of the slow gate is less well understood. It has been shown that proton translocation drives irreversible gating transitions associated with the slow gate. However, the relationship of the fast gate's pH dependence on the proton translocation and the molecular basis of the slow gate remain to be discovered.

  8. Carrier-mediated cocaine transport at the blood-brain barrier as a putative mechanism in addiction liability.

    Science.gov (United States)

    Chapy, Hélène; Smirnova, Maria; André, Pascal; Schlatter, Joël; Chiadmi, Fouad; Couraud, Pierre-Olivier; Scherrmann, Jean-Michel; Declèves, Xavier; Cisternino, Salvatore

    2014-10-31

    The rate of entry of cocaine into the brain is a critical factor that influences neuronal plasticity and the development of cocaine addiction. Until now, passive diffusion has been considered the unique mechanism known by which cocaine crosses the blood-brain barrier. We reassessed mechanisms of transport of cocaine at the blood-brain barrier using a human cerebral capillary endothelial cell line (hCMEC/D3) and in situ mouse carotid perfusion. Both in vivo and in vitro cocaine transport studies demonstrated the coexistence of a carrier-mediated process with passive diffusion. At pharmacological exposure level, passive diffusion of cocaine accounted for only 22.5% of the total cocaine influx in mice and 5.9% in hCMEC/D3 cells, whereas the carrier-mediated influx rate was 3.4 times greater than its passive diffusion rate in vivo. The functional identification of this carrier-mediated transport demonstrated the involvement of a proton antiporter that shared the properties of the previously characterized clonidine and nicotine transporter. The functionnal characterization suggests that the solute carrier (SLC) transporters Oct (Slc22a1-3), Mate (Slc47a1) and Octn (Slc22a4-5) are not involved in the cocaine transport in vivo and in vitro. Diphenhydramine, heroin, tramadol, cocaethylene, and norcocaine all strongly inhibited cocaine transport, unlike benzoylecgonine. Trans-stimulation studies indicated that diphenhydramine, nicotine, 3,4-methylenedioxyamphetamine (ecstasy) and the cathinone compound 3,4-methylenedioxypyrovalerone (MDPV) were also substrates of the cocaine transporter. Cocaine transport at the BBB involves a proton-antiporter flux that is quantitatively much more important than its passive diffusion. The molecular identification and characterization of this transporter will provide new tools to understand its role in addictive mechanisms. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e

  9. Osmotic tolerance of avian spermatozoa: Influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability

    Science.gov (United States)

    Blanco, J.M.; Long, J.A.; Gee, G.; Donoghue, A.M.; Wildt, D.E.

    2008-01-01

    Potential factors influencing sperm survival under hypertonic conditions were evaluated in the Sandhill crane (Grus canadensis) and turkey (Meleagridis gallopavo). Sperm osmotolerance (300-3000 mOsm/kg) was evaluated after: (1) equilibration times of 2, 10, 45 and 60 min at 4 ?C versus 21 ?C; (2) pre-equilibrating with dimethylacetamide (DMA) or dimethylsulfoxide (Me2SO) at either 4 ?C or 21 ?C; and (3) inhibition of the Na+/K+ and the Na+/H+ antiporter membrane ionic pumps. Sperm viability was assessed using the eosin-nigrosin live/dead stain. Species-specific differences occurred in response to hypertonic conditions with crane sperm remaining viable under extreme hypertonicity (3000 mOsm/kg), whereas turkey sperm viability was compromised with only slightly hypertonic (500 mOsm/kg) conditions. The timing of spermolysis under hypertonic conditions was also species-specific, with a shorter interval for turkey (2 min) than crane (10 min) sperm. Turkey sperm osmotolerance was slightly improved by lowering the incubation temperature from 21 to 4 ?C. Pre-equilibrating sperm with DMA reduced the incidence of hypertonic spermolysis only in the crane, at both room and refrigeration temperature. Inhibiting the Na+/K+ and the Na+/H+ antiporter membrane ion pumps did not impair resistance of crane and turkey spermatozoa to hypertonic stress; pump inhibition actually increased turkey sperm survival compared to control sperm. Results demonstrate marked species specificity in osmotolerance between crane and turkey sperm, as well as in the way temperature and time of exposure affect sperm survival under hypertonic conditions. Differences are independent of the role of osmotic pumps in these species.

  10. Characterization of Salt Overly Sensitive 1 (SOS1) gene homoeologs in quinoa (Chenopodium quinoa Willd.).

    Science.gov (United States)

    Maughan, P J; Turner, T B; Coleman, C E; Elzinga, D B; Jellen, E N; Morales, J A; Udall, J A; Fairbanks, D J; Bonifacio, A

    2009-07-01

    Salt tolerance is an agronomically important trait that affects plant species around the globe. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in germination and growth of plants in saline environments. Quinoa (Chenopodium quinoa Willd.) is a halophytic, allotetraploid grain crop of the family Amaranthaceae with impressive nutritional content and an increasing worldwide market. Many quinoa varieties have considerable salt tolerance, and research suggests quinoa may utilize novel mechanisms to confer salt tolerance. Here we report the cloning and characterization of two homoeologous SOS1 loci (cqSOS1A and cqSOS1B) from C. quinoa, including full-length cDNA sequences, genomic sequences, relative expression levels, fluorescent in situ hybridization (FISH) analysis, and a phylogenetic analysis of SOS1 genes from 13 plant taxa. The cqSOS1A and cqSOS1B genes each span 23 exons spread over 3477 bp and 3486 bp of coding sequence, respectively. These sequences share a high level of similarity with SOS1 homologs of other species and contain two conserved domains, a Nhap cation-antiporter domain and a cyclic-nucleotide binding domain. Genomic sequence analysis of two BAC clones (98 357 bp and 132 770 bp) containing the homoeologous SOS1 genes suggests possible conservation of synteny across the C. quinoa sub-genomes. This report represents the first molecular characterization of salt-tolerance genes in a halophytic species in the Amaranthaceae as well as the first comparative analysis of coding and non-coding DNA sequences of the two homoeologous genomes of C. quinoa.

  11. Angiotensin 2 directly increases rabbit renal brush-border membrane sodium transport: Presence of local signal transduction system

    Energy Technology Data Exchange (ETDEWEB)

    Morduchowicz, G.A.; Sheikh-Hamad, D.; Dwyer, B.E.; Stern, N.; Jo, O.D.; Yanagawa, N. (Sepulveda Veterans Administration, CA (USA))

    1991-05-01

    In the present study, the authors have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10(-11)-10(-7) M) was found to stimulate 22Na+ uptake by the isolated BBM vesicles directly. All did not affect the Na(+)-dependent BBM glucose uptake, and the effect of AII on BBM 22Na+ uptake was inhibited by amiloride, suggesting the involvement of Na+/H+ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na+/H+ antiport system. In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A2 (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-beta S or PTX abolished, the effects of AII on BBM PLA and 22Na+ uptake, suggesting the involvement of G-protein in AII's actions. On the other hand, inhibition of PLA by mepacrine prevented AII's effect on BBM 22Na+ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM 22Na+ uptake, suggesting the role of PLA activation in mediating AII's effect on BBM 22Na+ uptake. In summary, results of the present study show a direct stimulatory effect of AII on BBM Na+/H+ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.

  12. Environmental adaptability and stress tolerance of Laribacter hongkongensis: a genome-wide analysis

    Directory of Open Access Journals (Sweden)

    Lau Susanna KP

    2011-06-01

    Full Text Available Abstract Background Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea and it can reside in human, fish, frogs and water. In this study, we performed an in-depth annotation of the genes in its genome related to adaptation to the various environmental niches. Results L. hongkongensis possessed genes for DNA repair and recombination, basal transcription, alternative σ-factors and 109 putative transcription factors, allowing DNA repair and global changes in gene expression in response to different environmental stresses. For acid stress, it possessed a urease gene cassette and two arc gene clusters. For alkaline stress, it possessed six CDSs for transporters of the monovalent cation/proton antiporter-2 and NhaC Na+:H+ antiporter families. For heavy metals acquisition and tolerance, it possessed CDSs for iron and nickel transport and efflux pumps for other metals. For temperature stress, it possessed genes related to chaperones and chaperonins, heat shock proteins and cold shock proteins. For osmotic stress, 25 CDSs were observed, mostly related to regulators for potassium ion, proline and glutamate transport. For oxidative and UV light stress, genes for oxidant-resistant dehydratase, superoxide scavenging, hydrogen peroxide scavenging, exclusion and export of redox-cycling antibiotics, redox balancing, DNA repair, reduction of disulfide bonds, limitation of iron availability and reduction of iron-sulfur clusters are present. For starvation, it possessed phosphorus and, despite being asaccharolytic, carbon starvation-related CDSs. Conclusions The L. hongkongensis genome possessed a high variety of genes for adaptation to acid, alkaline, temperature, osmotic, oxidative, UV light and starvation stresses and acquisition of and tolerance to heavy metals.

  13. Novel Metal Cation Resistance Systems from Mutant Fitness Analysis of Denitrifying Pseudomonas stutzeri.

    Science.gov (United States)

    Vaccaro, Brian J; Lancaster, W Andrew; Thorgersen, Michael P; Zane, Grant M; Younkin, Adam D; Kazakov, Alexey E; Wetmore, Kelly M; Deutschbauer, Adam; Arkin, Adam P; Novichkov, Pavel S; Wall, Judy D; Adams, Michael W W

    2016-10-01

    Metal ion transport systems have been studied extensively, but the specificity of a given transporter is often unclear from amino acid sequence data alone. In this study, predicted Cu(2+) and Zn(2+) resistance systems in Pseudomonas stutzeri strain RCH2 are compared with those experimentally implicated in Cu(2+) and Zn(2+) resistance, as determined by using a DNA-barcoded transposon mutant library. Mutant fitness data obtained under denitrifying conditions are combined with regulon predictions to yield a much more comprehensive picture of Cu(2+) and Zn(2+) resistance in strain RCH2. The results not only considerably expand what is known about well-established metal ion exporters (CzcCBA, CzcD, and CusCBA) and their accessory proteins (CzcI and CusF), they also reveal that isolates with mutations in some predicted Cu(2+) resistance systems do not show decreased fitness relative to the wild type when exposed to Cu(2+) In addition, new genes are identified that have no known connection to Zn(2+) (corB, corC, Psest_3226, Psest_3322, and Psest_0618) or Cu(2+) resistance (Mrp antiporter subunit gene, Psest_2850, and Psest_0584) but are crucial for resistance to these metal cations. Growth of individual deletion mutants lacking corB, corC, Psest_3226, or Psest_3322 confirmed the observed Zn-dependent phenotypes. Notably, to our knowledge, this is the first time a bacterial homolog of TMEM165, a human gene responsible for a congenital glycosylation disorder, has been deleted and the resulting strain characterized. Finally, the fitness values indicate Cu(2+)- and Zn(2+)-based inhibition of nitrite reductase and interference with molybdenum cofactor biosynthesis for nitrate reductase. These results extend the current understanding of Cu(2+) and Zn(2+) efflux and resistance and their effects on denitrifying metabolism. In this study, genome-wide mutant fitness data in P. stutzeri RCH2 combined with regulon predictions identify several proteins of unknown function that are

  14. Comparative analysis of the root transcriptomes of cultivated sweetpotato (Ipomoea batatas [L.] Lam) and its wild ancestor (Ipomoea trifida [Kunth] G. Don).

    Science.gov (United States)

    Ponniah, Sathish K; Thimmapuram, Jyothi; Bhide, Ketaki; Kalavacharla, Venu Kal; Manoharan, Muthusamy

    2017-01-13

    The complex process of formation of storage roots (SRs) from adventitious roots affects sweetpotato yield. Identifying the genes that are uniquely expressed in the SR forming cultivated species, Ipomoea batatas (Ib), and its immediate ancestral species, Ipomoea trifida (It), which does not form SRs, may provide insights into the molecular mechanisms underlying SR formation in sweetpotato. Illumina paired-end sequencing generated ~208 and ~200 million reads for Ib and It, respectively. Trinity assembly of the reads resulted in 98,317 transcripts for Ib and 275,044 for It, after post-assembly removal of trans-chimeras. From these sequences, we identified 4,865 orthologous genes in both Ib and It, 60 paralogous genes in Ib and 2,286 paralogous genes in It. Among paralogous gene sets, transcripts encoding the transcription factor RKD, which may have a role in nitrogen regulation and starch formation, and rhamnogalacturonate lyase (RGL) family proteins, which produce the precursors of cell wall polysaccharides, were found only in Ib. In addition, transcripts encoding a K + efflux antiporter (KEA5) and the ERECTA protein kinase, which function in phytohormonal regulation and root proliferation, respectively, were also found only in Ib. qRT-PCR indicated that starch and sucrose metabolism genes, such as those encoding ADP-glucose pyrophosphorylase and beta-amylase, showed lower expression in It than Ib, whereas lignin genes such as caffeoyl-CoA O-methyltransferase (CoMT) and cinnamyl alcohol dehydrogenase (CAD) showed higher expression in It than Ib. A total of 7,067 and 9,650 unique microsatellite markers, 1,037,396 and 495,931 single nucleotide polymorphisms (SNPs) and 103,439 and 69,194 InDels in Ib and It, respectively, were also identified from this study. The detection of genes involved in the biosynthesis of RGL family proteins, the transcription factor RKD, and genes encoding a K + efflux antiporter (KEA5) and the ERECTA protein kinase only in I. batatas indicate

  15. Design Function and Structure of a Monomeric CLC Transporter

    Energy Technology Data Exchange (ETDEWEB)

    L Robertson; L Kolmakova-Partensky; C Miller

    2011-12-31

    Channels and transporters of the ClC family cause the transmembrane movement of inorganic anions in service of a variety of biological tasks, from the unusual - the generation of the kilowatt pulses with which electric fish stun their prey - to the quotidian - the acidification of endosomes, vacuoles and lysosomes. The homodimeric architecture of ClC proteins, initially inferred from single-molecule studies of an elasmobranch Cl{sup -} channel and later confirmed by crystal structures of bacterial Cl{sup -}/H{sup +} antiporters, is apparently universal. Moreover, the basic machinery that enables ion movement through these proteins - the aqueous pores for anion diffusion in the channels and the ion-coupling chambers that coordinate Cl{sup -} and H{sup +} antiport in the transporters - are contained wholly within each subunit of the homodimer. The near-normal function of a bacterial ClC transporter straitjacketed by covalent crosslinks across the dimer interface and the behaviour of a concatemeric human homologue argue that the transport cycle resides within each subunit and does not require rigid-body rearrangements between subunits. However, this evidence is only inferential, and because examples are known in which quaternary rearrangements of extramembrane ClC domains that contribute to dimerization modulate transport activity, we cannot declare as definitive a 'parallel-pathways picture in which the homodimer consists of two single-subunit transporters operating independently. A strong prediction of such a view is that it should in principle be possible to obtain a monomeric ClC. Here we exploit the known structure of a ClC Cl{sup -}/H{sup +} exchanger, ClC-ec1 from Escherichia coli, to design mutants that destabilize the dimer interface while preserving both the structure and the transport function of individual subunits. The results demonstrate that the ClC subunit alone is the basic functional unit for transport and that cross-subunit interaction is not

  16. Amino acid secondary transporters: toward a common transport mechanism.

    Science.gov (United States)

    Schweikhard, Eva S; Ziegler, Christine M

    2012-01-01

    Solute carriers (SLC) that transport amino acids are key players in health and diseases in humans. Their prokaryotic relatives are often involved in essential physiological processes in microorganisms, e.g. in homeostasis and acidic/osmotic stress response. High-resolution X-ray structures of the sequence-unrelated amino acid transporters unraveled a striking structural similarity between carriers, which were formerly assigned to different families. The highly conserved fold is characterized by two inverted structural repeats of five transmembrane helices each and indicates common mechanistic transport concepts if not an evolutionary link among a large number of amino acid transporters. Therefore, these transporters are classified now into the structural amino acid-polyamine-organocation superfamily (APCS). The APCS includes among others the mammalian SLC6 transporters and the heterodimeric SLC7/SLC3 transporters. However, it has to be noted that the APCS is not limited entirely to amino acid transporters but contains also transporters for, e.g. amino acid derivatives and sugars. For instance, the betaine-choline-carnitine transporter family of bacterial activity-regulated Na(+)- and H(+)-coupled symporters for glycine betaine and choline is also part of this second largest structural superfamily. The APCS fold provides different possibilities to transport the same amino acid. Arginine can be transported by an H(+)-coupled symport or by antiport mechanism in exchange against agmatine for example. The convergence of the mechanistic concept of transport under comparable physiological conditions allows speculating if structurally unexplored amino acid transporters, e.g. the members of the SLC36 and SLC38 family, belong to the APCS, too. In the kidney, which is an organ that depends critically on the regulated amino acid transport, these different SLC transporters have to work together to account for proper function. Here, we will summarize the basic concepts of Na

  17. Basic residues R260 and K357 affect the conformational dynamics of the major facilitator superfamily multidrug transporter LmrP.

    Directory of Open Access Journals (Sweden)

    Wei Wang

    Full Text Available Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H(+(Na(+ antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260 and inner leaflet (K357 of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently

  18. The genome draft of coconut (Cocos nucifera).

    Science.gov (United States)

    Xiao, Yong; Xu, Pengwei; Fan, Haikuo; Baudouin, Luc; Xia, Wei; Bocs, Stéphanie; Xu, Junyang; Li, Qiong; Guo, Anping; Zhou, Lixia; Li, Jing; Wu, Yi; Ma, Zilong; Armero, Alix; Issali, Auguste Emmanuel; Liu, Na; Peng, Ming; Yang, Yaodong

    2017-11-01

    antiporter gene family and ion channel gene families between C. nucifera and Arabidopsis thaliana indicated that significant gene expansion may have occurred in the coconut involving Na+/H+ antiporter, carnitine/acylcarnitine translocase, potassium-dependent sodium-calcium exchanger, and potassium channel genes. Despite its agronomic importance, C. nucifera is still under-studied. In this report, we present a draft genome of C. nucifera and provide genomic information that will facilitate future functional genomics and molecular-assisted breeding in this crop species. © The Author 2017. Published by Oxford University Press.

  19. The Physiopathological Role of the Exchangers Belonging to the SLC37 Family

    Directory of Open Access Journals (Sweden)

    Anna Rita Cappello

    2018-04-01

    Full Text Available The human SLC37 gene family includes four proteins SLC37A1-4, localized in the endoplasmic reticulum (ER membrane. They have been grouped into the SLC37 family due to their sequence homology to the bacterial organophosphate/phosphate (Pi antiporter. SLC37A1-3 are the less characterized isoforms. SLC37A1 and SLC37A2 are Pi-linked glucose-6-phosphate (G6P antiporters, catalyzing both homologous (Pi/Pi and heterologous (G6P/Pi exchanges, whereas SLC37A3 transport properties remain to be clarified. Furthermore, SLC37A1 is highly homologous to the bacterial glycerol 3-phosphate permeases, so it is supposed to transport also glycerol-3-phosphate. The physiological role of SLC37A1-3 is yet to be further investigated. SLC37A1 seems to be required for lipid biosynthesis in cancer cell lines, SLC37A2 has been proposed as a vitamin D and a phospho-progesterone receptor target gene, while mutations in the SLC37A3 gene appear to be associated with congenital hyperinsulinism of infancy. SLC37A4, also known as glucose-6-phosphate translocase (G6PT, transports G6P from the cytoplasm into the ER lumen, working in complex with either glucose-6-phosphatase-α (G6Pase-α or G6Pase-β to hydrolyze intraluminal G6P to Pi and glucose. G6PT and G6Pase-β are ubiquitously expressed, whereas G6Pase-α is specifically expressed in the liver, kidney and intestine. G6PT/G6Pase-α complex activity regulates fasting blood glucose levels, whereas G6PT/G6Pase-β is required for neutrophil functions. G6PT deficiency is responsible for glycogen storage disease type Ib (GSD-Ib, an autosomal recessive disorder associated with both defective metabolic and myeloid phenotypes. Several kinds of mutations have been identified in the SLC37A4 gene, affecting G6PT function. An increased autoimmunity risk for GSD-Ib patients has also been reported, moreover, SLC37A4 seems to be involved in autophagy.

  20. In vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient in the endomembrane system.

    Science.gov (United States)

    Martinière, Alexandre; Bassil, Elias; Jublanc, Elodie; Alcon, Carine; Reguera, Maria; Sentenac, Hervé; Blumwald, Eduardo; Paris, Nadine

    2013-10-01

    The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the trans-Golgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (ΔpH -1.5) and Arabidopsis thaliana root cells (ΔpH -2.1). The average pH in VSR compartments was intermediate between that of the trans-Golgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H(+) ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

  1. Using Haloarcula marismortui bacteriorhodopsin as a fusion tag for enhancing and visible expression of integral membrane proteins in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Min-Feng Hsu

    Full Text Available Membrane proteins are key targets for pharmacological intervention because of their vital functions. Structural and functional studies of membrane proteins have been severely hampered because of the difficulties in producing sufficient quantities of properly folded and biologically active proteins. Here we generate a high-level expression system of integral membrane proteins in Escherichia coli by using a mutated bacteriorhodopsin (BR from Haloarcula marismortui (HmBRI/D94N as a fusion partner. A purification strategy was designed by incorporating a His-tag on the target membrane protein for affinity purification and an appropriate protease cleavage site to generate the final products. The fusion system can be used to detect the intended target membrane proteins during overexpression and purification either with the naked eye or by directly monitoring their characteristic optical absorption. In this study, we applied this approach to produce two functional integral membrane proteins, undecaprenyl pyrophosphate phosphatase and carnitine/butyrobetaine antiporter with significant yield enhancement. This technology could facilitate the development of a high-throughput strategy to screen for conditions that improve the yield of correctly folded target membrane proteins. Other robust BRs can also be incorporated in this system.

  2. Kinetic simulation of malate-aspartate and citrate-pyruvate shuttles in association with Krebs cycle.

    Science.gov (United States)

    Korla, Kalyani; Vadlakonda, Lakshmipathi; Mitra, Chanchal K

    2015-01-01

    In the present work, we have kinetically simulated two mitochondrial shuttles, malate-aspartate shuttle (used for transferring reducing equivalents) and citrate-pyruvate shuttle (used for transferring carbon skeletons). However, the functions of these shuttles are not limited to the points mentioned above, and they can be used in different arrangements to meet different cellular requirements. Both the shuttles are intricately associated with Krebs cycle through the metabolites involved. The study of this system of shuttles and Krebs cycle explores the response of the system in different metabolic environments. Here, we have simulated these subsets individually and then combined them to study the interactions among them and to bring out the dynamics of these pathways in focus. Four antiports and a pyruvate pump were modelled along with the metabolic reactions on both sides of the inner mitochondrial membrane. Michaelis-Menten approach was extended for deriving rate equations of every component of the system. Kinetic simulation was carried out using ordinary differential equation solver in GNU Octave. It was observed that all the components attained steady state, sooner or later, depending on the system conditions. Progress curves and phase plots were plotted to understand the steady state behaviour of the metabolites involved. A comparative analysis between experimental and simulated data show fair agreement thus validating the usefulness and applicability of the model.

  3. Biodegradation of malachite green by strain Pseudomonas sp. K9 and cloning of the tmr2 gene associated with an ISPpu12.

    Science.gov (United States)

    Huan-Mei; Lian-Tai, Li; Cai-Fang, Yan; Jin-Jin, Sun; Yuan-Gao; Hong, Qing; Shun-Peng, Li

    2011-06-01

    A bacterial strain K9 capable of degrading malachite green was isolated from the sludge of the wastewater treatment system of a chemical plant. It was identified preliminarily as Pseudomonas sp. Strain K9 was also able to degrade other triphenylmethane dyes, such as Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase, was cloned from strain K9, and functionally expressed in E. coli. A 5946-bp DNA fragment including the tmr2 gene was cloned from the genomic DNA of strain K9 by chromosome walking. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase) and orf1 (encoding a putative MerR family regulator), orf2 (encoding a CDF family heavy metal/H(+) antiporter). This association was also found in another malachite green-degrading strain Pseudomonas sp. MDB-1, which indicated that the tmr2 gene might be a horizontally transferable gene.

  4. Effects of non-uniform root zone salinity on water use, Na+ recirculation, and Na+ and H+ flux in cotton.

    Science.gov (United States)

    Kong, Xiangqiang; Luo, Zhen; Dong, Hezhong; Eneji, A Egrinya; Li, Weijiang

    2012-03-01

    A new split-root system was established through grafting to study cotton response to non-uniform salinity. Each root half was treated with either uniform (100/100 mM) or non-uniform NaCl concentrations (0/200 and 50/150 mM). In contrast to uniform control, non-uniform salinity treatment improved plant growth and water use, with more water absorbed from the non- and low salinity side. Non-uniform treatments decreased Na(+) concentrations in leaves. The [Na(+)] in the '0' side roots of the 0/200 treatment was significantly higher than that in either side of the 0/0 control, but greatly decreased when the '0' side phloem was girdled, suggesting that the increased [Na(+)] in the '0' side roots was possibly due to transportation of foliar Na(+) to roots through phloem. Plants under non-uniform salinity extruded more Na(+) from the root than those under uniform salinity. Root Na(+) efflux in the low salinity side was greatly enhanced by the higher salinity side. NaCl-induced Na(+) efflux and H(+) influx were inhibited by amiloride and sodium orthovanadate, suggesting that root Na(+) extrusion was probably due to active Na(+)/H(+) antiport across the plasma membrane. Improved plant growth under non-uniform salinity was thus attributed to increased water use, reduced leaf Na(+) concentration, transport of excessive foliar Na(+) to the low salinity side, and enhanced Na(+) efflux from the low salinity root.

  5. Developing transgenic Jatropha using the SbNHX1 gene from an extreme halophyte for cultivation in saline wasteland.

    Science.gov (United States)

    Joshi, Mukul; Jha, Anupama; Mishra, Avinash; Jha, Bhavanath

    2013-01-01

    Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na⁺/H⁺ antiporter gene that compartmentalises excess Na⁺ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.

  6. The over-expression of Chrysanthemum crassum CcSOS1 improves the salinity tolerance of chrysanthemum.

    Science.gov (United States)

    An, Juan; Song, Aiping; Guan, Zhiyong; Jiang, Jiafu; Chen, Fadi; Lou, Wanghuai; Fang, Weimin; Liu, Zhaolei; Chen, Sumei

    2014-06-01

    Soil salinity represents a major constraint on plant growth. Here, we report that the over-expression of the Chrysanthemum crassum plasma membrane Na(+)/H(+) antiporter gene CcSOS1, driven by the CaMV 35S promoter, improved the salinity tolerance of chrysanthemum 'Jinba'. In salinity-stressed transgenic plants, both the proportion of the leaf area suffering damage and the electrical conductivity of the leaf were lower in the transgenic lines than in salinity-stressed wild type plants. After a 6 day exposure to 200 mM NaCl, the leaf content of both chlorophyll (a+b) and proline was higher in the transgenic than in the wild type plants. The activity of both superoxide dismutase and peroxidase was higher in the transgenic than in the wild type plants throughout the period of NaCl stress. The transgenic plants had a stronger control over the ingress of Na(+) into the plant, particularly with respect to the youngest leaves, and so maintained a more favorable K(+)/Na(+) ratio. The result suggests that a possible strategy for improving the salinity tolerance of chrysanthemum could target the restriction of Na(+) accumulation. This study is the first to report the transgenic expression of a Na(+) efflux carrier in chrysanthemum.

  7. Transport pathways in the malaria-infected erythrocyte: characterization and their use as potential targets for chemotherapy

    Directory of Open Access Journals (Sweden)

    Hagai Ginsburg

    1994-01-01

    Full Text Available The intraerythrocytic malarial parasite is involved in an extremely intensive anabolic activity while it resides in its metabolically quiescent host cell. The necessary fast uptake of nutrients and the discharge of waste product, are guaranteed by parasite-induced alterations of the constitutive transporters of the host cell and the production of new parallel pathways. The membrane of the host cell thus becomes permeable to phospholipids, purine bases and nucleosides, small non-electrolytes, anions and cations. When the new pathways are quantitatively unimportant, classical inhibitors of native transporters can be used to inhibit parasite growth. Several compounds were found to effectively inhibit the new pathways and consequently, parasite growth. The pathways have also been used to introduce cytotoxic agents. The parasitophorous membrane consists of channels which are highly permeable to small solutes and display no ion selectivity. Transport of some cations and anions across the parasite membrane is rapid and insensitive to classical inhibitors, and in some cases it is mediated by specific antiporters which respond to their respective inhibitors. Macromolecules have been shown to reach the parasitophorous space through a duct contiguous with the host cell membrane, and subsequently to be endocytosed at the parasite membrane. The simultaneous presence of the parasitophorous membrane channels and the duct, however, is incompatible with experimental evidences. No specific inhibitors were found as yet that would efficiently inhibit transport through the channels or the duct.

  8. The osmotolerant fructophilic yeast Zygosaccharomyces rouxii employs two plasma-membrane fructose uptake systems belonging to a new family of yeast sugar transporters.

    Science.gov (United States)

    Leandro, Maria José; Sychrová, Hana; Prista, Catarina; Loureiro-Dias, Maria C

    2011-02-01

    Owing to its high resistance to weak-acid preservatives and extreme osmotolerance, Zygosaccharomyces rouxii is one of the main spoilage yeasts of sweet foods and beverages. In contrast with Saccharomyces cerevisiae, Z. rouxii is a fructophilic yeast; it consumes fructose faster than glucose. So far, to our knowledge, no specific Z. rouxii proteins responsible for this fructophilic behaviour have been characterized. We have identified two genes encoding putative fructose transporters in the Z. rouxii CBS 732 genome. Heterologous expression of these two Z. rouxii ORFs in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic analysis of sugar transport showed that both proteins are functionally expressed at the plasma membrane: ZrFfz1 is a high-capacity fructose-specific facilitator (K(m)∼400 mM and V(max)∼13 mmol h(-1) g(-1)) and ZrFfz2 is a facilitator transporting glucose and fructose with similar capacity and affinity (K(m)∼200 mM and V(max)∼4 mmol h(-1) g(-1)). These two proteins together with the Zygosaccharomyces bailii Ffz1 fructose-specific transporter belong to a new family of sugar transport systems mediating the uptake of hexoses via the facilitated diffusion mechanism, and are more homologous to drug/H(+) antiporters (regarding their primary protein structure) than to other yeast sugar transporters of the Sugar Porter family.

  9. Weak Organic Acids Decrease Borrelia burgdorferi Cytoplasmic pH, Eliciting an Acid Stress Response and Impacting RpoN- and RpoS-Dependent Gene Expression

    Directory of Open Access Journals (Sweden)

    Daniel P. Dulebohn

    2017-09-01

    Full Text Available The spirochete Borrelia burgdorferi survives in its tick vector, Ixodes scapularis, or within various hosts. To transition between and survive in these distinct niches, B. burgdorferi changes its gene expression in response to environmental cues, both biochemical and physiological. Exposure of B. burgdorferi to weak monocarboxylic organic acids, including those detected in the blood meal of fed ticks, decreased the cytoplasmic pH of B. burgdorferi in vitro. A decrease in the cytoplasmic pH induced the expression of genes encoding enzymes that have been shown to restore pH homeostasis in other bacteria. These include putative coupled proton/cation exchangers, a putative Na+/H+ antiporter, a neutralizing buffer transporter, an amino acid deaminase and a proton exporting vacuolar-type VoV1 ATPase. Data presented in this report suggested that the acid stress response triggered the expression of RpoN- and RpoS-dependent genes including important virulence factors such as outer surface protein C (OspC, BBA66, and some BosR (Borreliaoxidative stress regulator-dependent genes. Because the expression of virulence factors, like OspC, are so tightly connected by RpoS to general cellular stress responses and cell physiology, it is difficult to separate transmission-promoting conditions in what is clearly a multifactorial and complex regulatory web.

  10. Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis.

    Science.gov (United States)

    Peters, J S; Berkowitz, G A

    1991-04-01

    Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H(+) movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg(2+) causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg(2+) was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg(2+) and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H(+) fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg(2+) modulated) H(+) flux linked to monovalent cation antiport, and ATPase-dependent H(+) efflux.

  11. Studies on the System Regulating Proton Movement across the Chloroplast Envelope 1

    Science.gov (United States)

    Peters, Jeanne S.; Berkowitz, Gerald A.

    1991-01-01

    Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H+ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg2+ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg2+ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg2+ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H+ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg2+ modulated) H+ flux linked to monovalent cation antiport, and ATPase-dependent H+ efflux. PMID:16668116

  12. Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and ZxVP1-1 confers enhanced salinity tolerance in chimeric sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Wu, Guo-Qiang; Feng, Rui-Jun; Wang, Suo-Min; Wang, Chun-Mei; Bao, Ai-Ke; Wei, Li; Yuan, Hui-Jun

    2015-01-01

    Salinity is one of the major abiotic stresses that limit the growth and productivity of sugar beet (Beta vulgaris L.). To improve sugar beet's salinity tolerance, the ZxNHX and ZxVP1-1 genes encoding tonoplast Na(+)/H(+) antiporter and H(+)-PPase from xerophyte Zygophyllum xanthoxylum were co-expressed by Agrobacterium tumefaciens-mediated transformation. It is showed here that co-expression of ZxNHX and ZxVP1-1 confers enhanced salinity tolerance to the transformed sugar beet plants compared with the wild-type (WT) plants. The chimeric plants grew well in the presence of high salinity (400 mM NaCl), whereas WT plants displayed chlorosis and died within 8 days. Compared to WT plants, the chimeric plants co-expressing ZxNHX and ZxVP1-1 accumulated more proline, Na(+) and K(+) in their leaves and petioles when exposed to high salinity, which caused lower solute potential, retained more water and thus subjected to lesser cell membrane damage. Interestingly, the chimeric plants accumulated higher sucrose, glucose and fructose contents in their storage roots than WT plants in the absence or presence of high salinity. Our results suggested that co-expression of ZxNHX and ZxVP1-1 improved the osmoregulatory capacity in chimeric sugar beet through increased compartmentalization of ions into the vacuoles by enhancing the activity of proton pumps and thus mitigated Na(+)-toxicity for plants.

  13. Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters.

    Science.gov (United States)

    Mishra, Avinash; Tanna, Bhakti

    2017-01-01

    Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile , and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters ( NHX, SOS, HKT, VTPase ), ion channels (Cl - , Ca 2+ , aquaporins), antioxidant encoding genes ( APX, CAT, GST, BADH, SOD ) and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes). It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering.

  14. Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters

    Directory of Open Access Journals (Sweden)

    Avinash Mishra

    2017-05-01

    Full Text Available Halophytes have demonstrated their capability to thrive under extremely saline conditions and thus considered as one of the best germplasm for saline agriculture. Salinity is a worldwide problem, and the salt-affected areas are increasing day-by-day because of scanty rainfall, poor irrigation system, salt ingression, water contamination, and other environmental factors. The salinity stress tolerance mechanism is a very complex phenomenon, and some pathways are coordinately linked for imparting salinity tolerance. Though a number of salt responsive genes have been reported from the halophytes, there is always a quest for promising stress-responsive genes that can modulate plant physiology according to the salt stress. Halophytes such as Aeluropus, Mesembryanthemum, Suaeda, Atriplex, Thellungiella, Cakile, and Salicornia serve as a potential candidate for the salt-responsive genes and promoters. Several known genes like antiporters (NHX, SOS, HKT, VTPase, ion channels (Cl−, Ca2+, aquaporins, antioxidant encoding genes (APX, CAT, GST, BADH, SOD and some novel genes such as USP, SDR1, SRP etc. were isolated from halophytes and explored for developing stress tolerance in the crop plants (glycophytes. It is evidenced that stress triggers salt sensors that lead to the activation of stress tolerance mechanisms which involve multiple signaling proteins, up- or down-regulation of several genes, and finally the distinctive or collective effects of stress-responsive genes. In this review, halophytes are discussed as an excellent platform for salt responsive genes which can be utilized for developing salinity tolerance in crop plants through genetic engineering.

  15. The Molecular Basis of pH Sensing, Signaling, and Homeostasis in Fungi.

    Science.gov (United States)

    Bignell, Elaine

    2012-01-01

    Fungi mount efficient responses to altered extracellular pH. Characterization of the underlying mechanisms is fundamentally important in terms of understanding the molecular basis of pH homeostasis in higher eukaryotic cells, and for optimizing industrial processes which utilize fungi such as the production of pharmaceutical agents and food-use enzymes. Fungal pH adaptation is also a key requisite for establishment of multiple plant, insect, animal, and human diseases. Due to the differential reliance, respectively, of human and fungal cells upon electroneutral Na(+)-H(+) antiporters and outwardly directed electrogenic proton pumps, fundamental differences in the circuitry of pH homeostasis and adaptation exist, and these might be exploitable from a therapeutic perspective. At the molecular level, fungal pH tolerance is mediated by distinct but complementary homeostatic responses and highly conserved intracellular signaling pathways. Although traditionally studied as independent regulatory entities, the advent of systems biology has fuelled a new awareness of the interconnectivity between these very different modes of regulation. This review focuses upon the most recent advances in molecular understanding of three specific aspects of fungal pH adaptation, namely, sensing, signaling, and homeostasis. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Crystal structure of a bacterial homologue of the bile acid sodium symporter ASBT

    Science.gov (United States)

    Hu, Nien-Jen; Iwata, So; Cameron, Alexander D.; Drew, David

    2011-01-01

    High cholesterol levels greatly increase the risk of cardiovascular disease. By its conversion into bile acids, about 50% of cholesterol is eliminated from the body. However bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine via the Apical Sodium dependent Bile acid Transporter (ASBT). It has been shown in animal models that plasma cholesterol levels are significantly lowered by specific inhibitors of ASBT1,2, thus ASBT is a target for hypercholesterolemia drugs. Here, we describe the crystal structure of a bacterial homologue of ASBT from Neisseria meningitidis (ASBTNM) at 2.2Å. ASBTNM contains two inverted structural repeats of five transmembrane helices. A Core domain of six helices harbours two sodium ions while the remaining helices form a Panel-like domain. Overall the architecture of the protein is remarkably similar to the sodium-proton antiporter NhaA3 despite no detectable sequence homology. A bile acid molecule is situated between the Core and Panel domains in a large hydrophobic cavity. Residues near to this cavity have been shown to affect the binding of specific inhibitors of human ASBT4. The position of the bile acid together with the molecular architecture suggests the rudiments of a possible transport mechanism. PMID:21976025

  17. Uncovering the Salt Response of Soybean by Unraveling Its Wild and Cultivated Functional Genomes Using Tag Sequencing

    Science.gov (United States)

    Ali, Zulfiqar; Zhang, Da Yong; Xu, Zhao Long; Xu, Ling; Yi, Jin Xin; He, Xiao Lan; Huang, Yi Hong; Liu, Xiao Qing; Khan, Asif Ali; Trethowan, Richard M.; Ma, Hong Xiang

    2012-01-01

    Soil salinity has very adverse effects on growth and yield of crop plants. Several salt tolerant wild accessions and cultivars are reported in soybean. Functional genomes of salt tolerant Glycine soja and a salt sensitive genotype of Glycine max were investigated to understand the mechanism of salt tolerance in soybean. For this purpose, four libraries were constructed for Tag sequencing on Illumina platform. We identify around 490 salt responsive genes which included a number of transcription factors, signaling proteins, translation factors and structural genes like transporters, multidrug resistance proteins, antiporters, chaperons, aquaporins etc. The gene expression levels and ratio of up/down-regulated genes was greater in tolerant plants. Translation related genes remained stable or showed slightly higher expression in tolerant plants under salinity stress. Further analyses of sequenced data and the annotations for gene ontology and pathways indicated that soybean adapts to salt stress through ABA biosynthesis and regulation of translation and signal transduction of structural genes. Manipulation of these pathways may mitigate the effect of salt stress thus enhancing salt tolerance. PMID:23209559

  18. The Aspergillus nidulans acuL gene encodes a mitochondrial carrier required for the utilization of carbon sources that are metabolized via the TCA cycle.

    Science.gov (United States)

    Flipphi, Michel; Oestreicher, Nathalie; Nicolas, Valérie; Guitton, Audrey; Vélot, Christian

    2014-07-01

    In Aspergillus nidulans, the utilization of acetate as sole carbon source requires several genes (acu). Most of them are also required for the utilization of fatty acids. This is the case for acuD and acuE, which encode the two glyoxylate cycle-specific enzymes, isocitrate lyase and malate synthase, respectively, but also for acuL that we have identified as AN7287, and characterized in this study. Deletion of acuL resulted in the same phenotype as the original acuL217 mutant. acuL encodes a 322-amino acid protein which displays all the structural features of a mitochondrial membrane carrier, and shares 60% identity with the Saccharomyces cerevisiae succinate/fumarate mitochondrial antiporter Sfc1p (also named Acr1p). Consistently, the AcuL protein was shown to localize in mitochondria, and partial cross-complementation was observed between the S. cerevisiae and A. nidulans homologues. Extensive phenotypic characterization suggested that the acuL gene is involved in the utilization of carbon sources that are catabolized via the TCA cycle, and therefore require gluconeogenesis. In addition, acuL proves to be co-regulated with acuD and acuE. Overall, our data suggest that AcuL could link the glyoxylate cycle to gluconeogenesis by exchanging cytoplasmic succinate for mitochondrial fumarate. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. The importance of orientation in proton transport of a polymer film based on an oriented self-organized columnar liquid-crystalline polyether

    Energy Technology Data Exchange (ETDEWEB)

    Tylkowski, Bartosz; Castelao, Nuria [Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, Av. Paiesos Catalans, 26, E-43007, Tarragona (Spain); Giamberini, Marta, E-mail: marta.giamberini@urv.net [Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, Av. Paiesos Catalans, 26, E-43007, Tarragona (Spain); Garcia-Valls, Ricard [Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, Av. Paiesos Catalans, 26, E-43007, Tarragona (Spain); Reina, Jose Antonio [Departament de Quimica Analitica i Quimica Organica, Universitat Rovira i Virgili, Carrer Marcel.li Domingo s/n, E-43007, Tarragona (Spain); Gumi, Tania [Departament d' Enginyeria Quimica, Universitat Rovira i Virgili, Av. Paiesos Catalans, 26, E-43007, Tarragona (Spain)

    2012-02-01

    We prepared membranes based on a liquid-crystalline side-chain polyether obtained by chemical modification of commercial poly(epichlorohydrin) (PECH) with dendrons. This polymer exhibited a columnar structure, which could form an ion channel in the inner part. The columns were successfully oriented by taking advantage of surface interactions between the polymer and hydrophilic substrates, as confirmed by X-ray diffraction analysis (XRD), environmental scanning electron microscopy (ESEM) and optical microscopy between crossed polars (POM). Column orientation was found to be crucial for effective transport: the oriented membranes exhibited proton transport comparable to that of Nafion Registered-Sign N117 and no water uptake. An increase in sodium ion concentration in the feed phase suggested a proton/cation antiport. On the contrary, no proton transport was detected on unoriented membranes based on the same liquid-crystalline side-chain polyether or on unmodified PECH. - Highlights: Black-Right-Pointing-Pointer We prepared oriented membranes based on a liquid crystalline columnar polyether. Black-Right-Pointing-Pointer In this structure, the inner polyether chain could work as an ion channel. Black-Right-Pointing-Pointer We obtained membranes by casting a chloroform solution in the presence of water. Black-Right-Pointing-Pointer Membranes showed good proton permeability due to the presence of oriented channels.

  20. Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB.

    Science.gov (United States)

    Eicher, Thomas; Seeger, Markus A; Anselmi, Claudio; Zhou, Wenchang; Brandstätter, Lorenz; Verrey, François; Diederichs, Kay; Faraldo-Gómez, José D; Pos, Klaas M

    2014-09-19

    Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system.

  1. Mutational analysis of the respiratory nitrate transporter NarK2 of Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Michelle M Giffin

    Full Text Available Mycobacterium tuberculosis induces nitrate reductase activity in response to decreasing oxygen levels. This is due to regulation of both the transcription and the activity of the nitrate transporter NarK2. A model of NarK2 structure is proposed containing 12 membrane spanning regions consistent with other members of the major facilitator superfamily. The role of the proton gradient was determined by exposing M. tuberculosis to uncouplers. Nitrite production decreased indicating that the importation of nitrate involved an H(+/nitrate symporter. The addition of nitrite before nitrate had no effect, suggesting no role for a nitrate/nitrite antiporter. In addition the NarK2 knockout mutant showed no defect in nitrite export. NarK2 is proposed to be a Type I H(+/nitrate symporter. Site directed mutagenesis was performed changing 23 amino acids of NarK2. This allowed the identification of important regions and amino acids of this transporter. Five of these mutants were inactive for nitrate transport, seven produced reduced activity and eleven mutants retained wild type activity. NarK2 is inactivated in the presence of oxygen by an unknown mechanism. However none of the mutants, including those with mutated cysteines, were altered in their response to oxygen levels. The assimilatory nitrate transporter NasA of Bacillus subtilis was expressed in the M. tuberculosis NarK2 mutant. It remained active during aerobic incubation showing that the point of oxygen control is NarK2.

  2. Development of salt tolerant plants through genetic engineering (abstract)

    International Nuclear Information System (INIS)

    Mukhtar, Z.; Khan, S.A.; Zafar, Y.

    2005-01-01

    Salinity stress is one of the most serious factors limiting the productivity of agricultural crops. Genetic engineering provides a useful tool for tailoring plants with enhanced salt tolerance characteristics. Many organisms have evolved mechanisms to survive and grow under such extreme environments. These organisms provide us with a useful source of genes which can be used to improve salt tolerance in plants. The present study aims at identification and cloning of useful halo tolerance conferring genes from fungi and plants and to develop salt tolerant transgenic plants. Here we describe the cloning and use of HSR1 gene (a yeast transcription factor known to confer salt tolerance) and Na/sup +//H/sup +/ antiporter gene AtNHX1 (3016 bp) from Arabidopsis thaliana, and transformation of tobacco with HSR1 and AtNHX1 genes through Agrobacterium method. A number of transgenic tobacco plants were regenerated from leaf explants transformed with Agrobacterium tumefaciens (LBA4404) having HSR1 and AtNHX1 genes by leaf disc method. The putative transgenic plants were analyzed by PCR and dot blot analysis. Screening of these transgenic plants at different salinity levels is in progress which will help identify the suitable plant lines and thus the promising genes which can be further exploited to engineer salt tolerant crop plants. (author)

  3. Agrobacterium rhizogenes rolB gene affects photosynthesis and chlorophyll content in transgenic tomato (Solanum lycopersicum L.) plants.

    Science.gov (United States)

    Bettini, Priscilla P; Marvasi, Massimiliano; Fani, Fabiola; Lazzara, Luigi; Cosi, Elena; Melani, Lorenzo; Mauro, Maria Luisa

    2016-10-01

    Insertion of Agrobacterium rhizogenes rolB gene into plant genome affects plant development, hormone balance and defence. However, beside the current research, the overall transcriptional response and gene expression of rolB as a modulator in plant is unknown. Transformed rolB tomato plant (Solanum lycopersicum L.) cultivar Tondino has been used to investigate the differential expression profile. Tomato is a well-known model organism both at the genetic and molecular level, and one of the most important commercial food crops in the world. Through the construction and characterization of a cDNA subtracted library, we have investigated the differential gene expression between transgenic clones of rolB and control tomato and have evaluated genes specifically transcribed in transgenic rolB plants. Among the selected genes, five genes encoding for chlorophyll a/b binding protein, carbonic anhydrase, cytochrome b 6 /f complex Fe-S subunit, potassium efflux antiporter 3, and chloroplast small heat-shock protein, all involved in chloroplast function, were identified. Measurement of photosynthesis efficiency by the level of three different photosynthetic parameters (F v /F m , rETR, NPQ) showed rolB significant increase in non-photochemical quenching and a, b chlorophyll content. Our results point to highlight the role of rolB on plant fitness by improving photosynthesis. Copyright © 2016 Elsevier GmbH. All rights reserved.

  4. Elevated carbon dioxide blunts mammalian cAMP signaling dependent on inositol 1,4,5-triphosphate receptor-mediated Ca2+ release.

    Science.gov (United States)

    Cook, Zara C; Gray, Michael A; Cann, Martin J

    2012-07-27

    Elevated CO(2) is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO(2) blunts G protein-activated cAMP signaling. The effect of CO(2) is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO(2) on cAMP levels required the activity of the IP(3) receptor. Consistent with these findings, CO(2) caused an increase in steady state cytoplasmic Ca(2+) concentrations not observed in the absence of the IP(3) receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na(+)/H(+) antiporter (NHE3) to demonstrate a functional relevance for CO(2)-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO(2) abrogated the inhibitory effect of cAMP on NHE3 function via an IP(3) receptor-dependent mechanism.

  5. Elevated Carbon Dioxide Blunts Mammalian cAMP Signaling Dependent on Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Release*

    Science.gov (United States)

    Cook, Zara C.; Gray, Michael A.; Cann, Martin J.

    2012-01-01

    Elevated CO2 is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO2 blunts G protein-activated cAMP signaling. The effect of CO2 is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO2 on cAMP levels required the activity of the IP3 receptor. Consistent with these findings, CO2 caused an increase in steady state cytoplasmic Ca2+ concentrations not observed in the absence of the IP3 receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na+/H+ antiporter (NHE3) to demonstrate a functional relevance for CO2-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO2 abrogated the inhibitory effect of cAMP on NHE3 function via an IP3 receptor-dependent mechanism. PMID:22654111

  6. Effects of Lysine deficiency and Lys-Lys dipeptide on cellular apoptosis and amino acids metabolism.

    Science.gov (United States)

    Yin, Jie; Li, Yuying; Han, Hui; Zheng, Jie; Wang, Lijian; Ren, Wenkai; Chen, Shuai; Wu, Fei; Fang, Rejun; Huang, Xingguo; Li, Chunyong; Tan, Bie; Xiong, Xia; Zhang, Yuzhe; Liu, Gang; Yao, Jiming; Li, Tiejun; Yin, Yulong

    2017-09-01

    Lysine (Lys) is a common limiting amino acids (AA) for humans and animals and plays an important role in cell proliferation and metabolism, while metabolism of Lys deficiency and its dipeptide is still obscure. Thus, this study mainly investigated the effects of Lys deficiency and Lys-Lys dipeptide on apoptosis and AA metabolism in vitro and in vivo models. Lys deficiency induced cell-cycle arrest and apoptosis and upregulated Lys transporters in vitro and in vivo. SLC7A11, a cystine-glutamate antiporter, was markedly upregulated by Lys deficiency and then further mediated cystine uptake and glutamate release, which was negatively regulated by cystine and glutamate transporters. Meanwhile, Lys deprivation upregulated pept1 expression, which might improve Lys-Lys dipeptide absorption to compensate for the reduced Lys availability. Lys-Lys dipeptide alleviated Lys deficiency induced cell-cycle arrest and apoptosis and influenced AA metabolism. Furthermore, the mammalian target of rapamycin signal might be involved in sensing cellular Lys starvation and Lys-Lys dipeptide. Altogether, these studies suggest that Lys deficiency impairs AA metabolism and causes apoptosis. Lys-Lys dipeptide serves as a Lys source and alleviates Lys deficiency induced cellular imbalance. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. H(2) enhances arabidopsis salt tolerance by manipulating ZAT10/12-mediated antioxidant defence and controlling sodium exclusion.

    Science.gov (United States)

    Xie, Yanjie; Mao, Yu; Lai, Diwen; Zhang, Wei; Shen, Wenbiao

    2012-01-01

    The metabolism of hydrogen gas (H(2)) in bacteria and algae has been extensively studied for the interesting of developing H(2)-based fuel. Recently, H(2) is recognized as a therapeutic antioxidant and activates several signalling pathways in clinical trials. However, underlying physiological roles and mechanisms of H(2) in plants as well as its signalling cascade remain unknown. In this report, histochemical, molecular, immunological and genetic approaches were applied to characterize the participation of H(2) in enhancing Arabidopsis salt tolerance. An increase of endogenous H(2) release was observed 6 hr after exposure to 150 mM NaCl. Arabidopsis pretreated with 50% H(2)-saturated liquid medium, mimicking the induction of endogenous H(2) release when subsequently exposed to NaCl, effectively decreased salinity-induced growth inhibition. Further results showed that H(2) pretreatment modulated genes/proteins of zinc-finger transcription factor ZAT10/12 and related antioxidant defence enzymes, thus significantly counteracting the NaCl-induced reactive oxygen species (ROS) overproduction and lipid peroxidation. Additionally, H(2) pretreatment maintained ion homeostasis by regulating the antiporters and H(+) pump responsible for Na(+) exclusion (in particular) and compartmentation. Genetic evidence suggested that SOS1 and cAPX1 might be the target genes of H(2) signalling. Overall, our findings indicate that H(2) acts as a novel and cytoprotective regulator in coupling ZAT10/12-mediated antioxidant defence and maintenance of ion homeostasis in the improvement of Arabidopsis salt tolerance.

  8. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    International Nuclear Information System (INIS)

    Ulrich-Baker, M.G.; Wang, P.; Fitzpatrick, L.; Johnson, L.R.

    1988-01-01

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na + -H + exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of [ 3 H]thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na + -H + antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity

  9. Amiloride inhibits rat mucosal ornithine decarboxylase activity and DNA synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Ulrich-Baker, M.G.; Wang, P.; Fitzpatrick, L.; Johnson, L.R. (Univ. of Texas Health Science Center, Houston (USA))

    1988-03-01

    Refeeding fasted rats induces a dramatic trophic response in gastrointestinal mucosa and is associated with elevations in both rate of DNA synthesis and ornithine decarboxylase (ODC) activity. The signal for these increases is unknown. Amiloride prevents cell alkalinization by blocking Na{sup +}-H{sup +} exchange at apical epithelial cell membranes. In study 1, rats were fasted 48 h, treated with amiloride (0.5 to 500 mg/kg), and refed for 4 h. Refeeding increased ODC activities in the jejunal mucosa (X8) and liver (X19) but not in the oxyntic gland mucosa. In the jejunum, but not the liver, the activation of ODC was completely abolished by 100 mg/kg amiloride. In study 2, the rate of DNA synthesis was determine by measuring the rate of ({sup 3}H)thymidine incorporation 16 h after refeeding. Refeeding resulted in significantly increased rates of DNA synthesis over fasted levels, and amiloride at 100 mg/kg significantly reduced the elevations in the jejenum and liver. In conclusion, amiloride inhibits the postprandial increases in jejunal ODC activity and DNA synthesis in the jejunum and liver. The results indicate that (1) the Na{sup +}-H{sup +} antiport is essential to the increased ODC activity in the jejunum and liver after a meal and (2) increases in DNA synthesis and their suppression by amiloride are not necessary linked to ODC activity.

  10. Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese.

    Science.gov (United States)

    Cremonesi, Paola; Vanoni, Laura; Morandi, Stefano; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

    2011-03-30

    A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10(3)CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Chloride Transport through Supramolecular Barrel-Rosette Ion Channels: Lipophilic Control and Apoptosis-Inducing Activity.

    Science.gov (United States)

    Saha, Tanmoy; Gautam, Amitosh; Mukherjee, Arnab; Lahiri, Mayurika; Talukdar, Pinaki

    2016-12-21

    Despite the great interest in artificial ion channel design, only a small number of channel-forming molecules are currently available for addressing challenging problems, particularly in the biological systems. Recent advances in chloride-mediated cell death, aided by synthetic ion carriers, encouraged us to develop chloride selective supramolecular ion channels. The present work describes vicinal diols, tethered to a rigid 1,3-diethynylbenzene core, as pivotal moieties for the barrel-rosette ion channel formation, and the activity of such channels was tuned by controlling the lipophilicity of designed monomers. Selective transport of chloride ions via an antiport mechanism and channel formation in the lipid bilayer membranes were confirmed for the most active molecule. A theoretical model of the supramolecular barrel-rosette, favored by a network of intermolecular hydrogen bonding, has been proposed. The artificial ion-channel-mediated transport of chloride into cells and subsequent disruption of cellular ionic homeostasis were evident. Perturbation of chloride homeostasis in cells instigates cell death by inducing the caspase-mediated intrinsic pathway of apoptosis.

  12. The effects of environmental deuterium on normal and neoplastic cultured cell development

    International Nuclear Information System (INIS)

    Bild, W.; Schuller, T.; Zhihai, Qin; Blankenstein, T.; Nastasa, V.; Haulica, I.

    2000-01-01

    The powdered culture media (RPMI - 1640) were reconstituted either with normal distilled water (150 ppm deuterium) either with deuterium - depleted water (DDW) in various concentrations (30, 60, 90 ppm) and sterilized by filtration with 0.2 μm filters. The cell lines used were NIH (normal mouse fibroblasts), RAG (mouse renal carcinoma) and TS/A (mouse mammary adenocarcinoma). In auxiliary tests, BAIBC mouse splenocytes in direct culture were used, stimulated for growth with concanavalin A or LPS (bacterial lipopolysaccharide). The estimation of the growth was made using the MTT assay or direct counting with trypan blue exclusion. The following results were obtained: Deuterium - depleted water had a stimulating effect on cell growth, the most important stimulating action being from the 90 ppm deuterium-water. The growth curves show, in a first phase, a stimulation of the rapid -growing neoplastic cells, followed by a slower growth of the normal cells. Amiloride 100 mM blocking of the Na + /K + membrane pump did not affect the cell growth curves, while the lansoprazole 100 mM blocking of the K + /H + ATP-ase brought the growth curves at the level of those with normal water. This might show an eventual involvement of the K + /H + antiport in the stimulating effects of the DDW. (authors)

  13. Characterization of the YdeO regulon in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Yuki Yamanaka

    Full Text Available Enterobacteria are able to survive under stressful conditions within animals, such as acidic conditions in the stomach, bile salts during transfer to the intestine and anaerobic conditions within the intestine. The glutamate-dependent (GAD system plays a major role in acid resistance in Escherichia coli, and expression of the GAD system is controlled by the regulatory cascade consisting of EvgAS > YdeO > GadE. To understand the YdeO regulon in vivo, we used ChIP-chip to interrogate the E. coli genome for candidate YdeO binding sites. All of the seven operons identified by ChIP-chip as being potentially regulated by YdeO were confirmed as being under the direct control of YdeO using RT-qPCR, EMSA, DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified four stress-response transcription factors, DctR, NhaR, GadE, and GadW and enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation of the GAD system and NhaR is an activator for the sodium/proton antiporter gene. In conjunction with co-transcribed Slp, DctR is involved in protection against metabolic endoproducts under acidic conditions. Taken all together, we suggest that YdeO is a key regulator of E. coli survival in both acidic and anaerobic conditions.

  14. Response of Desulfovibrio vulgaris to Alkaline Stress

    Energy Technology Data Exchange (ETDEWEB)

    Stolyar, S.; He, Q.; He, Z.; Yang, Z.; Borglin, S.E.; Joyner, D.; Huang, K.; Alm, E.; Hazen, T.C.; Zhou, J.; Wall, J.D.; Arkin, A.P.; Stahl, D.A.

    2007-11-30

    The response of exponentially growing Desulfovibrio vulgarisHildenborough to pH 10 stress was studied using oligonucleotidemicroarrays and a study set of mutants with genes suggested by microarraydata to be involved in the alkaline stress response deleted. The datashowed that the response of D. vulgaris to increased pH is generallysimilar to that of Escherichia coli but is apparently controlled byunique regulatory circuits since the alternative sigma factors (sigma Sand sigma E) contributing to this stress response in E. coli appear to beabsent in D. vulgaris. Genes previously reported to be up-regulated in E.coli were up-regulated in D. vulgaris; these genes included three ATPasegenes and a tryptophan synthase gene. Transcription of chaperone andprotease genes (encoding ATP-dependent Clp and La proteases and DnaK) wasalso elevated in D. vulgaris. As in E. coli, genes involved in flagellumsynthesis were down-regulated. The transcriptional data also identifiedregulators, distinct from sigma S and sigma E, that are likely part of aD. vulgaris Hildenborough-specific stress response system.Characterization of a study set of mutants with genes implicated inalkaline stress response deleted confirmed that there was protectiveinvolvement of the sodium/proton antiporter NhaC-2, tryptophanase A, andtwo putative regulators/histidine kinases (DVU0331 andDVU2580).

  15. The architecture of respiratory complex I.

    Science.gov (United States)

    Efremov, Rouslan G; Baradaran, Rozbeh; Sazanov, Leonid A

    2010-05-27

    Complex I is the first enzyme of the respiratory chain and has a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation by an unknown mechanism. Dysfunction of complex I has been implicated in many human neurodegenerative diseases. We have determined the structure of its hydrophilic domain previously. Here, we report the alpha-helical structure of the membrane domain of complex I from Escherichia coli at 3.9 A resolution. The antiporter-like subunits NuoL/M/N each contain 14 conserved transmembrane (TM) helices. Two of them are discontinuous, as in some transporters. Unexpectedly, subunit NuoL also contains a 110-A long amphipathic alpha-helix, spanning almost the entire length of the domain. Furthermore, we have determined the structure of the entire complex I from Thermus thermophilus at 4.5 A resolution. The L-shaped assembly consists of the alpha-helical model for the membrane domain, with 63 TM helices, and the known structure of the hydrophilic domain. The architecture of the complex provides strong clues about the coupling mechanism: the conformational changes at the interface of the two main domains may drive the long amphipathic alpha-helix of NuoL in a piston-like motion, tilting nearby discontinuous TM helices, resulting in proton translocation.

  16. Mechanisms of Sodium Transport in Plants—Progresses and Challenges

    Directory of Open Access Journals (Sweden)

    Monika Keisham

    2018-02-01

    Full Text Available Understanding the mechanisms of sodium (Na+ influx, effective compartmentalization, and efflux in higher plants is crucial to manipulate Na+ accumulation and assure the maintenance of low Na+ concentration in the cytosol and, hence, plant tolerance to salt stress. Na+ influx across the plasma membrane in the roots occur mainly via nonselective cation channels (NSCCs. Na+ is compartmentalized into vacuoles by Na+/H+ exchangers (NHXs. Na+ efflux from the plant roots is mediated by the activity of Na+/H+ antiporters catalyzed by the salt overly sensitive 1 (SOS1 protein. In animals, ouabain (OU-sensitive Na+, K+-ATPase (a P-type ATPase mediates sodium efflux. The evolution of P-type ATPases in higher plants does not exclude the possibility of sodium efflux mechanisms similar to the Na+, K+-ATPase-dependent mechanisms characteristic of animal cells. Using novel fluorescence imaging and spectrofluorometric methodologies, an OU-sensitive sodium efflux system has recently been reported to be physiologically active in roots. This review summarizes and analyzes the current knowledge on Na+ influx, compartmentalization, and efflux in higher plants in response to salt stress.

  17. Development of a primary microglia screening assay and its use to characterize inhibition of system xc- by erastin and its analogs

    Directory of Open Access Journals (Sweden)

    Mariana Figuera-Losada

    2017-03-01

    Full Text Available The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System xC- cystine/glutamate antiporter (SXC, a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC50 values in agreement with previous studies using established cell lines.

  18. Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses.

    Science.gov (United States)

    Mo, Chunyan; Wan, Shumin; Xia, Youquan; Ren, Ning; Zhou, Yang; Jiang, Xingyu

    2018-01-01

    Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR), and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10 , and Na + /H + antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

  19. A plant plasma membrane Ca2+ pump is required for normal pollen tube growth and fertilization.

    Science.gov (United States)

    Schiøtt, Morten; Romanowsky, Shawn M; Baekgaard, Lone; Jakobsen, Mia Kyed; Palmgren, Michael G; Harper, Jeffrey F

    2004-06-22

    Ca(2+) signals are thought to play important roles in plant growth and development, including key aspects of pollen tube growth and fertilization. The dynamics of a Ca(2+) signal are largely controlled by influx (through channels) and efflux (through pumps and antiporters). The Arabidopsis genome encodes 14 Ca(2+) pumps, 10 of which belong to a family of autoinhibited Ca(2+) ATPases (ACA) that are predicted to be activated by Ca(2+)/calmodulin. Here, we show that isoform ACA9 is expressed primarily in pollen and localized to the plasma membrane. Three independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] gene disruptions of ACA9 were found to result in partial male sterility. Complementation was observed by using a ACA9-yellow fluorescence protein (YFP) fusion that displayed plasma membrane localization. Mutant aca9 pollen displayed a reduced growth potential and a high frequency of aborted fertilization, resulting in a >80% reduction in seed set. These findings identify a plasma membrane Ca(2+) transporter as a key regulator of pollen development and fertilization in flowering plants.

  20. Genome structures and halophyte-specific gene expression of the extremophile Thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and Arabidopsis.

    Science.gov (United States)

    Oh, Dong-Ha; Dassanayake, Maheshi; Haas, Jeffrey S; Kropornika, Anna; Wright, Chris; d'Urzo, Matilde Paino; Hong, Hyewon; Ali, Shahjahan; Hernandez, Alvaro; Lambert, Georgina M; Inan, Gunsu; Galbraith, David W; Bressan, Ray A; Yun, Dae-Jin; Zhu, Jian-Kang; Cheeseman, John M; Bohnert, Hans J

    2010-11-01

    The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5' untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species.

  1. Partial purification of the ATP-driven calcium pump of Streptococcus sanguis

    Energy Technology Data Exchange (ETDEWEB)

    Lynn, A.R.; Rosen, B.P.

    1986-05-01

    ATP-dependent transport of calcium has been observed in several species of streptococci as uptake of /sup 45/Ca/sup 2 +/ into everted membrane vesicles. Membranes from Streptococcus sanguis and Streptococcus faecalis were solubilized with octyl-..beta..-D-glucoside or Triton X-100, and the extracts reconstituted into proteoliposomes containing Escherichia coli or soybean phospholipid. Calcium transport in reconstituted proteoliposomes was insensitive to the ionophores nigericin and valinomycin and was unaffected by the F/sub 0/F/sub 1/ inhibitor N,N'-dicyclohexylcarbodiimide. Uptake was inhibited by ortho-vanadate with a K/sub i/ in the micromolar range. These results demonstrate that the reconstituted transport activities are not the result of ATP-driven proton pumping via the F/sub 0/F/sub 1/ coupled to a calcium/proton antiporter and suggest that existence of a calcium translocating ATPase. Partial purification of the transport activity from Streptococcus sanguis has been achieved using density gradient centrifugation and FPLC.

  2. Active potassium extrusion regulated by intracellular pH in Streptococcus faecalis.

    Science.gov (United States)

    Kakinuma, Y; Igarashi, K

    1988-10-05

    Potassium extrusion in bacteria is thought to play a role in the regulation of the cytoplasmic pH; in several organisms, it has been ascribed to secondary antiport of K+ for protons. Streptococcus faecalis exhibited a distinctive pattern: potassium extrusion occurred only when the cytoplasmic pH was alkaline and required the generation of ATP. The key observation is that glycolyzing cells suspended in an alkaline medium extruded K+, even against a K+ concentration gradient, provided the medium contained a weak permeant base (e.g. diethanolamine or methylamine). The amines render the cytoplasmic pH alkaline; when conditions were arranged to keep the cytoplasm neutral, no K+ extrusion was seen. Potassium extrusion required the presence of either glucose or arginine and was unaffected by protonophores and by inhibition of the F1Fo-ATPase. When the medium contained [14C]methylamine, the cells accumulated the base to an extent stoichiometrically equivalent to the K+ lost. Concurrently, the cytoplasmic pH fell from 8.8 to 7.6, at which point K+ extrusion ceased. The results suggest that K+ extrusion is due to an ATP-driven transport system that expels K+ by exchange for H+ and is active only at alkaline cytoplasmic pH.

  3. Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.

    Science.gov (United States)

    Soysa, Radika; Venselaar, Hanka; Poston, Jacqueline; Ullman, Buddy; Hasne, Marie-Pierre

    2013-06-15

    The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247-Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.

  4. An overview of techniques for the measurement of calcium distribution, calcium fluxes, and cytosolic free calcium in mammalian cells

    International Nuclear Information System (INIS)

    Borle, A.B.

    1990-01-01

    An array of techniques can be used to study cell calcium metabolism that comprises several calcium compartments and many types of transport systems such as ion channels, ATP-dependent pumps, and antiporters. The measurement of total call calcium brings little information of value since 60 to 80% of total cell calcium is actually bound to the extracellular glycocalyx. Cell fractionation and differential centrifugation have been used to study intracellular Ca 2+ compartmentalization, but the methods suffer from the possibility of Ca 2+ loss or redistribution among cell fractions. Steady-state kinetic analyses of 45 Ca uptake or desaturation curves have been used to study the distribution of Ca 2+ among various kinetic pools in living cells and their rate of Ca 2+ exchange, but the analyses are constrained by many limitations. Nonsteady-state tracer studies can provide information about rapid changes in calcium influx or efflux in and out of the cell. Zero-time kinetics of 45 Ca uptake can detect instantaneous changes in calcium influx, while 45 Ca fractional efflux ratio, can detect rapid stimulations or inhibitions of calcium efflux out of cells. The best strategy to study cell calcium metabolism is to use several different methods that focus on a specific problem from widely different angles

  5. High-throughput single-molecule force spectroscopy for membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios [M E Mueller Institute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel (Switzerland); Ratera, Merce; Palacin, Manuel [Institute for Research in Biomedicine, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona (Spain); Bippes, Christian A; Mueller, Daniel J [BioTechnology Center, Technical University, Tatzberg 47, D-01307 Dresden (Germany)], E-mail: andreas.engel@unibas.ch, E-mail: dimitrios.fotiadis@mci.unibe.ch

    2008-09-24

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether {approx}400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with {approx}200 (AdiC) and {approx}400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  6. An overview of membrane transport proteins in Saccharomyces cerevisiae.

    Science.gov (United States)

    Andre, B

    1995-12-01

    All eukaryotic cells contain a wide variety of proteins embedded in the plasma and internal membranes, which ensure transmembrane solute transport. It is now established that a large proportion of these transport proteins can be grouped into families apparently conserved throughout organisms. This article presents the data of an in silicio analysis aimed at establishing a preliminary classification of membrane transport proteins in Saccharomyces cerevisiae. This analysis was conducted at a time when about 65% of all yeast genes were available in public databases. In addition to approximately 60 transport proteins whose function was at least partially known, approximately 100 deduced protein sequences of unknown function display significant sequence similarity to membrane transport proteins characterized in yeast and/or other organisms. While some protein families have been well characterized by classical genetic experimental approaches, others have largely if not totally escaped characterization. The proteins revealed by this in silicio analysis also include a putative K+ channel, proteins similar to aquaporins of plant and animal origin, proteins similar to Na+-solute symporters, a protein very similar to electroneural cation-chloride cotransporters, and a putative Na+-H+ antiporter. A new research area is anticipated: the functional analysis of many transport proteins whose existence was revealed by genome sequencing.

  7. The potential of Na+/Ca2+ exchange blockers in the treatment of cardiac disease.

    Science.gov (United States)

    Hobai, Ion A; O'Rourke, Brian

    2004-06-01

    The Na(+)/Ca(2+) exchanger (NCX), a surface membrane antiporter, is the primary pathway for Ca(2+) efflux from the cardiac cell and a determinant of both the electrical and contractile state of the heart. Enhanced expression of NCX has recently been recognised as one of the molecular mechanisms that contributes to reduced Ca(2+) release, impaired contractility and an increased risk of arrhythmias during the development of cardiac hypertrophy and failure. The NCX has also been implicated in the mechanism of arrhythmias and cellular injury associated with ischaemia and reperfusion. Hence, NCX blockade represents a potential therapeutic strategy for treating cardiac disease, however, its reversibility and electrogenic properties must be taken into consideration when predicting the outcome. NCX inhibition has been demonstrated to be protective against ischaemic injury and to have a positive inotropic and antiarrhythmic effect in failing heart cells. However, progress has been impaired by the absence of clinically useful agents. Two drugs, KB-R7943 and SEA-0400, have been developed as NCX blockers but both lack specificity. Selective peptide inhibitors have been well characterised but are active only when delivered to the intracellular space. Gene therapy strategies may circumvent the latter problem in the future. This review discusses the effects of NCX blockade, supporting its potential as a new cardiovascular therapeutic strategy.

  8. Na+/K+ pump interacts with the h-current to control bursting activity in central pattern generator neurons of leeches

    Science.gov (United States)

    Kueh, Daniel; Barnett, William H; Cymbalyuk, Gennady S; Calabrese, Ronald L

    2016-01-01

    The dynamics of different ionic currents shape the bursting activity of neurons and networks that control motor output. Despite being ubiquitous in all animal cells, the contribution of the Na+/K+ pump current to such bursting activity has not been well studied. We used monensin, a Na+/H+ antiporter, to examine the role of the pump on the bursting activity of oscillator heart interneurons in leeches. When we stimulated the pump with monensin, the period of these neurons decreased significantly, an effect that was prevented or reversed when the h-current was blocked by Cs+. The decreased period could also occur if the pump was inhibited with strophanthidin or K+-free saline. Our monensin results were reproduced in model, which explains the pump’s contributions to bursting activity based on Na+ dynamics. Our results indicate that a dynamically oscillating pump current that interacts with the h-current can regulate the bursting activity of neurons and networks. DOI: http://dx.doi.org/10.7554/eLife.19322.001 PMID:27588351

  9. Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

    Directory of Open Access Journals (Sweden)

    Saraiva R.M.

    1997-01-01

    Full Text Available Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+-dependent K+ currents (IKCa probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation

  10. A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects Escherichia coli from bile salt stress.

    Science.gov (United States)

    Paul, Stephanie; Alegre, Kamela O; Holdsworth, Scarlett R; Rice, Matthew; Brown, James A; McVeigh, Paul; Kelly, Sharon M; Law, Christopher J

    2014-05-01

    Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as Escherichia coli. Although the tripartite AcrAB-TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E. coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E. coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB-TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H(+) antiport. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  11. Digital force-feedback for protein unfolding experiments using atomic force microscopy

    International Nuclear Information System (INIS)

    Bippes, Christian A; Janovjak, Harald; Kedrov, Alexej; Muller, Daniel J

    2007-01-01

    Since its invention in the 1990s single-molecule force spectroscopy has been increasingly applied to study protein (un-)folding, cell adhesion, and ligand-receptor interactions. In most force spectroscopy studies, the cantilever of an atomic force microscope (AFM) is separated from a surface at a constant velocity, thus applying an increasing force to folded bio-molecules or bio-molecular bonds. Recently, Fernandez and co-workers introduced the so-called force-clamp technique. Single proteins were subjected to a defined constant force allowing their life times and life time distributions to be directly measured. Up to now, the force-clamping was performed by analogue PID controllers, which require complex additional hardware and might make it difficult to combine the force-feedback with other modes such as constant velocity. These points may be limiting the applicability and versatility of this technique. Here we present a simple, fast, and all-digital (software-based) PID controller that yields response times of a few milliseconds in combination with a commercial AFM. We demonstrate the performance of our feedback loop by force-clamp unfolding of single Ig27 domains of titin and the membrane proteins bacteriorhodopsin (BR) and the sodium/proton antiporter NhaA

  12. The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export.

    Science.gov (United States)

    Minamino, Tohru; Morimoto, Yusuke V; Hara, Noritaka; Aldridge, Phillip D; Namba, Keiichi

    2016-03-01

    The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+-protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

  13. A physiologically-oriented transcriptomic analysis of the midgut of Tenebrio molitor.

    Science.gov (United States)

    Moreira, Nathalia R; Cardoso, Christiane; Dias, Renata O; Ferreira, Clelia; Terra, Walter R

    2017-05-01

    Physiological data showed that T. molitor midgut is buffered at pH 5.6 at the two anterior thirds and at 7.9 at the posterior third. Furthermore, water is absorbed and secreted at the anterior and posterior midgut, respectively, driving a midgut counter flux of fluid. To look for the molecular mechanisms underlying these phenomena and nutrient absorption as well, a transcriptomic approach was used. For this, 11 types of transporters were chosen from the midgut transcriptome obtained by pyrosequencing (Roche 454). After annotation with the aid of databanks and manual curation, the sequences were validated by RT-PCR. The expression level of each gene at anterior, middle and posterior midgut and carcass (larva less midgut) was evaluated by RNA-seq taking into account reference sequences based on 454 contigs and reads obtained by Illumina sequencing. The data showed that sugar and amino acid uniporters and symporters are expressed along the whole midgut. In the anterior midgut are found transporters for NH 3 and NH 4 + that with a chloride channel may be responsible for acidifying the lumen. At the posterior midgut, bicarbonate-Cl - antiporter with bicarbonate supplied by carbonic anhydrase may alkalinize the lumen. Water absorption caused mainly by an anterior Na + -K + -2Cl - symporter and water secretion caused by a posterior K + -Cl - may drive the midgut counter flux. Transporters that complement the action of those described were also found. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. The Minimum Biological Energy Quantum

    Directory of Open Access Journals (Sweden)

    Volker Müller

    2017-10-01

    Full Text Available Some anaerobic archaea and bacteria live on substrates that do not allow the synthesis of one mol of ATP per mol of substrate via substrate level phosphorylation (SLP. Energy conservation in these cases is only possible by a chemiosmotic mechanism that involves the generation of an electrochemical ion gradient across the cytoplasmic membrane that then drives ATP synthesis via an ATP synthase. The minimal amount of energy required for ATP synthesis is thus dependent on the magnitude of the electrochemical ion gradient, the phosphorylation potential in the cell and the ion/ATP ratio of the ATP synthase. It was always thought that the minimum biological energy quantum is defined as the amount of energy required to translocate one ion across the cytoplasmic membrane. We will discuss the thermodynamics of the reactions involved in chemiosmosis and describe the limitations for ion transport and ATP synthesis that led to the proposal that at least −20 kJ/mol are required for ATP synthesis. We will challenge this hypothesis by arguing that the enzyme energizing the membrane may translocate net less than one ion: By using a primary pump connected to an antiporter module a stoichiometry below one can be obtained, implying that the minimum biological energy quantum that sustains life is even lower than assumed to date.

  15. Energizing porters by proton-motive force.

    Science.gov (United States)

    Nelson, N

    1994-11-01

    It is generally accepted that the chemistry of water was the most crucial determinant in shaping life on earth. Among the more important chemical features of water is its dissociation into protons and hydroxyl ions. The presence of relatively high proton concentrations in the ambient solution resulted in the evolution of proton pumps during the dawn of life on earth. These proton pumps maintained neutral pH inside the cells and generated electrochemical gradients of protons (proton-motive force) across their membranes. The existence of proton-motive force enabled the evolution of porters driven by it that are most probably among the more primitive porters in the world. The directionality of the substrate transport by the porters could be to both sides of the membranes because they can serve as proton symporters or antiporters. One of the most important subjects of this meeting is the mechanism by which proton-motive and other ion-motive forces drive the transport processes through porters. Is there a common mechanism of action for all proton-driven porters? Is there some common partial reaction by which we can identify the way that porters are energized by proton-motive force? Is there a common coupling between proton movement and uptake or secretion of certain molecules? Even a partial answer to one of these questions would advance our knowledge... or confusion. As my mentor Efraim Racker used to say: 'If you are not totally confused you do not understand the issue'.

  16. A nuclear calcium-sensing pathway is critical for gene regulation and salt stress tolerance in Arabidopsis.

    Science.gov (United States)

    Guan, Qingmei; Wu, Jianmin; Yue, Xiule; Zhang, Yanyan; Zhu, Jianhua

    2013-08-01

    Salt stress is an important environmental factor that significantly limits crop productivity worldwide. Studies on responses of plants to salt stress in recent years have identified novel signaling pathways and have been at the forefront of plant stress biology and plant biology in general. Thus far, research on salt stress in plants has been focused on cytoplasmic signaling pathways. In this study, we discovered a nuclear calcium-sensing and signaling pathway that is critical for salt stress tolerance in the reference plant Arabidopsis. Through a forward genetic screen, we found a nuclear-localized calcium-binding protein, RSA1 (SHORT ROOT IN SALT MEDIUM 1), which is required for salt tolerance, and identified its interacting partner, RITF1, a bHLH transcription factor. We show that RSA1 and RITF1 regulate the transcription of several genes involved in the detoxification of reactive oxygen species generated by salt stress and that they also regulate the SOS1 gene that encodes a plasma membrane Na(+)/H(+) antiporter essential for salt tolerance. Together, our results suggest the existence of a novel nuclear calcium-sensing and -signaling pathway that is important for gene regulation and salt stress tolerance.

  17. Overexpression of SlSOS2 (SlCIPK24) confers salt tolerance to transgenic tomato.

    Science.gov (United States)

    Huertas, Raúl; Olías, Raquel; Eljakaoui, Zakia; Gálvez, Francisco Javier; Li, Jun; De Morales, Paz Alvarez; Belver, Andrés; Rodríguez-Rosales, María Pilar

    2012-08-01

    The Ca(2+)-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na(+) and K(+) under saline conditions. We have identified and functionally characterized the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2. On the basis of protein sequence similarity and complementation studies in yeast and Arabidopsis, it can be concluded that SlSOS2 is the functional tomato homolog of Arabidopsis AtSOS2 and that SlSOS2 operates in a tomato SOS signal transduction pathway. The biotechnological potential of SlSOS2 to provide salt tolerance was evaluated by gene overexpression in tomato (Solanum lycopersicum L. cv. MicroTom). The better salt tolerance of transgenic plants relative to non-transformed tomato was shown by their faster relative growth rate, earlier flowering and higher fruit production when grown with NaCl. The increased salinity tolerance of SlSOS2-overexpressing plants was associated with higher sodium content in stems and leaves and with the induction and up-regulation of the plasma membrane Na(+)/H(+) (SlSOS1) and endosomal-vacuolar K(+), Na(+)/H(+) (LeNHX2 and LeNHX4) antiporters, responsible for Na(+) extrusion out of the root, active loading of Na(+) into the xylem, and Na(+) and K(+) compartmentalization. © 2012 Blackwell Publishing Ltd.

  18. Involvement of SlSOS2 in tomato salt tolerance

    Science.gov (United States)

    Belver, Andrés; Olías, Raquel; Huertas, Raúl; Rodríguez-Rosales, María Pilar

    2012-01-01

    The Ca2+-dependent SOS pathway has emerged as a key mechanism in the homeostasis of Na+ and K+ under saline conditions. We recently identified and functionally characterized by complementation studies in yeast and Arabidopsis the gene encoding the calcineurin-interacting protein kinase of the SOS pathway in tomato, SlSOS2.1 We also show evidences on the biotechnological potential of SlSOS2 conferring salt tolerance to transgenic tomato. The increased salinity tolerance of SlSOS2 overexpressing plants is associated with higher sodium content in stems and leaves. SlSOS2 overexpression upregulates the Na+/H+ exchange at the plasma membrane (SlSOS1) and K+,Na+/H+ antiport at the endosomal and vacuolar compartments (LeNHX2 and LeNHX4). Therefore, SlSOS2 seems to be involved in tomato salinity tolerance through regulation of Na+ extrusion from the root, active loading of Na+ into the xylem and Na+ and K+ compartmentalization. PMID:22825351

  19. High-throughput single-molecule force spectroscopy for membrane proteins

    International Nuclear Information System (INIS)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios; Ratera, Merce; Palacin, Manuel; Bippes, Christian A; Mueller, Daniel J

    2008-01-01

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ∼400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ∼200 (AdiC) and ∼400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications

  20. Knock-out of Arabidopsis AtNHX4 gene enhances tolerance to salt stress

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hong-Tao; Liu, Hua; Gao, Xiao-Shu [Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032 (China); Zhang, Hongxia, E-mail: hxzhang@sippe.ac.cn [Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032 (China)

    2009-05-08

    AtNHX4 belongs to the monovalent cation:proton antiporter-1 (CPA1) family in Arabidopsis. Several members of this family have been shown to be critical for plant responses to abiotic stress, but little is known on the biological functions of AtNHX4. Here, we provide the evidence that AtNHX4 plays important roles in Arabidopsis responses to salt stress. Expression of AtNHX4 was responsive to salt stress and abscisic acid. Experiments with CFP-AtNHX4 fusion protein indicated that AtNHX4 is vacuolar localized. The nhx4 mutant showed enhanced tolerance to salt stress, and lower Na{sup +} content under high NaCl stress compared with wild-type plants. Furthermore, heterologous expression of AtNHX4 in Escherichia coli BL21 rendered the transformants hypersensitive to NaCl. Deletion of the hydrophilic C-terminus of AtNHX4 dramatically increased the hypersensitivity of transformants, indicating that AtNHX4 may function in Na{sup +} homeostasis in plant cell, and its C-terminus plays a role in regulating the AtNHX4 activity.

  1. Comparative Study on Growth Performance of Transgenic (Over-Expressed OsNHX1 and Wild-Type Nipponbare under Different Salinity Regimes

    Directory of Open Access Journals (Sweden)

    Nurul Kahrani ISHAK

    2015-11-01

    Full Text Available Transgenic Nipponbare which over-expressed a Na+/H+ antiporter gene OsNHX1 was used to compare its growth performance, water status and photosynthetic efficiency with its wild type under varying salinity regimes. Chlorophyll content, quantum yield and photosynthetic rate were measured to assess the impact of salinity stress on photosynthetic efficiency for transgenic and wild-type Nipponbare. Effects of salinity on water status and gas exchange to both lines were studied by measuring water use efficiency, instantaneous transpiration rate and stomatal conductance. Dry shoot weight and leaf area were determined after three months of growth to assess the impacts of salinity on the growth of those two lines. Our study showed that both lines were affected by salinity stress, however, the transgenic line showed higher photosynthetic efficiency, better utilization of water, and better growth due to low transpiration rate and stomatal conductance. Reduction of photosynthetic efficiency exhibited by the wild-type Nipponbare was correlated to its poor growth under salinity stress.

  2. [Cold induced cDNA library construction of highland barley (Hordeum vulgare L. var. nudum Hk. f.) using suppression subtractive hybridization technology].

    Science.gov (United States)

    He, Tao; Jia, Jing Fen

    2008-12-01

    Cold-induced genes of highland barley (Hordeum vulgare L. var. nudum Hk. f.) were studied using suppression subtractive hybridization (SSH) technique. The cDNA from the materials treated with 4 degrees C was used as "tester", and that from the materials growing in green house (20+/-2 degrees C) as "driver". A subtractive library of highland barley including 640 cDNA clones was constructed in this study. Enzyme digestion of 32 clones chosen randomly from the library indicated that 87.5% of them contained inserts. The cDNA inserts of 16 clones were sequenced. Blast search analyses showed that these cDNAs were homologies to genes encoding the following proteins: metallothionein, protein kinase, ethylene signal transcription factor, bZIP transcription factor, zing finger transcription factor, ribulose-1,5-bisphosphate carboxylase, ribosomal protein, sodium: hydrogen antiporter, catalase, NADPH-cytochrome reductase, ascorbate peroxidase, DNA binding protein, and sugar transporter-like protein. These results indicated that the cDNA clones in the library were related to cold-induced genes, and suggested that the cold-tolerant mechanism of highland barley might be a complicated, interactive system involving multiple approaches and genes. Construction of subtractive cDNA library provided an advantage for further studies to isolate and clone cold-induced genes in highland barley.

  3. A Review of Temperature, pH, and Other Factors that Influence the Survival of Salmonella in Mayonnaise and Other Raw Egg Products

    Directory of Open Access Journals (Sweden)

    Thilini Piushani Keerthirathne

    2016-11-01

    Full Text Available Salmonellosis is one of the main causes of foodborne illnesses worldwide, with outbreaks predominately linked to contamination of eggs and raw egg products, such as mayonnaise. This review explores previous studies that have investigated Salmonella control mechanisms utilized in the production of raw egg mayonnaise and other food products. Apart from the use of pasteurized eggs, the main control mechanism identified is the pH of the raw egg products, which plays an important role in the consistency and stability while affecting the survival of Salmonella spp. However, currently there is no consensus regarding the critical pH limit for the control of Salmonella. The effectiveness of pH as a control mechanism is influenced by the type of acid used, with the effectiveness of lemon juice compared with vinegar highly debated. Additionally, Salmonella susceptibility to pH stresses may also be influenced by storage temperature (in some studies refrigeration temperatures protected Salmonella spp. from acidulants and is further complicated by the development of Salmonella cross-tolerance-induced responses, pH homeostasis achieved by the cellular antiport and symport systems, and acid tolerance response (ATR. These mechanisms all provide Salmonella with an added advantage to ensure survival under various pH conditions. Other confounding factors include the fat content, and the addition of NaCl, garlic and plant essential oils (PEOs from mint, cinnamon, cardamom and clove.

  4. Tissue P Systems With Channel States Working in the Flat Maximally Parallel Way.

    Science.gov (United States)

    Song, Bosheng; Perez-Jimenez, Mario J; Paun, Gheorghe; Pan, Linqiang

    2016-10-01

    Tissue P systems with channel states are a class of bio-inspired parallel computational models, where rules are used in a sequential manner (on each channel, at most one rule can be used at each step). In this work, tissue P systems with channel states working in a flat maximally parallel way are considered, where at each step, on each channel, a maximal set of applicable rules that pass from a given state to a unique next state, is chosen and each rule in the set is applied once. The computational power of such P systems is investigated. Specifically, it is proved that tissue P systems with channel states and antiport rules of length two are able to compute Parikh sets of finite languages, and such P systems with one cell and noncooperative symport rules can compute at least all Parikh sets of matrix languages. Some Turing universality results are also provided. Moreover, the NP-complete problem SAT is solved by tissue P systems with channel states, cell division and noncooperative symport rules working in the flat maximally parallel way; nevertheless, if channel states are not used, then such P systems working in the flat maximally parallel way can solve only tractable problems. These results show that channel states provide a frontier of tractability between efficiency and non-efficiency in the framework of tissue P systems with cell division (assuming P ≠ NP ).

  5. Application of plant biotechnology to address water and salt stress in developing countries (abstract)

    International Nuclear Information System (INIS)

    Masmoudi, K.

    2005-01-01

    Drought and salinity are major constraints on crop production and food security, and have adverse impact especially on socio-economic aspect in the Middle East and North Africa region. Studies of the physiological response of wheat to salt stress indicate that sequestering sodium that enters the leaf away from the cell cytosol, and enhancing osmotic adjustment capability, can ameliorate the negative impact of soil water salinity on plant growth. Sodium at high millimolar levels in the cytoplasm is toxic to plant and yeast cells, Sequestration of Na/sup +/ ions into the vacuole through the action of tonoplast proton pumps (an H/sup +/-ATPase in the case of yeast, and either an H/sup +/-pyrophosphatase (H/sup +/-PPase) or H/sup +/-ATPase in the case of plants) and an Na/sup +//H/sup +/ anti porter is one mechanism that confers salt tolerance to these organisms. The cloning and characterization of genes encoding these tonoplast transport proteins from crop plants may contribute to our understanding of how to enhance crop plant response to saline stress. We cloned wheat ortho logs of the Arabidopsis genes AtNHXI and AVP I using a wheat cDNA library, The full length sequence for the wheat Na/sup +//H/sup +/ anti porter (TNHX3) and the vacuolar H/sup +/-pyrophosphatase (TVP I) were deposited in Genbank database under the accession number AY296910 and AY296911, respectively. The deduced amino acid sequence of TNHXj is l homologous to the sequences of other NHX gene products cloned from wheat as well as barley and Arabidopsis. The vacuolar H/sup +/-PPase pump we cloned, TVP I is the first member of this gene family cloned from wheat. Function of TNHXj as a cation/proton antiporter was demonstrated using the nhxl yeast mutant. TNHXj was capable of suppressing the hygromycin sensitivity of nhxl. Functional characterization of the wheat H/sup +/-PPase TVP I was demonstrated using the yeast enal (plasma membrane Na/sup +/-efflux transporter) mutant. Expression of TVP I in enal

  6. A novel vanadium transporter of the Nramp family expressed at the vacuole of vanadium-accumulating cells of the ascidian Ascidia sydneiensis samea.

    Science.gov (United States)

    Ueki, Tatsuya; Furuno, Nobuaki; Michibata, Hitoshi

    2011-04-01

    Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified. We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function. Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO(2+) into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K(m) and V(max)) of AsNramp-mediated VO(2+) transport at pH 8.5 were 90nM and 9.1pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO(2+) under the same conditions. Excess Fe(2+), Cu(2+), Mn(2+), or Zn(2+) inhibited the transport of VO(2+). AsNramp was revealed to be a novel VO(2+)/H(+) antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H(+)-ATPase in vanadocytes. This is the first report of identification and functional analysis on a membrane transporter for vanadium ions. 2010 Elsevier B.V. All rights reserved.

  7. Inhibition of breast cancer-cell glutamate release with sulfasalazine limits cancer-induced bone pain.

    Science.gov (United States)

    Ungard, Robert G; Seidlitz, Eric P; Singh, Gurmit

    2014-01-01

    Cancer in bone is frequently a result of metastases from distant sites, particularly from the breast, lung, and prostate. Pain is a common and often severe pathological feature of cancers in bone, and is a significant impediment to the maintenance of quality of life of patients living with bone metastases. Cancer cell lines have been demonstrated to release significant amounts of the neurotransmitter and cell-signalling molecule l-glutamate via the system xC(-) cystine/glutamate antiporter. We have developed a novel mouse model of breast cancer bone metastases to investigate the impact of inhibiting cancer cell glutamate transporters on nociceptive behaviour. Immunodeficient mice were inoculated intrafemorally with the human breast adenocarcinoma cell line MDA-MB-231, then treated 14days later via mini-osmotic pumps inserted intraperitoneally with sulfasalazine, (S)-4-carboxyphenylglycine, or vehicle. Both sulfasalazine and (S)-4-carboxyphenylglycine attenuated in vitro cancer cell glutamate release in a dose-dependent manner via the system xC(-) transporter. Animals treated with sulfasalazine displayed reduced nociceptive behaviours and an extended time until the onset of behavioural evidence of pain. Animals treated with a lower dose of (S)-4-carboxyphenylglycine did not display this reduction in nociceptive behaviour. These results suggest that a reduction in glutamate secretion from cancers in bone with the system xC(-) inhibitor sulfasalazine may provide some benefit for treating the often severe and intractable pain associated with bone metastases. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  8. H(2 enhances arabidopsis salt tolerance by manipulating ZAT10/12-mediated antioxidant defence and controlling sodium exclusion.

    Directory of Open Access Journals (Sweden)

    Yanjie Xie

    Full Text Available BACKGROUND: The metabolism of hydrogen gas (H(2 in bacteria and algae has been extensively studied for the interesting of developing H(2-based fuel. Recently, H(2 is recognized as a therapeutic antioxidant and activates several signalling pathways in clinical trials. However, underlying physiological roles and mechanisms of H(2 in plants as well as its signalling cascade remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this report, histochemical, molecular, immunological and genetic approaches were applied to characterize the participation of H(2 in enhancing Arabidopsis salt tolerance. An increase of endogenous H(2 release was observed 6 hr after exposure to 150 mM NaCl. Arabidopsis pretreated with 50% H(2-saturated liquid medium, mimicking the induction of endogenous H(2 release when subsequently exposed to NaCl, effectively decreased salinity-induced growth inhibition. Further results showed that H(2 pretreatment modulated genes/proteins of zinc-finger transcription factor ZAT10/12 and related antioxidant defence enzymes, thus significantly counteracting the NaCl-induced reactive oxygen species (ROS overproduction and lipid peroxidation. Additionally, H(2 pretreatment maintained ion homeostasis by regulating the antiporters and H(+ pump responsible for Na(+ exclusion (in particular and compartmentation. Genetic evidence suggested that SOS1 and cAPX1 might be the target genes of H(2 signalling. CONCLUSIONS: Overall, our findings indicate that H(2 acts as a novel and cytoprotective regulator in coupling ZAT10/12-mediated antioxidant defence and maintenance of ion homeostasis in the improvement of Arabidopsis salt tolerance.

  9. Response of Ca2+-ATPase to clinorotaion of pea seedlings. O. M. Nedukha and E. L. Kordyum

    Science.gov (United States)

    Nedukha, Olena

    2016-07-01

    The present study was aimed to reveal of response of Ca2+-ATPase activity of cortex cells in distal elongation zone of Pisum sativum root to slow clinorotation. Pea seedlings were grown on a horizontal clinostat (2 rpm) and in the stationary control for 6 days. The electron-cytochemical method was used to examine the effects of imitated microgravity on the distribution of Ca2+-ATPase in outer layers of root cortex. The quantitative analysis of the density of cytochemical reaction products was measured using the Image J program. Electron microscopy showed the presence of electron-dense lead phosphate precipitated grains, the enzymatic activity reaction products on the plasma membrane, membranes of vesicular structures, endoplasmic reticulum (ER) and on organelles envelope in both of samples of the stationary control and clinorotated seedlings. We revealed the sensitivity of Ca2+-ATPase to clinorotation. The quantitative analysis of the area and density of enzymatic activity reaction products revealed that clinorotation led to the decrease of 3.4 times the density of reaction products on the plasma membrane and the increase of reaction products density on endomembranes and organelles membranes, in particular: in 2.2 times on mitochondria membranes; in 1.3 times - on membranes of ER; in 2.5 times - on tonoplast; by an order of magnitude greater - on contacting membranes of organelles with plasma membrane in comparison with such in cells of control samples. The data analysis can indicate an intensification of calcium pump on endomembranes, on envelopes of cytoplasmic organelles and nucleus. The obtained data suggest that the redistribution of Ca2+-ATPase activity in cells can be mediated by the activation of certain isoforms of enzyme or/and by an activation of Ca2+/H+ antiporter in plasma membrane that helps to maintain optimal calcium balance in plant cells under imitated microgravity.

  10. Polyamines Are Critical for the Induction of the Glutamate Decarboxylase-dependent Acid Resistance System in Escherichia coli *

    Science.gov (United States)

    Chattopadhyay, Manas K.; Tabor, Herbert

    2013-01-01

    As part of our studies on the biological functions of polyamines, we have used a mutant of Escherichia coli that lacks all the genes for polyamine biosynthesis for a global transcriptional analysis on the effect of added polyamines. The most striking early response to the polyamine addition is the increased expression of the genes for the glutamate-dependent acid resistance system (GDAR) that is important for the survival of the bacteria when passing through the acid environment of the stomach. Not only were the two genes for glutamate decarboxylases (gadA and gadB) and the gene for glutamate-γ-aminobutyrate antiporter (gadC) induced by the polyamine addition, but the various genes involved in the regulation of this system were also induced. We confirmed the importance of polyamines for the induction of the GDAR system by direct measurement of glutamate decarboxylase activity and acid survival. The effect of deletions of the regulatory genes on the GDAR system and the effects of overproduction of two of these genes were also studied. Strikingly, overproduction of the alternative σ factor rpoS and of the regulatory gene gadE resulted in very high levels of glutamate decarboxylase and almost complete protection against acid stress even in the absence of any polyamines. Thus, these data show that a major function of polyamines in E. coli is protection against acid stress by increasing the synthesis of glutamate decarboxylase, presumably by increasing the levels of the rpoS and gadE regulators. PMID:24097985

  11. Identification and characterization of orthologs of AtNHX5 and AtNHX6 in Brassica napus

    Directory of Open Access Journals (Sweden)

    Brett Andrew Ford

    2012-09-01

    Full Text Available Improving crop species by breeding for salt tolerance or introducing salt tolerant traits is one method of increasing crop yields in saline affected areas. The model plant species Arabidopsis thaliana has been extensively studied and there is substantial information available about the function and importance of many genes and proteins involved in salt tolerance. The identification and characterization of A. thaliana orthologs in species such as Brassica napus (oilseed rape can prove difficult due to the significant genomic changes that have occurred since their divergence approximately 20 million years ago. The recently released B. rapa genome provides an excellent resource for comparative studies of Arabidopsis and the cultivated Brassica species, and facilitates the identification of Brassica species orthologs which may be of agronomic importance. Sodium hydrogen antiporter (NHX proteins transport a sodium or potassium ion in exchange for a hydrogen ion in the other direction across a membrane. In A. thaliana there are eight members of the NHX family designated AtNHX1-8 that can be sub-divided into three clades (plasma membrane (PM, intracellular class I (IC-I and intracellular class II (IC-II based on their subcellular localization. In plants, many NHX proteins are primary determinants of salt tolerance and act by transporting Na+ out of the cytosol where it would otherwise accumulate to toxic levels. Significant work has been done analyzing both PM and IC-I clade members role in salt tolerance in a variety of plant species but relatively little analysis has been described for the IC-II clade. Here we describe the identification of B. napus orthologs of AtNHX5 and AtNHX6, using the Brassica rapa genome sequence, macro- and micro-synteny analysis, comparative expression and promoter motif analysis, and highlight the value of these multiple approaches for identifying true orthologs in closely related species with multiple paralogs.

  12. Hydrogen sulfide: role in ion channel and transporter modulation in the eye

    Directory of Open Access Journals (Sweden)

    Ya Fatou eNjie-Mbye

    2012-07-01

    Full Text Available Hydrogen sulfide (H2S, a colorless gas with a characteristic smell of rotten eggs, has been portrayed for decades as a toxic environmental pollutant. Since evidence of its basal production in mammalian tissues a decade ago, H2S has attracted substantial interest as a potential inorganic gaseous mediator with biological importance in cellular functions. Current research suggests that, next to its counterparts nitric oxide and carbon monoxide, H2S is an important multifunctional signaling molecule with pivotal regulatory roles in various physiological and pathophysiological processes as diverse as learning and memory, modulation of synaptic activities, cell survival, inflammation and maintenance of vascular tone in the central nervous and cardiovascular systems. In contrast, there are few reports of a regulatory role of H2S in the eye. Accumulating reports on the pharmacological role of H2S in ocular tissues indicate the existence of a functional trans-sulfuration pathway and a potential physiological role for H2S as a gaseous neuromodulator in the eye. Thus, understanding the role of H2S in vision-related processes is imperative to our expanding knowledge of this molecule as a gaseous mediator in ocular tissues. This review aims to provide a comprehensive and current understanding of the potential role of H2S as a signaling molecule in the eye. This objective is achieved by discussing the involvement of H2S in the regulation of (1 ion channels such as calcium (L-type, T-type and intracellular stores, potassium (KATP and small conductance channels and chloride channels, (2 glutamate transporters such as EAAT1/GLAST and the L-cystine/glutamate antiporter. The role of H2S as an important mediator in cellular functions and physiological processes that are triggered by its interaction with ion channels/transporters in the eye will also be discussed.

  13. Metabolic alkalosis.

    Science.gov (United States)

    Khanna, A; Kurtzman, N A

    2006-01-01

    Metabolic alkalosis is a primary pathophysiologic event characterized by the gain of bicarbonate or the loss of nonvolatile acid from extracellular fluid. The kidney preserves normal acid-base balance by two mechanisms: bicarbonate reclamation mainly in the proximal tubule and bicarbonate generation predominantly in the distal nephron. Bicarbonate reclamation is mediated mainly by a Na-H antiporter and to a smaller extent by the H-ATPase. The principal factors affecting HCO 3 reabsorption include effective arterial blood volume, glomerular filtration rate, chloride, and potassium. Bicarbonate regeneration is primarily affected by distal Na delivery and reabsorption, aldosterone, arterial pH, and arterial pCO2. To generate metabolic alkalosis, either a gain of base or a loss of acid, must occur. The loss of acid may be via the GI tract or by the kidney. Excess base may be gained by oral or parenteral HCO 3 administration or by lactate, acetate, or citrate administration. Factors that help maintain metabolic alkalosis include decreased glomerular filtration rate (GFR), volume contraction, hypokalemia, hypochloremia, and aldosterone excess. Clinical states associated with metabolic alkalosis are vomiting, mineralocorticoid excess, the adrenogenital syndrome, licorice ingestion, diuretic administration, and Bartter's and Gitelma's Syndromes. The effects of metabolic alkalosis on the body are varied and include effects on the central nervous system, myocardium, skeletal muscle, and the liver. Treatment of this disorder is simple, once the pathophysiology of the cause is delineated. Therapy consists of reversing the contributory factors promoting alkalosis and in severe cases, administration of carbonic anhydrase inhibitors, acid infusion, and low bicarbonate dialysis.

  14. In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

    Science.gov (United States)

    Pandey, Sonika; Patel, Manish Kumar; Mishra, Avinash; Jha, Bhavanath

    2016-01-01

    Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

  15. Water-mediated interactions enable smooth substrate transport in a bacterial efflux pump.

    Science.gov (United States)

    Vargiu, Attilio Vittorio; Ramaswamy, Venkata Krishnan; Malvacio, Ivana; Malloci, Giuliano; Kleinekathöfer, Ulrich; Ruggerone, Paolo

    2018-04-01

    Efflux pumps of the Resistance-Nodulation-cell Division superfamily confer multi-drug resistance to Gram-negative bacteria. The most-studied polyspecific transporter belonging to this class is the inner-membrane trimeric antiporter AcrB of Escherichia coli. In previous studies, a functional rotation mechanism was proposed for its functioning, according to which the three monomers undergo concerted conformational changes facilitating the extrusion of substrates. However, the molecular determinants and the energetics of this mechanism still remain unknown, so its feasibility must be proven mechanistically. A computational protocol able to mimic the functional rotation mechanism in AcrB was developed. By using multi-bias molecular dynamics simulations we characterized the translocation of the substrate doxorubicin driven by conformational changes of the protein. In addition, we estimated for the first time the free energy profile associated to this process. We provided a molecular view of the process in agreement with experimental data. Moreover, we showed that the conformational changes occurring in AcrB enable the formation of a layer of structured waters on the internal surface of the transport channel. This water layer, in turn, allows for a fairly constant hydration of the substrate, facilitating its diffusion over a smooth free energy profile. Our findings reveal a new molecular mechanism of polyspecific transport whereby water contributes by screening potentially strong substrate-protein interactions. We provided a mechanistic understanding of a fundamental process related to multi-drug transport. Our results can help rationalizing the behavior of other polyspecific transporters and designing compounds avoiding extrusion or inhibitors of efflux pumps. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

  16. Transport in Halobacterium Halobium: Light-Induced Cation-Gradients, Amino Acid Transport Kinetics, and Properties of Transport Carriers

    Science.gov (United States)

    Lanyi, Janos K.

    1977-01-01

    Cell envelope vesicles prepared from H. halobium contain bacteriorhodopsin and upon illumination protons are ejected. Coupled to the proton motive force is the efflux of Na(+). Measurements of Na-22 flux, exterior pH change, and membrane potential, Delta(psi) (with the dye 3,3'-dipentyloxadicarbocyanine) indicate that the means of Na(+) transport is sodium/proton exchange. The kinetics of the pH changes and other evidence suggests that the antiport is electrogenic (H(+)/Na(++ greater than 1). The resulting large chemical gradient for Na(+) (outside much greater than inside), as well as the membrane potential, will drive the transport of 18 amino acids. The I9th, glutamate, is unique in that its accumulation is indifferent to Delta(psi): this amino acid is transported only when a chemical gradient for Na(+) is present. Thus, when more and more NaCl is included in the vesicles glutamate transport proceeds with longer and longer lags. After illumination the gradient of H+() collapses within 1 min, while the large Na(+) gradient and glutamate transporting activity persists for 10- 15 min, indicating that proton motive force is not necessary for transport. A chemical gradient of Na(+), arranged by suspending vesicles loaded with KCl in NaCl, drives glutamate transport in the dark without other sources of energy, with V(sub max) and K(sub m) comparable to light-induced transport. These and other lines of evidence suggest that the transport of glutamate is facilitated by symport with Na(+), in an electrically neutral fashion, so that only the chemical component of the Na(+) gradient is a driving force.

  17. Converting the Yeast Arginine Can1 Permease to a Lysine Permease*

    Science.gov (United States)

    Ghaddar, Kassem; Krammer, Eva-Maria; Mihajlovic, Natalija; Brohée, Sylvain; André, Bruno; Prévost, Martine

    2014-01-01

    Amino acid uptake in yeast cells is mediated by about 16 plasma membrane permeases, most of which belong to the amino acid-polyamine-organocation (APC) transporter family. These proteins display various substrate specificity ranges. For instance, the general amino acid permease Gap1 transports all amino acids, whereas Can1 and Lyp1 catalyze specific uptake of arginine and lysine, respectively. Although Can1 and Lyp1 have different narrow substrate specificities, they are close homologs. Here we investigated the molecular rules determining the substrate specificity of the H+-driven arginine-specific permease Can1. Using a Can1-Lyp1 sequence alignment as a guideline and a three-dimensional Can1 structural model based on the crystal structure of the bacterial APC family arginine/agmatine antiporter, we introduced amino acid substitutions liable to alter Can1 substrate specificity. We show that the single substitution T456S results in a Can1 variant transporting lysine in addition to arginine and that the combined substitutions T456S and S176N convert Can1 to a Lyp1-like permease. Replacement of a highly conserved glutamate in the Can1 binding site leads to variants (E184Q and E184A) incapable of any amino acid transport, pointing to a potential role for this glutamate in H+ coupling. Measurements of the kinetic parameters of arginine and lysine uptake by the wild-type and mutant Can1 permeases, together with docking calculations for each amino acid in their binding site, suggest a model in which residues at positions 176 and 456 confer substrate selectivity at the ligand-binding stage and/or in the course of conformational changes required for transport. PMID:24448798

  18. Heterologous Production of an Energy-Conserving Carbon Monoxide Dehydrogenase Complex in the Hyperthermophile Pyrococcus furiosus

    Directory of Open Access Journals (Sweden)

    Gerrit Jan Schut

    2016-01-01

    Full Text Available Carbon monoxide (CO is an important intermediate in anaerobic carbon fixation pathways in acetogenesis and methanogenesis. In addition, some anaerobes can utilize CO as an energy source. In the hyperthermophilic archaeon Thermococcus onnurineus, which grows optimally at 80°C, CO oxidation and energy conservation is accomplished by a respiratory complex encoded by a 16-gene cluster containing a carbon monoxide dehydrogenase, a membrane-bound [NiFe]-hydrogenase and a Na+/H+ antiporter module. This complex oxidizes CO, evolves CO2 and H2, and generates a Na+ motive force that is used to conserve energy by a Na+-dependent ATP synthase. Herein we used a bacterial artificial chromosome to insert the 13.2 kb gene cluster encoding the CO-oxidizing respiratory complex of T. onnurineus into the genome of the heterotrophic archaeon, Pyrococcus furiosus, which grows optimally at 100°C. P. furiosus is normally unable to utilize CO, however, the recombinant strain readily oxidized CO and generated H2 at 80°C. Moreover, CO also served as an energy source and allowed the P. furiosus strain to grow with a limiting concentration of sugar or with peptides as the carbon source. Moreover, CO oxidation by P. furiosus was also coupled to the re-utilization, presumably for biosynthesis, of acetate generated by fermentation. The functional transfer of CO utilization between Thermococcus and Pyrococcus species demonstrated herein is representative of the horizontal gene transfer of an environmentally-relevant metabolic capability. The transfer of CO utilizing, hydrogen-producing genetic modules also has applications for biohydrogen production and a CO-based industrial platform for various thermophilic organisms.

  19. Components of calcium homeostasis in Archaeon Methanobacterium thermoautotrophicum

    International Nuclear Information System (INIS)

    Varecka, L.; Smigan, P.; Vancek, M.; Greksak, M.

    1998-01-01

    The cells of Archaea are interesting from several points of view. Among others there are: (a) the evolutionary relationship to procaryotes and eucaryotes and (b) the involvement of Na + and H + gradient in archaeal bio-energetics. The observations are presented which are devoted to the description of components of Ca 2+ homeostasis, an apparatus is vital for both procaryotic and eukaryotic organisms, in obligate anaerobe Methanobacterium thermoautotrophicum. This is, after the demonstration of the ATP-dependent Ca 2+ transport in Halobacterium halobium membrane vesicles, the first complex description of processes of Ca 2+ homeostasis in Archaea. The Ca 2+ influx and efflux was measured using radionuclide 4 5 Ca 2+ . The experiment were performed under strictly anaerobic conditions. The measurement of the membrane potential by means of 3 H-tetraphenyl phosphonium chloride showed that the presence of Na + depolarized the membrane from -110 to -60 mV. The growth of M. thermoautotrophicum and methanogenesis was suppressed but nor arrested by the presence EGTA suggesting that the Ca 2+ homeostasis may be involved in controlling these cellular functions. The results indicate the presence of three components involved in establishing the Ca 2+ homeostasis in cell of M. thermoautotrophicum. The first is the Ca 2+ -carrier mediating the CA 2+ influx driven by the proton motive force or the membrane potential. The Ca 2+ efflux is mediated by two transport systems, Na + /Ca 2+ and H + /Ca 2+ anti-porters. The evidence for the presence of the Ca 2+ -transporting ATPase was not obtained so far. (authors)

  20. Decrease of intracellular pH as possible mechanism of embryotoxicity of glycol ether alkoxyacetic acid metabolites

    International Nuclear Information System (INIS)

    Louisse, Jochem; Bai Yanqing; Verwei, Miriam; Sandt, Johannes J.M. van de; Blaauboer, Bas J.; Rietjens, Ivonne M.C.M.

    2010-01-01

    Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pH i ) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pH i in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pH i of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na + /H + -antiporter, corroborating an important role of the pH i in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pH i may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers.

  1. Calcium and proton transport in membrane vesicles from barley roots

    International Nuclear Information System (INIS)

    DuPont, F.M.; Windle, J.J.; Bush, D.S.; Jones, R.L.

    1990-01-01

    Ca 2+ uptake by membrane fractions from barley (Hordeum vulgare L. cv CM72) roots was characterized. Uptake of 45 Ca 2+ was measured in membrane vesicles obtained from continuous and discontinuous sucrose gradients. A single, large peak of Ca 2+ uptake coincided with the peak of proton transport by the tonoplast H + -ATPase. Depending on the concentration of Ca 2+ in the assay, Ca 2+ uptake was inhibited 50 to 75% by those combinations of ionophores and solutes that eliminated the pH gradient and membrane potential. However, 25 to 50% of the Ca 2+ uptake in the tonoplast-enriched fraction was not sensitive to ionophores but was inhibited by vanadate. The results suggest that 45 Ca uptake was driven by the low affinity, high capacity tonoplast Ca 2+ /nH + antiporter and also by a high affinity, lower capacity Ca 2+ -ATPase. The Ca 2+ -ATPase may be associated with tonoplast, Golgi or contaminating vesicles of unknown origin. No Ca 2+ transport was specifically associated with the distinct peak of endoplasmic reticulum that was identified by NADH cytochrome c reductase, choline phosphotransferase, and dolichol-P-mannosyl synthase activities. A small shoulder of Ca 2+ uptake in the plasma membrane region of the gradient was inhibited by vanadate and erythrosin B and may represent the activity of a separate plasma membrane Ca 2+ -ATPase. Vesicle volumes were estimated using electron spin resonance techniques, and intravesicular Ca 2+ concentrations were estimated to be as high as 5 millimolar. ATP-driven uptake of Ca 2+ created 800- to 2,000-fold concentration gradients within minutes. Problems in interpreting the effects of Ca 2+ on ATP-generated pH gradients are discussed and the suggestion is made that Ca 2+ dissipates pH gradients by a different mechanism than is responsible for Ca 2+ uptake into tonoplast vesicles

  2. Modulation of Lactobacillus plantarum gastrointestinal robustness by fermentation conditions enables identification of bacterial robustness markers.

    Directory of Open Access Journals (Sweden)

    Hermien van Bokhorst-van de Veen

    Full Text Available BACKGROUND: Lactic acid bacteria (LAB are applied worldwide in the production of a variety of fermented food products. Additionally, specific Lactobacillus species are nowadays recognized for their health-promoting effects on the consumer. To optimally exert such beneficial effects, it is considered of great importance that these probiotic bacteria reach their target sites in the gut alive. METHODOLOGY/PRINCIPAL FINDINGS: In the accompanying manuscript by Bron et al. the probiotic model organism Lactobacillus plantarum WCFS1 was cultured under different fermentation conditions, which was complemented by the determination of the corresponding molecular responses by full-genome transcriptome analyses. Here, the gastrointestinal (GI survival of the cultures produced was assessed in an in vitro assay. Variations in fermentation conditions led to dramatic differences in GI-tract survival (up to 7-log and high robustness could be associated with low salt and low pH during the fermentations. Moreover, random forest correlation analyses allowed the identification of specific transcripts associated with robustness. Subsequently, the corresponding genes were targeted by genetic engineering, aiming to enhance robustness, which could be achieved for 3 of the genes that negatively correlated with robustness and where deletion derivatives displayed enhanced survival compared to the parental strain. Specifically, a role in GI-tract survival could be confirmed for the lp_1669-encoded AraC-family transcription regulator, involved in capsular polysaccharide remodeling, the penicillin-binding protein Pbp2A involved in peptidoglycan biosynthesis, and the Na(+/H(+ antiporter NapA3. Moreover, additional physiological analysis established a role for Pbp2A and NapA3 in bile salt and salt tolerance, respectively. CONCLUSION: Transcriptome trait matching enabled the identification of biomarkers for bacterial (gut-robustness, which is important for our molecular

  3. Involvement of heme oxygenase-1 expression in neuroprotection by piceatannol, a natural analog and a metabolite of resveratrol, against glutamate-mediated oxidative injury in HT22 neuronal cells.

    Science.gov (United States)

    Son, Yong; Byun, Seung Jae; Pae, Hyun-Ock

    2013-08-01

    Neuronal cell death caused by oxidative stress is common in a variety of neural diseases and can be investigated in detail in cultured HT22 neuronal cells, where the amino acid glutamate at high concentrations causes glutathione depletion by inhibition of the glutamate/cystine antiporter system, intracellular accumulation of reactive oxygen species (ROS) and eventually oxidative stress-induced neuronal cell death. Using this paradigm, we have previously reported that resveratrol (3,5,4'-trans-trihydroxystilbene) protects HT22 neuronal cells from glutamate-induced oxidative stress by inducing heme oxygenase (HO)-1 expression. Piceatannol (3,5,4',3'-trans-trihydroxystilbene), which is a hydroxylated resveratrol analog and one of the resveratrol metabolites, is estimated to exert neuroprotective effect similar to that of resveratrol. The aim of this study, thus, is to determine whether piceatannol, similarly to resveratrol, would protect HT22 neuronal cells from glutamate-induced oxidative stress. Glutamate at high concentrations induced neuronal cell death and ROS formation. Piceatannol reduced glutamate-induced cell death and ROS formation. The observed cytoprotective effect was much higher when HT22 neuronal cells were pretreated with piceatannol for 6 or 12 h prior to glutamate treatment than when pretreated for 0.5 h. Piceatannol also increased HO-1 expression and HO activity via its activation of nuclear factor-E2-related factor 2 (Nrf2). Interestingly, neuroprotective effect of piceatannol was partly (but not completely) abolished by either down-regulation of HO-1 expression or blockage of HO-1 activity. Taken together, our results suggest that piceatannol, similar to resveratrol, is capable of protecting HT22 neuronal cells against glutamate-induced cell death, at least in part, by inducing Nrf2-dependent HO-1 expression.

  4. Comparative study of the active cadmium efflux systems operating at the plasma membrane and tonoplast of cucumber root cells.

    Science.gov (United States)

    Migocka, Magdalena; Papierniak, Anna; Kosatka, Ewelina; Klobus, Grazyna

    2011-10-01

    The strategies developed by plants to avoid the toxicity of cadmium (Cd) and other heavy metals involve active sequestration of metals into the apoplast and vacuoles. The protein systems excluding heavy metals from the cell cytosol localize to the plasma membrane and tonoplast and are energized either by ATP or by the electrochemical gradient generated by H(+)-ATPase or by V-ATPase and pyrophosphatase (PPase), respectively. In this work, a comparative study on the contribution of both the plasma membrane and tonoplast in the active detoxification of plant cells after treatment with Cd was performed. The studies using plants treated and untreated with Cd reveal that both, H(+)-coupled and MgATP-driven efflux of Cd across plasma membranes and tonoplast is markedly stimulated in the presence of Cd in the environment. Previous studies on plasma-membrane localized H(+)-coupled Cd efflux together with the present data demonstrating tonoplast H(+)/Cd(2+) antiport activity suggest that H(+)-coupled secondary transport of Cd displays a lower affinity for Cd when compared with Cd primary pumps driven by MgATP. In addition, it is shown that MgATP-energized Cd efflux across both membranes is significantly enhanced by cysteine, dithiothreitol, and glutathione. These results suggest that Cd is excluded from the cytosol through an energy-dependent system as a free ion as well as a complexed form. Although both membranes contribute in the active exclusion of ionized and complexed Cd from the cytosol, the overall calculation of Cd accumulation in the everted plasma membranes and vacuolar vesicles suggests that the tonoplast and vacuole have a major function in Cd efflux from the cytosol in the roots of cucumber subjected to Cd stress.

  5. Activity of tonoplast proton pumps and Na+/H+ exchange in potato cell cultures is modulated by salt.

    Science.gov (United States)

    Queirós, Filipa; Fontes, Natacha; Silva, Paulo; Almeida, Domingos; Maeshima, Masayoshi; Gerós, Hernâni; Fidalgo, Fernanda

    2009-01-01

    The efficient exclusion of excess Na from the cytoplasm and vacuolar Na(+) accumulation are the main mechanisms for the adaptation of plants to salt stress. This is typically carried out by transmembrane transport proteins that exclude Na(+) from the cytosol in exchange for H(+), a secondary transport process which is energy-dependent and driven by the proton-motive force generated by plasma-membrane and tonoplast proton pumps. Tonoplast enriched-vesicles from control and 150 mM NaCl-tolerant calli lines were used as a model system to study the activity of V-H(+)-PPase and V-H(+)-ATPase and the involvement of Na(+) compartmentalization into the vacuole as a mechanism of salt tolerance in Solanum tuberosum. Both ATP- and pyrophosphate (PP(i))-dependent H(+)-transport were higher in tonoplast vesicles from the salt-tolerant line than in vesicles from control cells. Western blotting of tonoplast proteins confirmed that changes in V-H(+)-PPase activity are correlated with increased protein amount. Conversely, immunodetection of the A-subunit of V-H(+)-ATPase revealed that a mechanism of post-translational regulation is probably involved. Na(+)-dependent dissipation of a pre-established pH gradient was used to measure Na(+)/H(+) exchange in tonoplast vesicles. The initial rates of proton efflux followed Michaelis-Menten kinetics and the V(max) of proton dissipation was 2-fold higher in NaCl-tolerant calli when compared to the control. H(+)-coupled exchange was specific for Na(+) and Li(+) and not for K(+). The increase of both the pH gradient across the tonoplast and the Na(+)/H(+) antiport activity in response to salt strongly suggests that Na(+) sequestration into the vacuole contributes to salt tolerance in potato.

  6. L-lactate transport in Ehrlich ascites-tumour cells.

    Science.gov (United States)

    Spencer, T L; Lehninger, A L

    1976-01-01

    Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids. PMID:7237

  7. Expression Patterns and Identified Protein-Protein Interactions Suggest That Cassava CBL-CIPK Signal Networks Function in Responses to Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    Chunyan Mo

    2018-03-01

    Full Text Available Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL protein and CBL-interacting protein kinase (CIPK is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL (MeCBL and 26 CIPK (MeCIPK genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBLs and CIPKs. Reverse-transcriptase polymerase chain reaction (RT-PCR analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle. The expression patterns of 7 CBL and 26 CIPK genes in response to NaCl, PEG, heat and cold stresses were analyzed by quantitative real-time PCR (qRT-PCR, and it was found that the expression of each was induced by multiple stimuli. Furthermore, we found that many pairs of CBLs and CIPKs could interact with each other via investigating the interactions between 8 CBL and 25 CIPK proteins using a yeast two-hybrid system. Yeast cells co-transformed with cassava MeCIPK24, MeCBL10, and Na+/H+ antiporter MeSOS1 genes exhibited higher salt tolerance compared to those with one or two genes. These results suggest that the cassava CBL-CIPK signal network might play key roles in response to abiotic stresses.

  8. The conserved nhaAR operon is drastically divergent between B2 and non-B2 Escherichia coli and is involved in extra-intestinal virulence.

    Science.gov (United States)

    Lescat, Mathilde; Reibel, Florence; Pintard, Coralie; Dion, Sara; Glodt, Jérémy; Gateau, Cecile; Launay, Adrien; Ledda, Alice; Cruveiller, Stephane; Cruvellier, Stephane; Tourret, Jérôme; Tenaillon, Olivier

    2014-01-01

    The Escherichia coli species is divided in phylogenetic groups that differ in their virulence and commensal distribution. Strains belonging to the B2 group are involved in extra-intestinal pathologies but also appear to be more prevalent as commensals among human occidental populations. To investigate the genetic specificities of B2 sub-group, we used 128 sequenced genomes and identified genes of the core genome that showed marked difference between B2 and non-B2 genomes. We focused on the gene and its surrounding region with the strongest divergence between B2 and non-B2, the antiporter gene nhaA. This gene is part of the nhaAR operon, which is in the core genome but flanked by mobile regions, and is involved in growth at high pH and high sodium concentrations. Consistently, we found that a panel of non-B2 strains grew faster than B2 at high pH and high sodium concentrations. However, we could not identify differences in expression of the nhaAR operon using fluorescence reporter plasmids. Furthermore, the operon deletion had no differential impact between B2 and non-B2 strains, and did not result in a fitness modification in a murine model of gut colonization. Nevertheless, sequence analysis and experiments in a murine model of septicemia revealed that recombination in nhaA among B2 strains was observed in strains with low virulence. Finally, nhaA and nhaAR operon deletions drastically decreased virulence in one B2 strain. This effect of nhaAR deletion appeared to be stronger than deletion of all pathogenicity islands. Thus, a population genetic approach allowed us to identify an operon in the core genome without strong effect in commensalism but with an important role in extra-intestinal virulence, a landmark of the B2 strains.

  9. Functional dissection of the proton pumping modules of mitochondrial complex I.

    Directory of Open Access Journals (Sweden)

    Stefan Dröse

    2011-08-01

    Full Text Available Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5 of the three subunits with homology to bacterial Mrp-type Na(+/H(+ antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.

  10. Fluorescence lifetime to image epidermal ionic concentrations

    Science.gov (United States)

    Behne, Martin J.; Barry, Nicholas P.; Moll, Ingrid; Gratton, Enrico; Mauro, Theodora M.

    2004-09-01

    Measurements of ionic concentrations in skin have traditionally been performed with an array of methods which either did not reveal detailed localization information, or only provided qualitative, not quantitative information. FLIM combines a number of advantages into a method ideally suited to visualize concentrations of ions such as H+ in intact, unperturbed epidermis and stratum corneum (SC). Fluorescence lifetime is dye concentration-independent, the method requires only low light intensities and is therefore not prone to photobleaching or phototoxic artifacts, and because multiphoton lasers of IR wavelength are used, light penetrates deep into intact tissue. The standard method to measure SC pH is the flat pH electrode, which provides reliable information only about surface pH changes, without further vertical or subcellular spatial resolution; i.e., specific microdomains such as the corneocyte interstices are not resolved, and the deeper SC is inaccessible without resorting to inherently disruptive stripping methods. Furthermore, the concept of a gradient of pH through the SC stems from such stripping experiments, but other confirmation for this concept is lacking. Our investigations into the SC pH distribution so far have revealed the crucial role of the Sodium/Hydrogen Antiporter NHE1 in generation of SC acidity, the colocalization of enzymatic lipid processing activity in the SC with acidic domains of the SC, and the timing and localization of emerging acidity in the SC of newborns. Together, these results have led to an improved understanding of the SC pH, its distribution, origin, and regulation. Future uses for this method include measurements of other ions important for epidermal processes, such as Ca2+, and a quantitative approach to topical drug penetration.

  11. [Pt(O,O'-acac)(γ-acac)(DMS)] alters SH-SY5Y cell migration and invasion by the inhibition of Na+/H+ exchanger isoform 1 occurring through a PKC-ε/ERK/mTOR Pathway.

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O'-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibits cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation.

  12. Chronic Inhibition of STAT3/STAT5 in Treatment-Resistant Human Breast Cancer Cell Subtypes: Convergence on the ROS/SUMO Pathway and Its Effects on xCT Expression and System xc- Activity

    Science.gov (United States)

    Linher-Melville, Katja; Nashed, Mina G.; Ungard, Robert G.; Haftchenary, Sina; Rosa, David A.; Gunning, Patrick T.; Singh, Gurmit

    2016-01-01

    Pharmacologically targeting activated STAT3 and/or STAT5 has been an active area of cancer research. The cystine/glutamate antiporter, system xc-, contributes to redox balance and export of intracellularly produced glutamate in response to up-regulated glutaminolysis in cancer cells. We have previously shown that blocking STAT3/5 using the small molecule inhibitor, SH-4-54, which targets the SH2 domains of both proteins, increases xCT expression, thereby increasing system xc- activity in human breast cancer cells. The current investigation demonstrates that chronic SH-4-54 administration, followed by clonal selection of treatment-resistant MDA-MB-231 and T47D breast cancer cells, elicits distinct subtype-dependent effects. xCT mRNA and protein levels, glutamate release, and cystine uptake are decreased relative to untreated passage-matched controls in triple-negative MDA-MB-231 cells, with the inverse occurring in estrogen-responsive T47D cells. This “ying-yang” effect is linked with a shifted balance between the phosphorylation status of STAT3 and STAT5, intracellular ROS levels, and STAT5 SUMOylation/de-SUMOylation. STAT5 emerged as a definitive negative regulator of xCT at the transcriptional level, while STAT3 activation is coupled with increased system xc- activity. We propose that careful classification of a patient’s breast cancer subtype is central to effectively targeting STAT3/5 as a therapeutic means of treating breast cancer, particularly given that xCT is emerging as an important biomarker of aggressive cancers. PMID:27513743

  13. Cotransport of sodium and chloride by the adult mammalian choroid plexus

    Energy Technology Data Exchange (ETDEWEB)

    Johanson, C.E.; Sweeney, S.M.; Parmelee, J.T.; Epstein, M.H. (Brown Univ./Rhode Island Hospital, Providence (USA))

    1990-02-01

    Cerebrospinal fluid formation stems primarily from the transport of Na and Cl in choroid plexus (CP). To characterize properties and modulation of choroidal transporters, we tested diuretics and other agents for ability to alter ion transport in vitro. Adult Sprague-Dawley rats were the source of CPs preincubated with drug for 20 min and then transferred to cerebrospinal fluid (CSF) medium containing 22Na or 36Cl with (3H)mannitol (extracellular correction). Complete base-line curves were established for cellular uptake of Na and Cl at 37 degrees C. The half-maximal uptake occurred at 12 s, so it was used to assess drug effects on rate of transport (nmol Na or Cl/mg CP). Bumetanide (10(-5) and 10(-4) M) decreased uptake of Na and Cl with maximal inhibition (up to 45%) at 10(-5) M. Another cotransport inhibitor, furosemide (10(-4) M), reduced transport of Na by 25% and Cl by 33%. However, acetazolamide (10(-4) M) and atriopeptin III (10(-7) M) significantly lowered uptake of Na (but not Cl), suggesting effect(s) other than on cotransport. The disulfonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 10(-4) M), known to inhibit Cl-HCO3 exchange, substantially reduced the transport of 36Cl. Bumetanide plus DIDS (both 10(-4) M) caused additive inhibition of 90% of Cl uptake, which provides strong evidence for the existence of both cotransport and antiport Cl carriers. Overall, this in vitro analysis, uncomplicated by variables of blood flow and neural tone, indicates the presence in rat CP of the cotransport of Na and Cl in addition to the established Na-H and Cl-HCO3 exchangers.

  14. Sublethal Concentrations of Carbapenems Alter Cell Morphology and Genomic Expression of Klebsiella pneumoniae Biofilms

    Science.gov (United States)

    Van Laar, Tricia A.; Chen, Tsute; You, Tao

    2015-01-01

    Klebsiella pneumoniae, a Gram-negative bacterium, is normally associated with pneumonia in patients with weakened immune systems. However, it is also a prevalent nosocomial infectious agent that can be found in infected surgical sites and combat wounds. Many of these clinical strains display multidrug resistance. We have worked with a clinical strain of K. pneumoniae that was initially isolated from a wound of an injured soldier. This strain demonstrated resistance to many commonly used antibiotics but sensitivity to carbapenems. This isolate was capable of forming biofilms in vitro, contributing to its increased antibiotic resistance and impaired clearance. We were interested in determining how sublethal concentrations of carbapenem treatment specifically affect K. pneumoniae biofilms both in morphology and in genomic expression. Scanning electron microscopy showed striking morphological differences between untreated and treated biofilms, including rounding, blebbing, and dimpling of treated cells. Comparative transcriptome analysis using RNA sequencing (RNA-Seq) technology identified a large number of open reading frames (ORFs) differentially regulated in response to carbapenem treatment at 2 and 24 h. ORFs upregulated with carbapenem treatment included genes involved in resistance, as well as those coding for antiporters and autoinducers. ORFs downregulated included those coding for metal transporters, membrane biosynthesis proteins, and motility proteins. Quantitative real-time PCR validated the general trend of some of these differentially regulated ORFs. Treatment of K. pneumoniae biofilms with sublethal concentrations of carbapenems induced a wide range of phenotypic and gene expression changes. This study reveals some of the mechanisms underlying how sublethal amounts of carbapenems could affect the overall fitness and pathogenic potential of K. pneumoniae biofilm cells. PMID:25583711

  15. Molecular aspects of calcium signalling at the crossroads of unikont and bikont eukaryote evolution--the ciliated protozoan Paramecium in focus.

    Science.gov (United States)

    Plattner, Helmut

    2015-03-01

    The ciliated protozoan, Paramecium tetraurelia has a high basic Ca(2+) leakage rate which is counteracted mainly by export through a contractile vacuole complex, based on its V-type H(+)-ATPase activity. In addition Paramecium cells dispose of P-type Ca(2+)-ATPases, i.e. a plasmamembrane and a sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (PMCA, SERCA). Antiporter systems are to be expected, as inferred from indirect evidence. Among the best known cytosolic Ca(2+)-binding proteins, calmodulin activates Ca(2+) influx channels in the somatic cell membrane, but inactivates Ca(2+) influx channels in cilia, where it, thus, ends ciliary reversal induced by depolarization via channels in the somatic cell membrane. Centrin inactivates Ca(2+) signals after stimulation by its high capacity/low affinity binding sites, whereas its high affinity sites regulate some other functions. Cortical Ca(2+) stores (alveolar sacs) are activated during stimulated trichocyst exocytosis and thereby mediate store-operated Ca(2+) entry (SOCE). Ca(2+) release channels (CRCs) localised to alveoli and underlying SOCE are considered as Ryanodine receptor-like proteins (RyR-LPs) which are members of a CRC family with 6 subfamilies. These also encompass genuine inositol 1,4,5-trisphosphate receptors (IP3Rs) and intermediates between the two channel types. All IP3R/RyR-type CRCs possess six carboxyterminal transmembrane domains (TMD), with a pore domain between TMD 5 and 6, endowed with a characteristic selectivity filter. There are reasons to assume a common ancestor molecule for such channels and diversification further on in evolution. The distinct distribution of specific CRCs in the different vesicles undergoing intracellular trafficking suggests constitutive formation of very locally restricted Ca(2+) signals during vesicle-vesicle interaction. In summary, essential steps of Ca(2+) signalling already occur at this level of evolution, including an unexpected multitude of CRCs. For dis

  16. NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I*

    Science.gov (United States)

    Rhein, Virginie F.; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

    2016-01-01

    Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 45 proteins. One arm lies in the inner membrane, and the other extends about 100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH, the primary electron acceptor FMN, and seven iron-sulfur clusters that form a pathway for electrons linking FMN to the terminal electron acceptor, ubiquinone, which is bound in a tunnel in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Seven of the subunits, forming the core of the membrane arm, are translated from mitochondrial genes, and the remaining subunits, the products of nuclear genes, are imported from the cytosol. Their assembly is coordinated by at least thirteen extrinsic assembly factor proteins that are not part of the fully assembled complex. They assist in insertion of co-factors and in building up the complex from smaller sub-assemblies. One such factor, NDUFAF5, belongs to the family of seven-β-strand S-adenosylmethionine-dependent methyltransferases. However, similar to another family member, RdmB, it catalyzes the introduction of a hydroxyl group, in the case of NDUFAF5, into Arg-73 in the NDUFS7 subunit of human complex I. This modification occurs early in the pathway of assembly of complex I, before the formation of the juncture between peripheral and membrane arms. PMID:27226634

  17. NDUFAF7 methylates arginine 85 in the NDUFS2 subunit of human complex I.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2013-11-15

    Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 subunits. One arm is embedded in the inner membrane with the other protruding ∼100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH and the primary electron acceptor FMN, and it provides a scaffold for seven iron-sulfur clusters that form an electron pathway linking FMN to the terminal electron acceptor, ubiquinone, which is bound in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, probably energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Complex I is put together from preassembled subcomplexes. Their compositions have been characterized partially, and at least 12 extrinsic assembly factor proteins are required for the assembly of the complex. One such factor, NDUFAF7, is predicted to belong to the family of S-adenosylmethionine-dependent methyltransferases characterized by the presence in their structures of a seven-β-strand protein fold. In the present study, the presence of NDUFAF7 in the mitochondrial matrix has been confirmed, and it has been demonstrated that it is a protein methylase that symmetrically dimethylates the ω-N(G),N(G') atoms of residue Arg-85 in the NDUFS2 subunit of complex I. This methylation step occurs early in the assembly of complex I and probably stabilizes a 400-kDa subcomplex that forms the initial nucleus of the peripheral arm and its juncture with the membrane arm.

  18. NDUFAF7 Methylates Arginine 85 in the NDUFS2 Subunit of Human Complex I*

    Science.gov (United States)

    Rhein, Virginie F.; Carroll, Joe; Ding, Shujing; Fearnley, Ian M.; Walker, John E.

    2013-01-01

    Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 44 subunits. One arm is embedded in the inner membrane with the other protruding ∼100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH and the primary electron acceptor FMN, and it provides a scaffold for seven iron-sulfur clusters that form an electron pathway linking FMN to the terminal electron acceptor, ubiquinone, which is bound in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, probably energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Complex I is put together from preassembled subcomplexes. Their compositions have been characterized partially, and at least 12 extrinsic assembly factor proteins are required for the assembly of the complex. One such factor, NDUFAF7, is predicted to belong to the family of S-adenosylmethionine-dependent methyltransferases characterized by the presence in their structures of a seven-β-strand protein fold. In the present study, the presence of NDUFAF7 in the mitochondrial matrix has been confirmed, and it has been demonstrated that it is a protein methylase that symmetrically dimethylates the ω-NG,NG′ atoms of residue Arg-85 in the NDUFS2 subunit of complex I. This methylation step occurs early in the assembly of complex I and probably stabilizes a 400-kDa subcomplex that forms the initial nucleus of the peripheral arm and its juncture with the membrane arm. PMID:24089531

  19. NDUFAF5 Hydroxylates NDUFS7 at an Early Stage in the Assembly of Human Complex I.

    Science.gov (United States)

    Rhein, Virginie F; Carroll, Joe; Ding, Shujing; Fearnley, Ian M; Walker, John E

    2016-07-08

    Complex I (NADH ubiquinone oxidoreductase) in mammalian mitochondria is an L-shaped assembly of 45 proteins. One arm lies in the inner membrane, and the other extends about 100 Å into the matrix of the organelle. The extrinsic arm contains binding sites for NADH, the primary electron acceptor FMN, and seven iron-sulfur clusters that form a pathway for electrons linking FMN to the terminal electron acceptor, ubiquinone, which is bound in a tunnel in the region of the junction between the arms. The membrane arm contains four antiporter-like domains, energetically coupled to the quinone site and involved in pumping protons from the matrix into the intermembrane space contributing to the proton motive force. Seven of the subunits, forming the core of the membrane arm, are translated from mitochondrial genes, and the remaining subunits, the products of nuclear genes, are imported from the cytosol. Their assembly is coordinated by at least thirteen extrinsic assembly factor proteins that are not part of the fully assembled complex. They assist in insertion of co-factors and in building up the complex from smaller sub-assemblies. One such factor, NDUFAF5, belongs to the family of seven-β-strand S-adenosylmethionine-dependent methyltransferases. However, similar to another family member, RdmB, it catalyzes the introduction of a hydroxyl group, in the case of NDUFAF5, into Arg-73 in the NDUFS7 subunit of human complex I. This modification occurs early in the pathway of assembly of complex I, before the formation of the juncture between peripheral and membrane arms. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. 22Na+ and 86Rb+ transport in vascular smooth muscle of SHR, Wistar Kyoto, and Wistar rats

    International Nuclear Information System (INIS)

    Kuriyama, S.; Denny, T.N.; Aviv, A.

    1988-01-01

    To gain further insight into differences in cellular Na+ and K+ regulation between the spontaneously hypertensive rat (SHR), Wistar Kyoto (WKY), and American Wistar (W) rats, 22Na+ and 86Rb+ washouts were performed under steady-state conditions in cultured vascular smooth muscle cells from the three rat strains. SHR vascular smooth muscle cells showed significantly higher bumetanide sensitive 86Rb+ washout rate constant (x 10(-4)/min; mean +/- SEM) than WKY cells (-38.6 +/- 2.84 and -23.8 +/- 3.58, respectively; p less than 0.005). SHR vascular smooth muscle cells also exhibited significantly higher values than WKY cells in the total 22Na+ washout rate constant (x 10(-2)/min) (-61.0 +/- 1.57 vs. -53.8 +/- 1.24; p less than 0.005). The amiloride sensitive component of the 22Na+ washout rate constant accounted for these differences (-18.6 +/- 1.04 for SHR and -12.1 +/- 2.00 for WKY; p less than 0.05). There were no apparent differences in cellular Na+ concentrations between WKY and SHR cells. In general, the 86Rb+ and 22Na+ washout parameters of W rat cells were quite similar to those of cells from SHR. We conclude that the bumetanide-sensitive 86Rb+ washout (the Na+ K+-cotransport), the overall, and the amiloride-sensitive 22Na+ washout (the latter primarily represents the Na+/H+ antiport) are higher in SHR than WKY rat vascular smooth muscle cells. These findings indicate innate differences in cellular Na+ and K+ transport in vascular smooth muscle cells of the SHR and WKY rat. The mechanisms responsible for these differences are yet to be determined

  1. Regulation of gene expression is associated with tolerance of the Arctic copepod Calanus glacialis to CO2-acidified sea water.

    Science.gov (United States)

    Bailey, Allison; De Wit, Pierre; Thor, Peter; Browman, Howard I; Bjelland, Reidun; Shema, Steven; Fields, David M; Runge, Jeffrey A; Thompson, Cameron; Hop, Haakon

    2017-09-01

    Ocean acidification is the increase in seawater p CO 2 due to the uptake of atmospheric anthropogenic CO 2 , with the largest changes predicted to occur in the Arctic seas. For some marine organisms, this change in p CO 2 , and associated decrease in pH, represents a climate change-related stressor. In this study, we investigated the gene expression patterns of nauplii of the Arctic copepod Calanus glacialis cultured at low pH levels. We have previously shown that organismal-level performance (development, growth, respiration) of C. glacialis nauplii is unaffected by low pH. Here, we investigated the molecular-level response to lowered pH in order to elucidate the physiological processes involved in this tolerance. Nauplii from wild-caught C. glacialis were cultured at four pH levels (8.05, 7.9, 7.7, 7.5). At stage N6, mRNA was extracted and sequenced using RNA-seq. The physiological functionality of the proteins identified was categorized using Gene Ontology and KEGG pathways. We found that the expression of 151 contigs varied significantly with pH on a continuous scale (93% downregulated with decreasing pH). Gene set enrichment analysis revealed that, of the processes downregulated, many were components of the universal cellular stress response, including DNA repair, redox regulation, protein folding, and proteolysis. Sodium:proton antiporters were among the processes significantly upregulated, indicating that these ion pumps were involved in maintaining cellular pH homeostasis. C. glacialis significantly alters its gene expression at low pH, although they maintain normal larval development. Understanding what confers tolerance to some species will support our ability to predict the effects of future ocean acidification on marine organisms.

  2. Iron and thiols as two major players in carcinogenesis: friends or foes?

    Science.gov (United States)

    Toyokuni, Shinya

    2014-01-01

    Iron is the most abundant metal in the human body and mainly works as a cofactor for proteins such as hemoglobin and various enzymes. No independent life forms on earth can survive without iron. However, excess iron is intimately associated with carcinogenesis by increasing oxidative stress via its catalytic activity to generate hydroxyl radicals. Biomolecules with redox-active sulfhydryl function(s) (thiol compounds) are necessary for the maintenance of mildly reductive cellular environments to counteract oxidative stress, and for the execution of redox reactions for metabolism and detoxification. Involvement of glutathione S-transferase and thioredoxin has long attracted the attention of cancer researchers. Here, I update recent findings on the involvement of iron and thiol compounds during carcinogenesis and in cancer cells. It is now recognized that the cystine/glutamate transporter (antiporter) is intimately associated with ferroptosis, an iron-dependent, non-apoptotic form of cell death, observed in cancer cells, and also with cancer stem cells; the former with transporter blockage but the latter with its stabilization. Excess iron in the presence of oxygen appears the most common known mutagen. Ironically, the persistent activation of antioxidant systems via genetic alterations in Nrf2 and Keap1 also contributes to carcinogenesis. Therefore, it is difficult to conclude the role of iron and thiol compounds as friends or foes, which depends on the quantity/distribution and induction/flexibility, respectively. Avoiding further mutation would be the most helpful strategy for cancer prevention, and myriad of efforts are being made to sort out the weaknesses of cancer cells.

  3. Cardiac glycoside activities link Na(+)/K(+) ATPase ion-transport to breast cancer cell migration via correlative SAR.

    Science.gov (United States)

    Magpusao, Anniefer N; Omolloh, George; Johnson, Joshua; Gascón, José; Peczuh, Mark W; Fenteany, Gabriel

    2015-02-20

    The cardiac glycosides ouabain and digitoxin, established Na(+)/K(+) ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na(+)/K(+) ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na(+)/K(+) ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein-small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na(+)/K(+) transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1-M6 correlates with the Na(+) pump activity and cell migration. Other Na(+)/K(+) ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na(+)/K(+) ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

  4. Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling

    Directory of Open Access Journals (Sweden)

    Morrill Gene A

    2013-01-01

    Full Text Available Abstract Background Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions. Results [125I]Insulin binding (Kd = 54 ± 6 nM at the oocyte plasma membrane activates membrane serine protease(s, followed by the loss of low affinity ouabain binding sites, with a concomitant 3–4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na+/Ca2+ exchange, increased endocytosis, decreased Na+ conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi. Hyperpolarization is largely due to Na+-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na+/glucose cotransport. The Na+ sym- and antiporter systems are driven by the Na+ free energy gradient generated by Na+/K+-ATPase. Shifts in α and/or β Na+-pump subunits to caveolar (lipid raft membrane regions may activate Na/K-ATPase and contribute to the Na+ free energy gradient and the increase in both Na+/glucose co-transport and pHi. Conclusions Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na+ channels and mobilizes fully functional Na+-pumps, generating a Na+ free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

  5. Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na+ and Ca2+ fluxes, Na+/K+ pump activity, and intracellular pH

    International Nuclear Information System (INIS)

    Mendoza, S.A.; Schneider, J.A.; Lopez-Rivas, A.; Sinnett-Smith, J.W.; Rozengurt, E.

    1986-01-01

    The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive 86 Rb + uptake (a measure of Na + /K + pump activity) in quiescent Swiss 3T3 cells. This effect occurred within seconds after the addition of the peptide and appeared to be mediated by an increase in Na + entry into the cells. The effect of bombesin on Na + entry and Na + /K + pump activity was concentration dependent with half-maximal stimulation occurring at 0.3-0.4 nM. The structurally related peptides litorin, gastrin-releasing peptide, and neuromedin B also stimulated ouabain-sensitive 86 Rb + uptake; the relative potencies of these peptides in stimulating the Na + /K + pump were comparable to their potencies in increasing DNA synthesis. Bombesin increased Na + influx, at least in part, through an Na + /H + antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na + . In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of 45 Ca 2+ from quiescent Swiss 3T3 cells. This Ca 2+ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca 2+ . The peptides also caused a rapid increase in cytosolic free calcium concentration. Prolonged pretreatment of Swiss 3T3 cells with phorbol dibutyrate, which causes a loss of protein kinase C activity, greatly decreased the stimulation of 86 Rb + uptake and Na + entry by bombesin implicating this phosphotransferase system in the mediation of part of these responses to bombesin. Since some activation of monovalent ion transport by bombesin was seen in phorbol dibutyrate-pretreated cells, it is likely that the peptide also stimulates monovalent ion transport by a second mechanism

  6. Characterization of thyroid hormone effects on Na-K pump and membrane potential of cultured rat skeletal myotubes

    International Nuclear Information System (INIS)

    Brodie, C.; Sampson, S.R.

    1988-01-01

    The purpose of this study was to characterize the effects of thyroid hormone on the Na-K pump and resting membrane potential (EM) of rat skeletal myotubes in culture. Myotubes were obtained from fetal (19-21 day) or neonatal rats (1-2 day) by serial trypsinization and maintained in culture for up to 10 days. Cells were treated with T4 or T3 on day 6 or 7, and measurements were made of EM, [ 3 H]ouabain binding, and ouabain-sensitive 86 Rb uptake at various times thereafter. Hormone treatment increased the values of all three variables within 24 h, plateau levels being attained by 48-72 h. Cycloheximide and actinomycin D totally blocked the effects of thyroid hormone when added together to the cells, thus suggesting that protein synthesis is necessary for the effects of these hormones. Scatchard analysis showed that the new receptors have lower ouabain affinity than those in control. Blockade of spontaneously occurring action potentials with tetrodotoxin, which blocks voltage-dependent Na channels, or Na/H antiporter with amiloride, abolished the hormone effects seen after 24 h and significantly reduced those obtained after 48 h of hormone treatment. The results demonstrate that thyroid hormone-induced increased amount and activity of the electrogenic Na-K pump in cultured myotubes occurs, at least in part, in response to an initial effect to increase Na influx. Moreover, the findings are consistent with the concept that the Na-K pump plays an important role in regulation of EM in this preparation

  7. Histamine Derived from Probiotic Lactobacillus reuteri Suppresses TNF via Modulation of PKA and ERK Signaling

    Science.gov (United States)

    Thomas, Carissa M.; Hong, Teresa; van Pijkeren, Jan Peter; Hemarajata, Peera; Trinh, Dan V.; Hu, Weidong; Britton, Robert A.; Kalkum, Markus; Versalovic, James

    2012-01-01

    Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases. PMID:22384111

  8. Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling.

    Directory of Open Access Journals (Sweden)

    Carissa M Thomas

    Full Text Available Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA, histidine/histamine antiporter (hdcP, and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H(2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

  9. Renal Collectrin Protects against Salt-Sensitive Hypertension and Is Downregulated by Angiotensin II.

    Science.gov (United States)

    Chu, Pei-Lun; Gigliotti, Joseph C; Cechova, Sylvia; Bodonyi-Kovacs, Gabor; Chan, Fang; Ralph, Donna Lee; Howell, Nancy; Kalantari, Kambiz; Klibanov, Alexander L; Carey, Robert M; McDonough, Alicia A; Le, Thu H

    2017-06-01

    Collectrin, encoded by the Tmem27 gene, is a transmembrane glycoprotein with approximately 50% homology with angiotensin converting enzyme 2, but without a catalytic domain. Collectrin is most abundantly expressed in the kidney proximal tubule and collecting duct epithelia, where it has an important role in amino acid transport. Collectrin is also expressed in endothelial cells throughout the vasculature, where it regulates L-arginine uptake. We previously reported that global deletion of collectrin leads to endothelial dysfunction, augmented salt sensitivity, and hypertension. Here, we performed kidney crosstransplants between wild-type (WT) and collectrin knockout ( Tmem27 Y/- ) mice to delineate the specific contribution of renal versus extrarenal collectrin on BP regulation and salt sensitivity. On a high-salt diet, WT mice with Tmem27 Y/- kidneys had the highest systolic BP and were the only group to exhibit glomerular mesangial hypercellularity. Additional studies showed that, on a high-salt diet, Tmem27 Y/- mice had lower renal blood flow, higher abundance of renal sodium-hydrogen antiporter 3, and lower lithium clearance than WT mice. In WT mice, administration of angiotensin II for 2 weeks downregulated collectrin expression in a type 1 angiotensin II receptor-dependent manner. This downregulation coincided with the onset of hypertension, such that WT and Tmem27 Y/- mice had similar levels of hypertension after 2 weeks of angiotensin II administration. Altogether, these data suggest that salt sensitivity is determined by intrarenal collectrin, and increasing the abundance or activity of collectrin may have therapeutic benefits in the treatment of hypertension and salt sensitivity. Copyright © 2017 by the American Society of Nephrology.

  10. ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and in type 2 diabetes mice.

    Science.gov (United States)

    Huang, Yun-Ying; Huang, Xiong-Qin; Zhao, Li-Yan; Sun, Fang-Yun; Chen, Wen-Liang; Du, Jie-Yi; Yuan, Feng; Li, Jie; Huang, Xue-Lian; Liu, Jie; Lv, Xiao-Fei; Guan, Yong-Yuan; Chen, Jian-Wen; Wang, Guan-Lei

    2014-11-01

    Palmitate, a common saturated free fatty acid (FFA), has been demonstrated to induce preadipocyte apoptosis in the absence of adipogenic stimuli, suggesting that preadipocytes may be prone to apoptosis under adipogenic insufficient conditions, like type 2 diabetes mellitus (T2DM). ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, is critical for cell fate choices of proliferation versus apoptosis under diseased conditions. However, it is unknown whether ClC-3 is related with preadipocyte apoptosis induced by palmitate or T2DM. Palmitate, but not oleate, induced apoptosis and increase in ClC-3 protein expression and endoplasmic reticulum (ER) stress in 3T3-L1 preadipocyte. ClC-3 specific siRNA attenuated palmitate-induced apoptosis and increased protein levels of Grp78, ATF4, CHOP and phosphorylation of JNK1/2, whereas had no effects on increased phospho-PERK and phospho-eIF2α protein expression. Moreover, the enhanced apoptosis was shown in preadipocytes from high-sucrose/fat, low-dose STZ induced T2DM mouse model with hyperglycemia, hyperlipidemia (elevated serum TG and FFA levels) and insulin resistance. ClC-3 knockout significantly attenuated preadipocyte apoptosis and the above metabolic disorders in T2DM mice. These data demonstrated that ClC-3 deficiency prevent preadipocytes against palmitate-induced apoptosis via suppressing ER stress, and also suggested that ClC-3 may play a role in regulating cellular apoptosis and disorders of glucose and lipid metabolism during T2DM.

  11. Protein export through the bacterial flagellar type III export pathway.

    Science.gov (United States)

    Minamino, Tohru

    2014-08-01

    For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V. All rights reserved.

  12. Stepwise Functional Evolution in a Fungal Sugar Transporter Family.

    Science.gov (United States)

    Gonçalves, Carla; Coelho, Marco A; Salema-Oom, Madalena; Gonçalves, Paula

    2016-02-01

    Sugar transport is of the utmost importance for most cells and is central to a wide range of applied fields. However, despite the straightforward in silico assignment of many novel transporters, including sugar porters, to existing families, their exact biological role and evolutionary trajectory often remain unclear, mainly because biochemical characterization of membrane proteins is inherently challenging, but also owing to their uncommonly turbulent evolutionary histories. In addition, many important shifts in membrane carrier function are apparently ancient, which further limits our ability to reconstruct evolutionary trajectories in a reliable manner. Here, we circumvented some of these obstacles by examining the relatively recent emergence of a unique family of fungal sugar facilitators, related to drug antiporters. The former transporters, named Ffz, were previously shown to be required for fructophilic metabolism in yeasts. We first exploited the wealth of fungal genomic data available to define a comprehensive but well-delimited family of Ffz-like transporters, showing that they are only present in Dikarya. Subsequently, a combination of phylogenetic analyses and in vivo functional characterization was used to retrace important changes in function, while highlighting the evolutionary events that are most likely to have determined extant distribution of the gene, such as horizontal gene transfers (HGTs). One such HGT event is proposed to have set the stage for the onset of fructophilic metabolism in yeasts, a trait that according to our results may be the metabolic hallmark of close to 100 yeast species that thrive in sugar rich environments. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. The modulation of leaf metabolism plays a role in salt tolerance of Cymodocea nodosa exposed to hypersaline stress in mesocosms

    Directory of Open Access Journals (Sweden)

    Amalia ePiro

    2015-06-01

    Full Text Available By the proteomic approach we tested the physiological responses of the euryhaline seagrass Cymodocea nodosa to deliberate manipulation of salinity in a mesocosm system. Plants were subjected to a chronic hypersaline condition (43 psu to compare their proteins expression and plant photochemistry responses after 15 and 30 days of exposure with those of plants cultured under normal/ambient saline conditions (37 psu. Results showed a general decline in the expression level of leaf proteins in hypersaline stressed plants, with more intense reductions on the long-lasting exposure. Specifically, the carbon-fixing enzyme RuBisCo displayed a lower expression level in stressed plants relative to controls; while contrarily, the key enzymes involve in the regulation of glycolisis, the cytosolic glyceraldehyde-3-phopsphate dehydrogenase, the enolase 2 and triose-phosphate isomerase, showed significant higher expression levels. Responses that suggest a shift of the carbon metabolism in stressed plants. Hypersaline stress also induced a significant alteration of the photosynthetic physiology of the C. nodosa by means of the down-regulation of structural proteins and enzymes of both PSII and PSI; however we found an over-expression of the cytochrome b559 alpha subunit of the PSII initial complex, which is a receptor for the PSII core proteins involved in biogenesis or repair processes and therefore potentially involved in the absence of effects at the photochemical level of stressed plants. As expected hypersalinity also affects the vacuolar metabolism increasing the leaf cell turgor pressure and enhancing the up-take of Na+ by the over-expression of the tonoplast specific intrinsic protein pyrophosphate-energized inorganic pyrophosphatase (H(+-PPase that is coupled with the Na+/H+-antiporter. The modulation of carbon metabolism and the enhancement of vacuole capacity in Na+ sequestration and osmolarity changes are discussed in relation to salt tolerance of C

  14. In planta Transformed Cumin (Cuminum cyminum L. Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

    Directory of Open Access Journals (Sweden)

    Sonika Pandey

    Full Text Available Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13, overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid, and lower electrolytic leakage, lipid peroxidation (MDA content and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

  15. Dexamethasone alleviates tumor-associated brain damage and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Zheng Fan

    Full Text Available Children and adults with the most aggressive form of brain cancer, malignant gliomas or glioblastoma, often develop cerebral edema as a life-threatening complication. This complication is routinely treated with dexamethasone (DEXA, a steroidal anti-inflammatory drug with pleiotropic action profile. Here we show that dexamethasone reduces murine and rodent glioma tumor growth in a concentration-dependent manner. Low concentrations of DEXA are already capable of inhibiting glioma cell proliferation and at higher levels induce cell death. Further, the expression of the glutamate antiporter xCT (system Xc-; SLC7a11 and VEGFA is up-regulated after DEXA treatment indicating early cellular stress responses. However, in human gliomas DEXA exerts differential cytotoxic effects, with some human glioma cells (U251, T98G resistant to DEXA, a finding corroborated by clinical data of dexamethasone non-responders. Moreover, DEXA-resistant gliomas did not show any xCT alterations, indicating that these gene expressions are associated with DEXA-induced cellular stress. Hence, siRNA-mediated xCT knockdown in glioma cells increased the susceptibility to DEXA. Interestingly, cell viability of primary human astrocytes and primary rodent neurons is not affected by DEXA. We further tested the pharmacological effects of DEXA on brain tissue and showed that DEXA reduces tumor-induced disturbances of the microenvironment such as neuronal cell death and tumor-induced angiogenesis. In conclusion, we demonstrate that DEXA inhibits glioma cell growth in a concentration and species-dependent manner. Further, DEXA executes neuroprotective effects in brains and reduces tumor-induced angiogenesis. Thus, our investigations reveal that DEXA acts pleiotropically and impacts tumor growth, tumor vasculature and tumor-associated brain damage.

  16. Increased Plasma Citrulline in Mice Marks Diet-Induced Obesity and May Predict the Development of the Metabolic Syndrome

    Science.gov (United States)

    Sailer, Manuela; Dahlhoff, Christoph; Giesbertz, Pieter; Eidens, Mena K.; de Wit, Nicole; Rubio-Aliaga, Isabel; Boekschoten, Mark V.; Müller, Michael; Daniel, Hannelore

    2013-01-01

    In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the “diabetic fingerprint” of plasma amino acid changes observed in humans. Additionally, citrulline may serve as an early indicator of the obesity-dependent metabolic impairments. PMID

  17. 31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

    Science.gov (United States)

    Civan, M M; Williams, S R; Gadian, D G; Rozengurt, E

    1986-01-01

    Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.

  18. Ceftriaxone- and N-acetylcysteine-induced brain tolerance to ischemia: Influence on glutamate levels in focal cerebral ischemia.

    Directory of Open Access Journals (Sweden)

    Weronika Krzyżanowska

    Full Text Available One of the major players in the pathophysiology of cerebral ischemia is disrupted homeostasis of glutamatergic neurotransmission, resulting in elevated extracellular glutamate (Glu concentrations and excitotoxicity-related cell death. In the brain, Glu concentrations are regulated by Glu transporters, including Glu transporter-1 (GLT-1 and cystine/Glu antiporter (system xc-. Modulation of these transporters by administration of ceftriaxone (CEF, 200 mg/kg, i.p. or N-acetylcysteine (NAC, 150 mg/kg, i.p. for 5 days before focal cerebral ischemia may induce brain tolerance to ischemia by significantly limiting stroke-related damage and normalizing Glu concentrations. In the present study, focal cerebral ischemia was induced by 90-minute middle cerebral artery occlusion (MCAO. We compared the effects of CEF and NAC pretreatment on Glu concentrations in extracellular fluid and cellular-specific expression of GLT-1 and xCT with the effects of two reference preconditioning methods, namely, ischemic preconditioning and chemical preconditioning in rats. Both CEF and NAC significantly reduced Glu levels in the frontal cortex and hippocampus during focal cerebral ischemia, and this decrease was comparable with the Glu level achieved with the reference preconditioning strategies. The results of immunofluorescence staining of GLT-1 and xCT on astrocytes, neurons and microglia accounted for the observed changes in extracellular Glu levels to a certain extent. Briefly, after MCAO, the expression of GLT-1 on astrocytes decreased, but pretreatment with CEF seemed to prevent this downregulation. In addition, every intervention used in this study seemed to reduce xCT expression on astrocytes and neurons. The results of this study indicate that modulation of Glu transporter expression may restore Glu homeostasis. Moreover, our results suggest that CEF and NAC may induce brain tolerance to ischemia by influencing GLT-1 and system xc- expression levels. These

  19. Effects of ceftriaxone on GLT1 isoforms, xCT and associated signaling pathways in P rats exposed to ethanol.

    Science.gov (United States)

    Rao, P S S; Saternos, Hannah; Goodwani, Sunil; Sari, Youssef

    2015-07-01

    Several studies have demonstrated a correlation between extracellular glutamate concentration in the mesolimbic reward pathway and alcohol craving. Extracellular glutamate concentration is regulated by several glutamate transporters. Glial glutamate transporter 1 (GLT1) is one of them that regulates the majority of extracellular glutamate concentration. In addition, cystine/glutamate antiporter (xCT) is another transporter that regulates extracellular glutamate. We focus in this study to determine the effects of ceftriaxone, β-lactam antibiotic, on glial proteins such as GLT1 isoforms, xCT, glutamate aspartate transporter (GLAST), and several associated signaling pathways as well as ethanol intake in P rats. Additionally, to examine the onset of signaling pathways associated with GLT1 upregulation following ceftriaxone treatment, we tested 2- versus 5-day daily dosing of ceftriaxone. Ceftriaxone treatment (100 mg/kg), 2 and 5 days, resulted in about five fold reduction in ethanol intake by P rats. The reduction in ethanol intake was associated with significantly enhanced expression of GLT1, GLT1a, GLT1b, and xCT in the nucleus accumbens (NAc) and prefrontal cortex (PFC) of 5-day ceftriaxone-treated P rats. Two-day-treated P rats showed marked changes in expression of these glutamate transporters in the PFC but not in the NAc. Importantly, ceftriaxone-treated P rats (2 and 5 days) demonstrated enhanced phosphorylation of Akt and nuclear translocation of nuclear factor kappaB (NFκB) in the NAc and PFC compared to control animals. These findings demonstrate that ceftriaxone treatment induced upregulation of GLT1, GLT1 isoforms, and xCT in association with activation of the Akt-NFκB signaling pathway.

  20. Enrichment of fusobacteria from the rumen that can utilize lysine as an energy source for growth.

    Science.gov (United States)

    Russell, James B

    2005-06-01

    Ruminal lysine degradation is a wasteful process that deprives the animal of an essential amino acid. Mixed ruminal bacteria did not deaminate lysine (50 mM) at a rapid rate, but lysine degrading bacteria could be enriched if Trypticase (5 mg/mL) was also added. Lysine degrading isolates produced acetate, butyrate and ammonia, were non-motile, stained Gram-negative and could also utilize lactate, glucose, maltose or galactose as an energy source for growth. Lactate was converted to acetate and propionate, and 16S rDNA indicated that their closest relatives were Fusobacterium necrophorum. Growing cultures produced ammonia at rates as high as 2400 nmol/mg protein/mL/min. Washed cell suspensions took up (14)C lysine (3 microM) at an initial rate of 6 nmol/mg protein/min, and glucose addition did not affect the transport. Cells washed aerobically had the same transport rate as those handled anaerobically, but only if the transport buffer contained sodium. The affinity constant for sodium was 8 mM, and sodium could not be replaced by lithium. Cells treated with the sodium/proton antiporter, monensin (5 microM), did not take up lysine, but a protonophore that inhibited growth (tetrachlorosalicylanilide, 10 microM) had no effect. An artificial membrane potential created by potassium diffusion did not increase the rate of lysine transport, and an Eadie-Hofstee plot indicated the transport rate was directly proportional to the lysine concentration. Decreasing the pH from 6.7 to 5.5 caused an 85% decrease in the rate of lysine transport. The addition of F. necrophorum JB2 (130 microg protein/mL) to mixed ruminal bacteria increased lysine degradation 10-fold, but only if the pH was 6.7 and monensin was not present. Further work will be needed to see if dietary lysine enriches fusobacteria in vivo.

  1. Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps

    Science.gov (United States)

    Olivares Pacheco, Jorge; Alvarez-Ortega, Carolina; Alcalde Rico, Manuel

    2017-01-01

    ABSTRACT It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H+ accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic “reaccommodation” might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations. PMID:28743808

  2. Genome structures and halophyte-specific gene expression of the extremophile thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and arabidopsis

    KAUST Repository

    Oh, Dongha

    2010-09-10

    The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5\\' untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species. © American Society of Plant Biologists.

  3. Acid and base stress and transcriptomic responses in Bacillus subtilis.

    Science.gov (United States)

    Wilks, Jessica C; Kitko, Ryan D; Cleeton, Sarah H; Lee, Grace E; Ugwu, Chinagozi S; Jones, Brian D; BonDurant, Sandra S; Slonczewski, Joan L

    2009-02-01

    Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K(+)/H(+) antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids.

  4. Transport of BMAA into Neurons and Astrocytes by System xc.

    Science.gov (United States)

    Albano, Rebecca; Lobner, Doug

    2018-01-01

    The study of the mechanism of β-N-methylamino-L-alanine (BMAA) neurotoxicity originally focused on its effects at the N-methyl-D-aspartate (NMDA) receptor. In recent years, it has become clear that its mechanism of action is more complicated. First, there are certain cell types, such as motor neurons and cholinergic neurons, where the dominate mechanism of toxicity is through action at AMPA receptors. Second, even in cortical neurons where the primary mechanism of toxicity appears to be activation of NMDA receptors, there are other mechanisms involved. We found that along with NMDA receptors, activation of mGLuR5 receptors and effects on the cystine/glutamate antiporter (system x c -) were involved in the toxicity. The effects on system x c - are of particular interest. System x c - mediates the transport of cystine into the cell in exchange for releasing glutamate into the extracellular fluid. By releasing glutamate, system x c - can potentially cause excitotoxicity. However, through providing cystine to the cell, it regulates the levels of cellular glutathione (GSH), the main endogenous intracellular antioxidant, and in this way may protect cells against oxidative stress. We have previously published that BMAA inhibits cystine uptake leading to GSH depletion and had indirect evidence that BMAA is transported into the cells by system x c -. We now present direct evidence that BMAA is transported into both astrocytes and neurons through system x c -. The fact that BMAA is transported by system x c - also provides a mechanism for BMAA to enter brain cells potentially leading to misincorporation into proteins and protein misfolding.

  5. Distinctive subdomains in the resorbing surface of osteoclasts.

    Directory of Open Access Journals (Sweden)

    Kinga A Szewczyk

    Full Text Available We employed a novel technique to inspect the substrate-apposed surface of activated osteoclasts, the cells that resorb bone, in the scanning electron microscope. The surface revealed unexpected complexity. At the periphery of the cells were circles and crescents of individual or confluent nodules. These corresponded to the podosomes and actin rings that form a 'sealing zone', encircling the resorptive hemivacuole into which protons and enzymes are secreted. Inside these rings and crescents the osteoclast surface was covered with strips and patches of membrane folds, which were flattened against the substrate surface and surrounded by fold-free membrane in which many orifices could be seen. Corresponding regions of folded and fold-free membrane were found by transmission electron microscopy in osteoclasts incubated on bone. We correlated these patterns with the distribution of several proteins crucial to resorption. The strips and patches of membrane folds corresponded in distribution to vacuolar H+-ATPase, and frequently co-localized with F-actin. Cathepsin K localized to F-actin-free foci towards the center of cells with circular actin rings, and at the retreating pole of cells with actin crescents. The chloride/proton antiporter ClC-7 formed a sharply-defined band immediately inside the actin ring, peripheral to vacuolar H+-ATPase. The sealing zone of osteoclasts is permeable to molecules with molecular mass up to 10,000. Therefore, ClC-7 might be distributed at the periphery of the resorptive hemivacuole in order to prevent protons from escaping laterally from the hemivacuole into the sealing zone, where they would dissolve the bone mineral. Since the activation of resorption is attributable to recognition of the αVβ3 ligands bound to bone mineral, such leakage would, by dissolving bone mineral, release the ligands and so terminate resorption. Therefore, ClC-7 might serve not only to provide the counter-ions that enable proton pumping, but

  6. RSL3 and Erastin differentially regulate redox signaling to promote Smac mimetic-induced cell death.

    Science.gov (United States)

    Dächert, Jasmin; Schoeneberger, Hannah; Rohde, Katharina; Fulda, Simone

    2016-09-27

    Redox mechanisms play an important role in the control of various signaling pathways. Here, we report that Second mitochondrial activator of caspases (Smac) mimetic-induced cell death is regulated by redox signaling. We show that RSL3, a glutathione (GSH) peroxidase (GPX) 4 inhibitor, or Erastin, an inhibitor of the cystine/glutamate antiporter, cooperate with the Smac mimetic BV6 to induce reactive oxygen species (ROS)-dependent cell death in acute lymphoblastic leukemia (ALL) cells. Addition of the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to rescue ROS-induced cell death, demonstrating that RSL3/BV6- or Erastin/BV6-induced cell death occurs in a caspase-independent manner. Interestingly, the iron chelator Deferoxamine (DFO) significantly inhibits RSL3/BV6-induced cell death, whereas it is unable to rescue cell death by Erastin/BV6, showing that RSL3/BV6-, but not Erastin/BV6-mediated cell death depends on iron. ROS production is required for both RSL3/BV6- and Erastin/BV6-induced cell death, since the ROS scavenger α-tocopherol (α-Toc) rescues RSL3/BV6- and Erastin/BV6-induced cell death. By comparison, genetic or pharmacological inhibition of lipid peroxidation by GPX4 overexpression or ferrostatin (Fer)-1 significantly decreases RSL3/BV6-, but not Erastin/BV6-induced cell death, despite inhibition of lipid peroxidation upon exposure to RSL3/BV6 or Erastin/BV6. Of note, inhibition of lipid peroxidation by Fer-1 protects from RSL3/BV6-, but not from Erastin/BV6-stimulated ROS production, indicating that other forms of ROS besides lipophilic ROS occur during Erastin/BV6-induced cell death. Taken together, RSL3/BV6 and Erastin/BV6 differentially regulate redox signaling and cell death in ALL cells. While RSL3/BV6 cotreatment induces ferroptotic cell death, Erastin/BV6 stimulates oxidative cell death independently of iron. These findings have important implications for the therapeutic targeting of redox signaling to

  7. The biological effects of deuterium depletion. A possible new tool in cancer therapy

    International Nuclear Information System (INIS)

    Somlyai, G.; Jancso, G.; Jakli, Gy.; Berkenyi, T.; Laskay, G.; Gyoengyi, Z.

    2000-01-01

    It is known that the deuterium/hydrogen mass ratio is the largest of stable isotopes of the same element, causing differences in the physical and chemical behaviour between the two hydrogen isotopes. The possible role of naturally occurring deuterium - whose concentration is over 16 mmol/l in surface water, 12-14 mmol/l in living organisms - in biological systems was first investigated in the early 90s. The results revealed that deuterium depleted water (DDW): i) inhibits cell proliferation of different cell lines in vitro (MDA and MCF-7: human breast, PC-3: human prostate, M14: human melanoma, HT-29: human colon, L 929 : mouse fibroblast, A4: murine haemopoietic); ii) as drinking water causes partial or complete tumour regression in xenotransplanted mice (MDA, MCF-7, PC-3); iii) can induce complete or partial tumour regression in dogs and cats with different tumours; iv) induced apoptosis in vitro and vivo; v) has a significant influence on the e-mye, Ha-ras and p53 genes by reducing their expression; vi) shows efficacy in Phase II double blind clinical trial with human prostate cancer. It is generally accepted that the earliest event in the response of mammalian cells to mitogens is the elevation of pH i , which may be the proliferative trigger. It is also known that the binding site for protons to be transported by plasma membrane H 4 -ATPasc of yeast does not accept deuterons with the same case as H 4 or perhaps not at all. It is therefore reasonable to assume that when the cell eliminates the H 4 to govern the pH i by activating the Na + /H 4 antiport system the D/H ratio increases in the intracellular space. We suggest that the cell cycle regulating system is somehow able to recognize the change in the D/H ratio and when this ratio reaches a certain threshold this will trigger the molecular mechanism which causes the cell to enter the S phase. The decrease of D concentration caused by DDW can interfere with the signal transduction pathways thus leading to

  8. Inhibitors of GLUT/SLC2A Enhance the Action of BCNU and Temozolomide against High-Grade Gliomas

    Directory of Open Access Journals (Sweden)

    Alberto Azzalin

    2017-04-01

    Full Text Available Glucose transport across glioblastoma membranes plays a crucial role in maintaining the enhanced glycolysis typical of high-grade gliomas and glioblastoma. We tested the ability of two inhibitors of the glucose transporters GLUT/SLC2A superfamily, indinavir (IDV and ritonavir (RTV, and of one inhibitor of the Na/glucose antiporter type 2 (SGLT2/SLC5A2 superfamily, phlorizin (PHZ, in decreasing glucose consumption and cell proliferation of human and murine glioblastoma cells. We found in vitro that RTV, active on at least three different GLUT/SLC2A transporters, was more effective than IDV, a specific inhibitor of GLUT4/SLC2A4, both in decreasing glucose consumption and lactate production and in inhibiting growth of U87MG and Hu197 human glioblastoma cell lines and primary cultures of human glioblastoma. PHZ was inactive on the same cells. Similar results were obtained when cells were grown in adherence or as 3D multicellular tumor spheroids. RTV treatment but not IDV treatment induced AMP-activated protein kinase (AMPKα phosphorylation that paralleled the decrease in glycolytic activity and cell growth. IDV, but not RTV, induced an increase in GLUT1/SLC2A1 whose activity could compensate for the inhibition of GLUT4/SLC2A4 by IDV. RTV and IDV pass poorly the blood brain barrier and are unlikely to reach sufficient liquoral concentrations in vivo to inhibit glioblastoma growth as single agents. Isobologram analysis of the association of RTV or IDV and 1,3-bis(2-chloroethyl-1-nitrosourea (BCNU or 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide (TMZ indicated synergy only with RTV on inhibition of glioblastoma cells. Finally, we tested in vivo the combination of RTV and BCNU on established GL261 tumors. This drug combination increased the overall survival and allowed a five-fold reduction in the dose of BCNU.

  9. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na(+) loading in xylem and confers salt tolerance in transgenic tobacco.

    Science.gov (United States)

    Yadav, Narendra Singh; Shukla, Pushp Sheel; Jha, Anupama; Agarwal, Pradeep K; Jha, Bhavanath

    2012-10-11

    Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na(+)/H(+) antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC), chlorophyll, K(+)/Na(+) ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT) plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS) and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na(+) content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na(+) content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na(+) loading to xylem from root and leaf tissues. Transgenic lines also showed increased K(+) and Ca(2+) content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na(+) efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na(+) content in different organs and also affect the other transporters activity indirectly. These

  10. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco

    Science.gov (United States)

    2012-01-01

    Background Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. Results The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC), chlorophyll, K+/Na+ ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT) plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS) and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na+ content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na+ content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na+ loading to xylem from root and leaf tissues. Transgenic lines also showed increased K+ and Ca2+ content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Conclusions Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na+ efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na+ content in different organs and also affect the other transporters activity indirectly

  11. A stress inducible SUMO conjugating enzyme gene (SaSce9 from a grass halophyte Spartina alterniflora enhances salinity and drought stress tolerance in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Karan Ratna

    2012-10-01

    Full Text Available Abstract Background SUMO (Small Ubiquitin related Modifier conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene ‘SaSce9’ from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance. Results SaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of ~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93% of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22. Conclusions These results highlight the prospect of improving abiotic

  12. Drug transport mechanism of the AcrB efflux pump.

    Science.gov (United States)

    Pos, Klaas M

    2009-05-01

    In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude cytotoxic substances from the cell directly into the medium bypassing periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump that extrudes multiple antibiotics, dyes, bile salts and detergents. The inner membrane component AcrB, a member of the Resistance Nodulation cell Division (RND) family, is the major site for substrate recognition and energy transduction of the entire tripartite system. The drug/proton antiport processes in this secondary transporter are suggested to be spatially separated, a feature frequently observed for primary transporters like membrane-bound ATPases. The recently elucidated asymmetric structure of the AcrB trimer reveals three different monomer conformations proposed to represent consecutive states in a directional transport cycle. Each monomer shows a distinct tunnel system with entrances located at the boundary of the outer leaflet of the inner membrane and the periplasm through the periplasmic porter (pore) domain towards the funnel of the trimer and TolC. In one monomer a hydrophobic pocket is present which has been shown to bind the AcrB substrates minocyclin and doxorubicin. The energy conversion from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (D407/D408/K940) residing in the hydrophobic transmembrane domain appear to be transduced by transmembrane helix 8 and associated with the conformational changes seen in the periplasmic domain. From the asymmetric structure a possible peristaltic pump transport mechanism based on a functional rotation of the AcrB trimer has been postulated. The novel drug transport model combines the alternate access pump mechanism with the rotating site catalysis of F(1)F(o) ATPase as

  13. The AcrB efflux pump: conformational cycling and peristalsis lead to multidrug resistance.

    Science.gov (United States)

    Seeger, Markus A; Diederichs, Kay; Eicher, Thomas; Brandstätter, Lorenz; Schiefner, André; Verrey, François; Pos, Klaas M

    2008-09-01

    Antimicrobial resistance of human pathogenic bacteria is an emerging problem for global public health. This resistance is often associated with the overproduction of membrane transport proteins that are capable to pump chemotherapeutics, antibiotics, detergents, dyes and organic solvents out of the cell. In Gram-negative bacteria such as Escherichia coli and Pseudomonas aeruginosa, tripartite multidrug efflux systems extrude a large variety of cytotoxic substances from the cell membrane directly into the medium bypassing the periplasm and the outer membrane. In E. coli, the tripartite efflux system AcrA/AcrB/TolC is the pump in charge of the efflux of multiple antibiotics, dyes, bile salts and detergents. The trimeric outer membrane factor (OMF) TolC forms a beta-barrel pore in the outer membrane and exhibits a long periplasmic alpha-helical conduit. The periplasmic membrane fusion protein (MFP) AcrA serves as a linker between TolC and the trimeric resistance nodulation cell division (RND) pump AcrB, located in the inner membrane acting as a proton/drug antiporter. The newly elucidated asymmetric structure of trimeric AcrB reveals three different monomer conformations representing consecutive states in a transport cycle. The monomers show tunnels with occlusions at different sites leading from the lateral side through the periplasmic porter (pore) domains towards the funnel of the trimer and TolC. The structural changes create a hydrophobic pocket in one monomer, which is not present in the other two monomers. Minocyclin and doxorubicin, both AcrB substrates, specifically bind to this pocket substantiating its role as drug binding pocket. The energy transduction from the proton motive force into drug efflux includes proton binding in (and release from) the transmembrane part. The conformational changes observed within a triad of essential, titratable residues (Asp407/Asp408/Lys940) residing in the hydrophobic transmembrane domain appear to be transduced by

  14. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

    Science.gov (United States)

    Xie, Junjun; Lei, Bo; Niu, Mengliang; Huang, Yuan; Kong, Qiusheng; Bie, Zhilong

    2015-01-01

    MicroRNAs (miRNAs), a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch.) and N15 (Cucurbita. moschata Duch.), with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs) were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small RNAs in the

  15. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

    Directory of Open Access Journals (Sweden)

    Junjun Xie

    Full Text Available MicroRNAs (miRNAs, a class of small non-coding RNAs, recognize their mRNA targets based on perfect sequence complementarity. MiRNAs lead to broader changes in gene expression after plants are exposed to stress. High-throughput sequencing is an effective method to identify and profile small RNA populations in non-model plants under salt stresses, significantly improving our knowledge regarding miRNA functions in salt tolerance. Cucurbits are sensitive to soil salinity, and the Cucurbita genus is used as the rootstock of other cucurbits to enhance salt tolerance. Several cucurbit crops have been used for miRNA sequencing but salt stress-related miRNAs in cucurbit species have not been reported. In this study, we subjected two Cucurbita germplasm, namely, N12 (Cucurbita. maxima Duch. and N15 (Cucurbita. moschata Duch., with different sodium accumulation patterns, to Illumina sequencing to determine small RNA populations in root tissues after 4 h of salt treatment and control. A total of 21,548,326 and 19,394,108 reads were generated from the control and salt-treated N12 root tissues, respectively. By contrast, 19,108,240 and 20,546,052 reads were obtained from the control and salt-treated N15 root tissues, respectively. Fifty-eight conserved miRNA families and 33 novel miRNAs were identified in the two Cucurbita germplasm. Seven miRNAs (six conserved miRNAs and one novel miRNAs were up-regulated in salt-treated N12 and N15 samples. Most target genes of differentially expressed novel miRNAs were transcription factors and salt stress-responsive proteins, including dehydration-induced protein, cation/H+ antiporter 18, and CBL-interacting serine/threonine-protein kinase. The differential expression of miRNAs between the two Cucurbita germplasm under salt stress conditions and their target genes demonstrated that novel miRNAs play an important role in the response of the two Cucurbita germplasm to salt stress. The present study initially explored small

  16. Complete genome sequence of Francisella tularensis subspecies holarctica FTNF002-00.

    Directory of Open Access Journals (Sweden)

    Ravi D Barabote

    Full Text Available Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD(23 deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997-1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also approximately 5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1-1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H(+ antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities.

  17. Plasma L-cystine/L-glutamate imbalance increases tumor necrosis factor-alpha from CD14+ circulating monocytes in patients with advanced cirrhosis.

    Directory of Open Access Journals (Sweden)

    Eiji Kakazu

    Full Text Available BACKGROUND AND AIMS: The innate immune cells can not normally respond to the pathogen in patients with decompensated cirrhosis. Previous studies reported that antigen-presenting cells take up L-Cystine (L-Cys and secrete substantial amounts of L-Glutamate (L-Glu via the transport system Xc- (4F2hc+xCT, and that this exchange influences the immune responses. The aim of this study is to investigate the influence of the plasma L-Cys/L-Glu imbalance observed in patients with advanced cirrhosis on the function of circulating monocytes. METHODS: We used a serum-free culture medium consistent with the average concentrations of plasma amino acids from patients with advanced cirrhosis (ACM, and examined the function of CD14+ monocytes or THP-1 under ACM that contained 0-300 nmol/mL L-Cys with LPS. In patients with advanced cirrhosis, we actually determined the TNF-alpha and xCT mRNA of monocytes, and evaluated the correlation between the plasma L-Cys/L-Glu ratio and TNF-alpha. RESULTS: The addition of L-Cys significantly increased the production of TNF alpha from monocytes under ACM. Monocytes with LPS and THP-1 expressed xCT and a high level of extracellular L-Cys enhanced L-Cys/L-Glu antiport, and the intracellular GSH/GSSG ratio was decreased. The L-Cys transport was inhibited by excess L-Glu. In patients with advanced cirrhosis (n = 19, the TNF-alpha and xCT mRNA of monocytes were increased according to the Child-Pugh grade. The TNF-alpha mRNA of monocytes was significantly higher in the high L-Cys/L-Glu ratio group than in the low ratio group, and the plasma TNF-alpha was significantly correlated with the L-Cys/L-Glu ratio. CONCLUSIONS: A plasma L-Cys/L-Glu imbalance, which appears in patients with advanced cirrhosis, increased the TNF-alpha from circulating monocytes via increasing the intracellular oxidative stress. These results may reflect the immune abnormality that appears in patients with decompensated cirrhosis.

  18. Molecular pharmacology of kidney and inner ear CLC-K chloride channels

    Directory of Open Access Journals (Sweden)

    Antonella eGradogna

    2010-10-01

    Full Text Available CLC-K channels belong to the CLC gene family, which comprises both Cl- channels and Cl-/H+ antiporters. They form homodimers which additionally co-assemble with the small protein barttin. In the kidney, they are involved in NaCl reabsorption ; in the inner ear they are important for endolymph production. Mutations in CLC-Kb lead to renal salt loss (Bartter’s syndrome; mutations in barttin lead additionally to deafness. CLC-K channels are interesting potential drug targets. CLC-K channel blockers have potential as alternative diuretics, whereas CLC-K activators could be used for the treatment of patients with Bartter’s syndrome. Several small organic acids inhibit CLC-K channels from the outside by binding to a site in the external vestibule of the ion conducting pore. Benzofuran derivatives with affinities better than 10 µM have been discovered. Niflumic acid (NFA exhibits a complex interaction with CLC-K channels. Below ~ 1 mM, NFA activates CLC-Ka, whereas at higher concentrations NFA inhibits channel activity. The co-planarity of the rings of the NFA molecule is essential for its activating action. Mutagenesis has led to the identification of potential regions of the channel that interact with NFA. CLC-K channels are also modulated by pH and [Ca2+]ext. The inhibition at low pH has been shown to be mediated by a His-residue at the beginning of helix Q, the penultimate transmembrane helix. Two acidic residues from opposite subunits form two symmetrically related intersubunit Ca2+ binding sites, whose occupation increases channel activity.The relatively high affinity CLC-K blockers may already serve as leads for the development of useful drugs. On the other hand, the CLC-K potentiator NFA has a quite low affinity, and, being a non-steroidal anti-inflammatory drug, can be expected to exert significant side effects. More specific and more potent activators will be needed and it will be important to understand the molecular mechanisms that

  19. Enhanced productivity of gamma-amino butyric acid by cascade modifications of a whole-cell biocatalyst.

    Science.gov (United States)

    Yang, Xinwei; Ke, Chongrong; Zhu, Jiangming; Wang, Yan; Zeng, Wenchao; Huang, Jianzhong

    2018-04-01

    We previously developed a gamma-amino butyric acid (GABA)-producing strain of Escherichia coli, leading to production of 614.15 g/L GABA at 45 °C from L-glutamic acid (L-Glu) with a productivity of 40.94 g/L/h by three successive whole-cell conversion cycles. However, the increase in pH caused by the accumulation of GABA resulted in inactivation of the biocatalyst and consequently led to relatively lower productivity. In this study, by overcoming the major problem associated with the increase in pH during the production process, a more efficient biocatalyst was obtained through cascade modifications of the previously reported E. coli strain. First, we introduced four amino acid mutations to the codon-optimized GadB protein from Lactococcus lactis to shift its decarboxylation activity toward a neutral pH, resulting in 306.65 g/L of GABA with 99.14 mol% conversion yield and 69.8% increase in GABA productivity. Second, we promoted transportation of L-Glu and GABA by removing the genomic region encoding the C-plug of GadC (a glutamate/GABA antiporter) to allow its transport path to remain open at a neutral pH, which improved the GABA productivity by 16.8% with 99.3 mol% conversion of 3 M L-Glu. Third, we enhanced the expression of soluble GadB by introducing the GroESL molecular chaperones, leading to 20.2% improvement in GABA productivity, with 307.40 g/L of GABA and a 61.48 g/L/h productivity obtained in one cycle. Finally, we inhibited the degradation of GABA by inactivation of gadA and gadB from the E. coli genome, which resulted in almost no GABA degradation after 40 h. After the cascade system modifications, the engineered recombinant E. coli strain achieved a 44.04 g/L/h productivity with a 99.6 mol% conversion of 3 M L-Glu in a 5-L bioreactor, about twofold increase in productivity compared to the starting strain. This increase represents the highest GABA productivity by whole-cell bioconversion using L-Glu as a substrate in one cycle observed

  20. [Pt(O,O’-acac)(γ-acac)(DMS)] Alters SH-SY5Y Cell Migration and Invasion by the Inhibition of Na+/H+ Exchanger Isoform 1 Occurring through a PKC-ε/ERK/mTOR Pathway

    Science.gov (United States)

    Muscella, Antonella; Vetrugno, Carla; Calabriso, Nadia; Cossa, Luca Giulio; De Pascali, Sandra Angelica; Fanizzi, Francesco Paolo; Marsigliante, Santo

    2014-01-01

    We previously showed that [Pt(O,O’-acac)(γ-acac)(DMS)] ([Pt(acac)2(DMS)]) exerted substantial cytotoxic effects in SH-SY5Y neuroblastoma cells, and decreased metalloproteases (MMPs) production and cells migration in MCF-7 breast cancer cells. The ubiquitously distributed sodium-hydrogen antiporter 1 (NHE1) is involved in motility and invasion of many solid tumours. The present study focuses on the effects of [Pt(acac)2(DMS)] in SH-SY5Y cell migration and also on the possibility that NHE1 may be involved in such effect. After sublethal [Pt(acac)2(DMS)] treatment cell migration was examined by wounding assay and cell invasion by transwell assay. NHE1 activity was measured in BCECF-loaded SH-SY5Y as the rate of Na+-dependent intracellular pH recovery in response to an acute acid pulse. Gelatin zymography for MMP-2/9 activities, Western blottings of MMPs, MAPKs, mTOR, S6 and PKCs and small interfering RNAs to PKC-ε/-δ mRNA were performed. Sublethal concentrations of [Pt(acac)2(DMS)] decreases NHE1 activity, inhibites cell migration and invasion and decreases expression and activity of MMP-2 and -9. [Pt(acac)2(DMS)] administered to SH-SY5Y cells provokes the increment of ROS, generated by NADPH oxidase, responsible for the PKC-ε and PKC-δ activation. Whilst PKC-δ activates p38/MAPK, responsible for the inhibition of MMP-2 and -9 secretion, PKC-ε activates a pathway made of ERK1/2, mTOR and S6K responsible for the inhibition of NHE1 activity and cell migration. In conclusion, we have shown a drastic impairment in tumour cell metastatization in response to inhibition of NHE1 and MMPs activities by [Pt(acac)2(DMS)] occurring through a novel mechanism mediated by PKC-δ/-ε activation. PMID:25372487

  1. Histamine H2 Receptor-Mediated Suppression of Intestinal Inflammation by Probiotic Lactobacillus reuteri.

    Science.gov (United States)

    Gao, Chunxu; Major, Angela; Rendon, David; Lugo, Monica; Jackson, Vanessa; Shi, Zhongcheng; Mori-Akiyama, Yuko; Versalovic, James

    2015-12-15

    Probiotics and commensal intestinal microbes suppress mammalian cytokine production and intestinal inflammation in various experimental model systems. Limited information exists regarding potential mechanisms of probiotic-mediated immunomodulation in vivo. In this report, we demonstrate that specific probiotic strains of Lactobacillus reuteri suppress intestinal inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse colitis model. Only strains that possess the hdc gene cluster, including the histidine decarboxylase and histidine-histamine antiporter genes, can suppress colitis and mucosal cytokine (interleukin-6 [IL-6] and IL-1β in the colon) gene expression. Suppression of acute colitis in mice was documented by diminished weight loss, colonic injury, serum amyloid A (SAA) protein concentrations, and reduced uptake of [(18)F]fluorodeoxyglucose ([(18)F]FDG) in the colon by positron emission tomography (PET). The ability of probiotic L. reuteri to suppress colitis depends on the presence of a bacterial histidine decarboxylase gene(s) in the intestinal microbiome, consumption of a histidine-containing diet, and signaling via the histamine H2 receptor (H2R). Collectively, luminal conversion of l-histidine to histamine by hdc(+) L. reuteri activates H2R, and H2R signaling results in suppression of acute inflammation within the mouse colon. Probiotics are microorganisms that when administered in adequate amounts confer beneficial effects on the host. Supplementation with probiotic strains was shown to suppress intestinal inflammation in patients with inflammatory bowel disease and in rodent colitis models. However, the mechanisms of probiosis are not clear. Our current studies suggest that supplementation with hdc(+) L. reuteri, which can convert l-histidine to histamine in the gut, resulted in suppression of colonic inflammation. These findings link luminal conversion of dietary components (amino acid metabolism) by gut microbes and probiotic

  2. Vibrio anguillarum Is Genetically and Phenotypically Unaffected by Long-Term Continuous Exposure to the Antibacterial Compound Tropodithietic Acid.

    Science.gov (United States)

    Rasmussen, Bastian Barker; Grotkjær, Torben; D'Alvise, Paul W; Yin, Guangliang; Zhang, Faxing; Bunk, Boyke; Spröer, Cathrin; Bentzon-Tilia, Mikkel; Gram, Lone

    2016-08-01

    Minimizing the use of antibiotics in the food production chain is essential for limiting the development and spread of antibiotic-resistant bacteria. One alternative intervention strategy is the use of probiotic bacteria, and bacteria of the marine Roseobacter clade are capable of antagonizing fish-pathogenic vibrios in fish larvae and live feed cultures for fish larvae. The antibacterial compound tropodithietic acid (TDA), an antiporter that disrupts the proton motive force, is key in the antibacterial activity of several roseobacters. Introducing probiotics on a larger scale requires understanding of any potential side effects of long-term exposure of the pathogen to the probionts or any compounds they produce. Here we exposed the fish pathogen Vibrio anguillarum to TDA for several hundred generations in an adaptive evolution experiment. No tolerance or resistance arose during the 90 days of exposure, and whole-genome sequencing of TDA-exposed lineages and clones revealed few mutational changes, compared to lineages grown without TDA. Amino acid-changing mutations were found in two to six different genes per clone; however, no mutations appeared unique to the TDA-exposed lineages or clones. None of the virulence genes of V. anguillarum was affected, and infectivity assays using fish cell lines indicated that the TDA-exposed lineages and clones were less invasive than the wild-type strain. Thus, long-term TDA exposure does not appear to result in TDA resistance and the physiology of V. anguillarum appears unaffected, supporting the application of TDA-producing roseobacters as probiotics in aquaculture. It is important to limit the use of antibiotics in our food production, to reduce the risk of bacteria developing antibiotic resistance. We showed previously that marine bacteria of the Roseobacter clade can prevent or reduce bacterial diseases in fish larvae, acting as probiotics. Roseobacters produce the antimicrobial compound tropodithietic acid (TDA), and we

  3. Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria - An amazing defence tool against hyperosmotic stress

    Directory of Open Access Journals (Sweden)

    Daniela eTrono

    2015-12-01

    Full Text Available In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about fifteen years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP. Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous and tissues/organs (etiolated and green have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane towards the matrix, so collapsing membrane potential (ΔΨ, the main component of the protonmotive force (Δp in plant mitochondria; moreover, cooperation between PmitoKATP and the K+/H+ antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate

  4. 'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interaction.

    Directory of Open Access Journals (Sweden)

    L David Kuykendall

    Full Text Available Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential 'accessory' genes for nitrogen fixation (nif, nodulation and host specificity (nod. A related bacterium, psyllid-vectored 'Ca. Liberibacter asiaticus,' is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the 'Ca. Liberibacter asiaticus' genome. Only two 'Ca. Liberibacter asiaticus' proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na(+/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and 'Ca. Liberibacter asiaticus' orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (≤ E-10 with 'Ca. Liberibacter asiaticus' proteins, often present as multiple orthologs of single 'Ca. Liberibacter asiaticus' proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies mutational

  5. The Glutaminase-Dependent System Confers Extreme Acid Resistance to New Species and Atypical Strains of Brucella

    Directory of Open Access Journals (Sweden)

    Luca Freddi

    2017-11-01

    Full Text Available Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB and of the glutamate (Glu/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the “classical” species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q, relying on the deamination of glutamine (Gln into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH ≤2.5, in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC–glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis.

  6. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco

    Directory of Open Access Journals (Sweden)

    Yadav Narendra

    2012-10-01

    Full Text Available Abstract Background Soil salinity adversely affects plant growth and development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1 gene encodes a plasma membrane Na+/H+ antiporter that plays an important role in imparting salt stress tolerance to plants. Here, we report the cloning and characterisation of the SbSOS1 gene from Salicornia brachiata, an extreme halophyte. Results The SbSOS1 gene is 3774 bp long and encodes a protein of 1159 amino acids. SbSOS1 exhibited a greater level of constitutive expression in roots than in shoots and was further increased by salt stress. Overexpressing the S. brachiata SbSOS1 gene in tobacco conferred high salt tolerance, promoted seed germination and increased root length, shoot length, leaf area, fresh weight, dry weight, relative water content (RWC, chlorophyll, K+/Na+ ratio, membrane stability index, soluble sugar, proline and amino acid content relative to wild type (WT plants. Transgenic plants exhibited reductions in electrolyte leakage, reactive oxygen species (ROS and MDA content in response to salt stress, which probably occurred because of reduced cytosolic Na+ content and oxidative damage. At higher salt stress, transgenic tobacco plants exhibited reduced Na+ content in root and leaf and higher concentrations in stem and xylem sap relative to WT, which suggests a role of SbSOS1 in Na+ loading to xylem from root and leaf tissues. Transgenic lines also showed increased K+ and Ca2+ content in root tissue compared to WT, which reflect that SbSOS1 indirectly affects the other transporters activity. Conclusions Overexpression of SbSOS1 in tobacco conferred a high degree of salt tolerance, enhanced plant growth and altered physiological and biochemical parameters in response to salt stress. In addition to Na+ efflux outside the plasma membrane, SbSOS1 also helps to maintain variable Na+ content in different organs and also affect the other

  7. Genetically modified plants for salinity stress tolerance (abstract)

    International Nuclear Information System (INIS)

    Sopory, S.K.; Singia-Pareek, S.I.; Kumar, S.; Rajgopal, D.; Aggarwal, P.; Kumar, D.; Reddy, K.M.

    2005-01-01

    Several recent reports have indicated that the area under salinity is on the increase and currently very few genotypes of important crop plants are available for cultivation under these conditions. In this regard, identification of novel stress responsive genes and transgenic approach offers an important strategy to develop salt tolerant plants. Using an efficient PCR-based cDNA subtraction method a large number of genes upregulated under salinity and dehydration stress have been identified also in rice and Pennisetum. Functional analysis of some of these genes is being done using transgenic approach. Earlier, we reported on the role of one of the stress regulated genes, glyoxalse I in conferring salinity tolerance. We now show that by manipulating the expression of both the genes of the glyoxalse pathway, glyoxalse I and II together, the ability of the double transgenic plants to tolerate salinity stress is greatly enhanced as compared to the single transgenic plants harbouring either the glyoxalse I or glyoxalse II. The cDNA for glyoxalse II was cloned from rice and mobilized into pCAMBIA vector having hptII gene as the selection marker. The seedlings of the T1 generation transgenic plants survived better under high salinity compared to the wild type plants; the double transgenics had higher limits of tolerance as compared to the lines transformed with single gene. A similar trend was seen even when plants were grown in pots under glass house conditions and raised to maturity under the continued presence of NaCl. In this, the transgenic plants were able to grow, flower and set seeds. The overexpression of glyoxalse pathway was also found to confer stress tolerance in rice. We have also isolated a gene encoding vacuolar sodium/proton antiporter from Pennisetum and over expressed in Brassica juncea and rice. The transgenic plants were able to tolerate salinity stress. Our work along with many others' indicates the potential of transgenic technology in developing

  8. Metabolic Compensation of Fitness Costs Is a General Outcome for Antibiotic-Resistant Pseudomonas aeruginosa Mutants Overexpressing Efflux Pumps.

    Science.gov (United States)

    Olivares Pacheco, Jorge; Alvarez-Ortega, Carolina; Alcalde Rico, Manuel; Martínez, José Luis

    2017-07-25

    It is generally assumed that the acquisition of antibiotic resistance is associated with a fitness cost. We have shown that overexpression of the MexEF-OprN efflux pump does not decrease the fitness of a resistant Pseudomonas aeruginosa strain compared to its wild-type counterpart. This lack of fitness cost was associated with a metabolic rewiring that includes increased expression of the anaerobic nitrate respiratory chain when cells are growing under fully aerobic conditions. It was not clear whether this metabolic compensation was exclusive to strains overexpressing MexEF-OprN or if it extended to other resistant strains that overexpress similar systems. To answer this question, we studied a set of P. aeruginosa mutants that independently overexpress the MexAB-OprM, MexCD-OprJ, or MexXY efflux pumps. We observed increased expression of the anaerobic nitrate respiratory chain in all cases, with a concomitant increase in NO 3 consumption and NO production. These efflux pumps are proton/substrate antiporters, and their overexpression may lead to intracellular H + accumulation, which may in turn offset the pH homeostasis. Indeed, all studied mutants showed a decrease in intracellular pH under anaerobic conditions. The fastest way to eliminate the excess of protons is by increasing oxygen consumption, a feature also displayed by all analyzed mutants. Taken together, our results support metabolic rewiring as a general mechanism to avoid the fitness costs derived from overexpression of P. aeruginosa multidrug efflux pumps. The development of drugs that block this metabolic "reaccommodation" might help in reducing the persistence and spread of antibiotic resistance elements among bacterial populations. IMPORTANCE It is widely accepted that the acquisition of resistance confers a fitness cost in such a way that in the absence of antibiotics, resistant populations will be outcompeted by susceptible ones. Based on this assumption, antibiotic cycling regimes have been

  9. Involvement of Calmodulin and Calmodulin-like Proteins in Plant Responses to Abiotic Stresses

    Directory of Open Access Journals (Sweden)

    B W Poovaiah

    2015-08-01

    Full Text Available Transient changes in intracellular Ca2+ concentration have been well recognized to act as cell signals coupling various environmental stimuli to appropriate physiological responses with accuracy and specificity in plants. Calmodulin (CaM and calmodulin-like proteins (CMLs are major Ca2+ sensors, playing critical roles in interpreting encrypted Ca2+ signals. Ca2+-loaded CaM/CMLs interact and regulate a broad spectrum of target proteins such as channels/pumps/antiporters for various ions, transcription factors, protein kinases, protein phosphatases, metabolic enzymes and proteins with unknown biochemical functions. Many of the target proteins of CaM/CMLs directly or indirectly regulate plant responses to environmental stresses. Basic information about stimulus-induced Ca2+ signal and overview of Ca2+ signal perception and transduction are briefly discussed in the beginning of this review. How CaM/CMLs are involved in regulating plant responses to abiotic stresses are emphasized in this review. Exciting progress has been made in the past several years, such as the elucidation of Ca2+/CaM-mediated regulation of AtSR1/CAMTA3 and plant responses to chilling and freezing stresses, Ca2+/CaM-mediated regulation of CAT3, MAPK8 and MKP1 in homeostasis control of ROS signals, discovery of CaM7 as a DNA-binding transcription factor regulating plant response to light signals. However, many key questions in Ca2+/CaM-mediated signaling warrant further investigation. Ca2+/CaM-mediated regulation of most of the known target proteins is presumed based on their interaction. The downstream targets of CMLs are mostly unknown, and how specificity of Ca2+ signaling could be realized through the actions of CaM/CMLs and their target proteins is largely unknown. Future breakthroughs in Ca2+/CaM-mediated signaling will not only improve our understanding of how plants respond to environmental stresses, but also provide the knowledge base to improve stress-tolerance of crops.

  10. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

    Directory of Open Access Journals (Sweden)

    Sofie Ignoul

    Full Text Available BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432 have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y, endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4 and not with late endosomal/lysosomal markers (LAMP-1, Rab7. Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6

  11. Acid and Base Stress and Transcriptomic Responses in Bacillus subtilis▿†

    Science.gov (United States)

    Wilks, Jessica C.; Kitko, Ryan D.; Cleeton, Sarah H.; Lee, Grace E.; Ugwu, Chinagozi S.; Jones, Brian D.; BonDurant, Sandra S.; Slonczewski, Joan L.

    2009-01-01

    Acid and base environmental stress responses were investigated in Bacillus subtilis. B. subtilis AG174 cultures in buffered potassium-modified Luria broth were switched from pH 8.5 to pH 6.0 and recovered growth rapidly, whereas cultures switched from pH 6.0 to pH 8.5 showed a long lag time. Log-phase cultures at pH 6.0 survived 60 to 100% at pH 4.5, whereas cells grown at pH 7.0 survived base induced adaptation to a more extreme acid or base, respectively. Expression indices from Affymetrix chip hybridization were obtained for 4,095 protein-encoding open reading frames of B. subtilis grown at external pH 6, pH 7, and pH 9. Growth at pH 6 upregulated acetoin production (alsDS), dehydrogenases (adhA, ald, fdhD, and gabD), and decarboxylases (psd and speA). Acid upregulated malate metabolism (maeN), metal export (czcDO and cadA), oxidative stress (catalase katA; OYE family namA), and the SigX extracytoplasmic stress regulon. Growth at pH 9 upregulated arginine catabolism (roc), which generates organic acids, glutamate synthase (gltAB), polyamine acetylation and transport (blt), the K+/H+ antiporter (yhaTU), and cytochrome oxidoreductases (cyd, ctaACE, and qcrC). The SigH, SigL, and SigW regulons were upregulated at high pH. Overall, greater genetic adaptation was seen at pH 9 than at pH 6, which may explain the lag time required for growth shift to high pH. Low external pH favored dehydrogenases and decarboxylases that may consume acids and generate basic amines, whereas high external pH favored catabolism-generating acids. PMID:19114526

  12. N-Acetyl-cysteine causes analgesia by reinforcing the endogenous activation of type-2 metabotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Bernabucci Matteo

    2012-10-01

    Full Text Available Abstract Background Pharmacological activation of type-2 metabotropic glutamate receptors (mGlu2 receptors causes analgesia in experimental models of inflammatory and neuropathic pain. Presynaptic mGlu2 receptors are activated by the glutamate released from astrocytes by means of the cystine/glutamate antiporter (System xc- or Sxc-. We examined the analgesic activity of the Sxc- activator, N-acetyl-cysteine (NAC, in mice developing inflammatory or neuropathic pain. Results A single injection of NAC (100 mg/kg, i.p. reduced nocifensive behavior in the second phase of the formalin test. NAC-induced analgesia was abrogated by the Sxc- inhibitor, sulphasalazine (8 mg/kg, i.p. or by the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.. NAC still caused analgesia in mGlu3−/− mice, but was inactive in mGlu2−/− mice. In wild-type mice, NAC retained the analgesic activity in the formalin test when injected daily for 7 days, indicating the lack of tolerance. Both single and repeated injections of NAC also caused analgesia in the complete Freund’s adjuvant (CFA model of chronic inflammatory pain, and, again, analgesia was abolished by LY341495. Data obtained in mice developing neuropathic pain in response to chronic constriction injury (CCI of the sciatic nerve were divergent. In this model, a single injection of NAC caused analgesia that was reversed by LY341495, whereas repeated injections of NAC were ineffective. Thus, tolerance to NAC-induced analgesia developed in the CCI model, but not in models of inflammatory pain. The CFA and CCI models differed with respect to the expression levels of xCT (the catalytic subunit of Sxc- and activator of G-protein signaling type-3 (AGS3 in the dorsal portion of the lumbar spinal cord. CFA-treated mice showed no change in either protein, whereas CCI mice showed an ipislateral reduction in xCT levels and a bilateral increase in AGS3 levels in the spinal cord. Conclusions These data demonstrate that

  13. Oxygen limitation modulates pH regulation of catabolism and hydrogenases, multidrug transporters, and envelope composition in Escherichia coli K-12

    Directory of Open Access Journals (Sweden)

    Radmacher Michael D

    2006-10-01

    Full Text Available Abstract Background In Escherichia coli, pH regulates genes for amino-acid and sugar catabolism, electron transport, oxidative stress, periplasmic and envelope proteins. Many pH-dependent genes are co-regulated by anaerobiosis, but the overall intersection of pH stress and oxygen limitation has not been investigated. Results The pH dependence of gene expression was analyzed in oxygen-limited cultures of E. coli K-12 strain W3110. E. coli K-12 strain W3110 was cultured in closed tubes containing LBK broth buffered at pH 5.7, pH 7.0, and pH 8.5. Affymetrix array hybridization revealed pH-dependent expression of 1,384 genes and 610 intergenic regions. A core group of 251 genes showed pH responses similar to those in a previous study of cultures grown with aeration. The highly acid-induced gene yagU was shown to be required for extreme-acid resistance (survival at pH 2. Acid also up-regulated fimbriae (fimAC, periplasmic chaperones (hdeAB, cyclopropane fatty acid synthase (cfa, and the "constitutive" Na+/H+ antiporter (nhaB. Base up-regulated core genes for maltodextrin transport (lamB, mal, ATP synthase (atp, and DNA repair (recA, mutL. Other genes showed opposite pH responses with or without aeration, for example ETS components (cyo,nuo, sdh and hydrogenases (hya, hyb, hyc, hyf, hyp. A hypF strain lacking all hydrogenase activity showed loss of extreme-acid resistance. Under oxygen limitation only, acid down-regulated ribosome synthesis (rpl,rpm, rps. Acid up-regulated the catabolism of sugar derivatives whose fermentation minimized acid production (gnd, gnt, srl, and also a cluster of 13 genes in the gadA region. Acid up-regulated drug transporters (mdtEF, mdtL, but down-regulated penicillin-binding proteins (dacACD, mreBC. Intergenic regions containing regulatory sRNAs were up-regulated by acid (ryeA, csrB, gadY, rybC. Conclusion pH regulates a core set of genes independently of oxygen, including yagU, fimbriae, periplasmic chaperones, and nha

  14. Contribution of positron emission tomography for the study of response variability to opioid drugs

    International Nuclear Information System (INIS)

    Auvity, Sylvain

    2017-01-01

    the neuro-pharmacokinetic component of opioid pharmacology. 11 C-diphenhydramine was developed in rats as a specific probe to estimate the H + /antiporter function at the blood-brain barrier. This newly identified carrier transport System was shown to control the brain exposure of many CNS drugs, including oxycodone. Then, we used 11 C-buprenorphine PET imaging and validated a co-injection strategy to quantify the parameters that describe the brain kinetics of buprenorphine in nonhuman primates. Throughout this project, several methods have been evaluated or developed in order to address original hypothesis regarding the pharmacology of opioid drugs. The choice for noninvasive and quantitative methods such as PET imaging allows for clinical translation. These tools may thus be useful to investigate the neuro-immune and neuro-pharmacokinetic component of the variability of response to opioid drugs in humans. (author) [fr

  15. Escherichia coli YqjA, a Member of the Conserved DedA/Tvp38 Membrane Protein Family, Is a Putative Osmosensing Transporter Required for Growth at Alkaline pH.

    Science.gov (United States)

    Kumar, Sujeet; Doerrler, William T

    2015-07-01

    The ability to persist and grow under alkaline conditions is an important characteristic of many bacteria. In order to survive at alkaline pH, Escherichia coli must maintain a stable cytoplasmic pH of about 7.6. Membrane cation/proton antiporters play a major role in alkaline pH homeostasis by catalyzing active inward proton transport. The DedA/Tvp38 family is a highly conserved membrane protein family of unknown function present in most sequenced genomes. YqjA and YghB are members of the E. coli DedA family with 62% amino acid identity and partially redundant functions. We have shown that E. coli with ΔyqjA and ΔyghB mutations cannot properly maintain the proton motive force (PMF) and is compromised in PMF-dependent drug efflux and other PMF-dependent functions. Furthermore, the functions of YqjA and YghB are dependent upon membrane-embedded acidic amino acids, a hallmark of several families of proton-dependent transporters. Here, we show that the ΔyqjA mutant (but not ΔyghB) cannot grow under alkaline conditions (ranging from pH 8.5 to 9.5), unlike the parent E. coli. Overexpression of yqjA restores growth at alkaline pH, but only when more than ∼100 mM sodium or potassium is present in the growth medium. Increasing the osmotic pressure by the addition of sucrose enhances the ability of YqjA to support growth under alkaline conditions in the presence of low salt concentrations, consistent with YqjA functioning as an osmosensor. We suggest that YqjA possesses proton-dependent transport activity that is stimulated by osmolarity and that it plays a significant role in the survival of E. coli at alkaline pH. The ability to survive under alkaline conditions is important for many species of bacteria. Escherichia coli can grow at pH 5.5 to 9.5 while maintaining a constant cytoplasmic pH of about 7.6. Under alkaline conditions, bacteria rely upon proton-dependent transporters to maintain a constant cytoplasmic pH. The DedA/Tvp38 protein family is a highly conserved

  16. Ion selectivity of the cation transport system of isolated intact cattle rod outer segments: evidence for a direct communication between the rod plasma membrane and the rod disk membranes.

    Science.gov (United States)

    Schnetkamp, P P

    1980-05-08

    The ion selectivity of cation transport through the plasma membrane of isolated intact cattle rod outer segments (rods) is investigated by means of 45Ca-exchange experiments and light-scattering experiments. These techniques appear to provide complementary information: the 45Ca experiments (45Ca fluxes in rods) describe electroneutral antiport, whereas the light-scattering experiments (shrinkage and swelling of rods upon hypertonic shocks with various electrolytes) reveal electrogenic uniport. Electroneutral symport of ions (salt transport) does not take place without addition of external ionophores and application of salts of weak acids. 1. Intact rods recover from a hypertonic shock in the presence of FCCP when lithium, sodium and potassium acetate are applied, but not when ammonium chloride, calcium and magnesium acetate are used. This indicates that the plasma membrane of isolated intact cattle rods is relatively permeable to net transport of Na+, Li+ and K+, and relatively impermeable to net transport of Cl-, Mg2+ and Ca2+ under conditions that do not give rise to diffusion potentials. 2. Rapid (t1/2 exchange diffusion of internal 45Ca with external Na+, Ca2+, Sr2+ and Ba2+, respectively. 3. All tested cations lower the rate of 45Ca uptake. The latter can be described by a single rate constant indicating a homogeneous rod preparation and a homogeneous endogenous Ca2+ pool. However, only those cations which stimulate 45Ca efflux from preloaded rods lower the final equilibrium of 45Ca uptake. Except for the effects of K+, Rb+ and Cs+ the reduction of the rate of 45Ca uptake by external cations appears to arise from competition for a common site on the plasms membrane. The observed affinities for this site do not correlate with actual transport (as indicated by the ability to stimulate 45Ca efflux). 4. K+ increases the affinity of the exchange diffusion system to Ca2+ from 1 microM to 0.15 microM and changes the relative affinities with respect to Ca2+ for the

  17. Endomembrane Cation Transporters and Membrane Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Sze, Heven [Univ. of Maryland, College Park, MD (United States). Dept. of Cell Biology & Molecular Genetics

    2017-04-01

    Multicellular, as well as unicellular, organisms have evolved mechanisms to regulate ion and pH homeostasis in response to developmental cues and to a changing environment. The working hypothesis is that the balance of fluxes mediated by diverse transporters at the plasma membrane and in subcellular organelles determines ionic cellular distribution, which is critical for maintenance of membrane potential, pH control, osmolality, transport of nutrients, and protein activity. An emerging theme in plant cell biology is that cells respond and adapt to diverse cues through changes of the dynamic endomembrane system. Yet we know very little about the transporters that might influence the operation of the secretory system in plants. Here we focus on transporters that influence alkali cation and pH homeostasis, mainly in the endomembrane/ secretory system. The endomembrane system of eukaryote cells serves several major functions: i) sort cargo (e.g. enzymes, transporters or receptors) to specific destinations, ii) modulate the protein and lipid composition of membrane domains through remodeling, and iii) determine and alter the properties of the cell wall through synthesis and remodeling. We had uncovered a novel family of predicted cation/H+ exchangers (CHX) and K+ efflux antiporters (KEA) that are prevalent in higher plants, but rare in metazoans. We combined phylogenetic and transcriptomic analyses with molecular genetic, cell biological and biochemical studies, and have published the first reports on functions of plant CHXs and KEAs. CHX studied to date act at the endomembrane system where their actions are distinct from the better-studied NHX (Na/K-H+ exchangers). Arabidopsis thaliana CHX20 in guard cells modulate stomatal opening, and thus is significant for vegetative survival. Other CHXs ensure reproductive success on dry land, as they participate in organizing pollen walls, targeting of pollen tubes to the ovule or promoting

  18. Calcium in plant cells

    Directory of Open Access Journals (Sweden)

    V. V. Schwartau

    2014-04-01

    Full Text Available The paper gives the review on the role of calcium in many physiological processes of plant organisms, including growth and development, protection from pathogenic influences, response to changing environmental factors, and many other aspects of plant physiology. Initial intake of calcium ions is carried out by Ca2+-channels of plasma membrane and they are further transported by the xylem owing to auxins’ attractive ability. The level of intake and selectivity of calcium transport to ove-ground parts of the plant is controlled by a symplast. Ca2+enters to the cytoplasm of endoderm cells through calcium channels on the cortical side of Kaspary bands, and is redistributed inside the stele by the symplast, with the use of Ca2+-АТPases and Ca2+/Н+-antiports. Owing to regulated expression and activity of these calcium transporters, calclum can be selectively delivered to the xylem. Important role in supporting calcium homeostasis is given to the vacuole which is the largest depo of calcium. Regulated quantity of calcium movement through the tonoplast is provided by a number of potential-, ligand-gated active transporters and channels, like Ca2+-ATPase and Ca2+/H+ exchanger. They are actively involved in the inactivation of the calcium signal by pumping Ca2+ to the depo of cells. Calcium ATPases are high affinity pumps that efficiently transfer calcium ions against the concentration gradient in their presence in the solution in nanomolar concentrations. Calcium exchangers are low affinity, high capacity Ca2+ transporters that are effectively transporting calcium after raising its concentration in the cell cytosol through the use of protons gradients. Maintaining constant concentration and participation in the response to stimuli of different types also involves EPR, plastids, mitochondria, and cell wall. Calcium binding proteins contain several conserved sequences that provide sensitivity to changes in the concentration of Ca2+ and when you

  19. A carbohydrate pulse experiment to demonstrate the sugar metabolization by S. mutans

    Directory of Open Access Journals (Sweden)

    T.P. Paulino

    2006-07-01

    carbohydrate addition (decreasing  1.4 units  of pH. The  initial  acidification  in the presence of xilose  may  occur  due  to the mechanism of sugar uptake by this organism, which involves the antiport with H+. In media without the addition  of  carbohydrate,  the  acidification  may  be  due  to  the  metabolization  of  intracellular  reserves  of sugars. Fluoride affects negatively the acidogenic capacity of S. mutans for all metabolized sugars.