WorldWideScience

Sample records for antiporters

  1. Cystine/glutamate antiporter blockage induces myelin degeneration.

    Science.gov (United States)

    Soria, Federico N; Zabala, Alazne; Pampliega, Olatz; Palomino, Aitor; Miguelez, Cristina; Ugedo, Luisa; Sato, Hideyo; Matute, Carlos; Domercq, María

    2016-08-01

    The cystine/glutamate antiporter is a membrane transport system responsible for the uptake of extracellular cystine and release of intracellular glutamate. It is the major source of cystine in most cells, and a key regulator of extrasynaptic glutamate in the CNS. Because cystine is the limiting factor in the biosynthesis of glutathione, and glutamate is the most abundant neurotransmitter, the cystine/glutamate antiporter is a central player both in antioxidant defense and glutamatergic signaling, two events critical to brain function. However, distribution of cystine/glutamate antiporter in CNS has not been well characterized. Here, we analyzed expression of the catalytic subunit of the cystine/glutamate antiporter, xCT, by immunohistochemistry in histological sections of the forebrain and spinal cord. We detected labeling in neurons, oligodendrocytes, microglia, and oligodendrocyte precursor cells, but not in GFAP(+) astrocytes. In addition, we examined xCT expression and function by qPCR and cystine uptake in primary rat cultures of CNS, detecting higher levels of antiporter expression in neurons and oligodendrocytes. Chronic inhibition of cystine/glutamate antiporter caused high toxicity to cultured oligodendrocytes. In accordance, chronic blockage of cystine/glutamate antiporter as well as glutathione depletion caused myelin disruption in organotypic cerebellar slices. Finally, mice chronically treated with sulfasalazine, a cystine/glutamate antiporter inhibitor, showed a reduction in the levels of myelin and an increase in the myelinated fiber g-ratio. Together, these results reveal that cystine/glutamate antiporter is expressed in oligodendrocytes, where it is a key factor to the maintenance of cell homeostasis. GLIA 2016. GLIA 2016;64:1381-1395. PMID:27247047

  2. Plant and Yeast NHX Antiporters: Roles in MembraneTrafficking

    Institute of Scientific and Technical Information of China (English)

    Quan-Sheng Qiu

    2012-01-01

    The plant NHX gene family encodes Na+/H+ antiporters which are crucial for salt tolerance,potassium homeostasis and cellular pH regulation.Understanding the role of NHX antiporters in membrane trafficking is becoming an increasingly interesting subject of study.Membrane trafficking is a central cellular process during which proteins,lipids and polysaccharides are continuously exchanged among membrane compartments.Yeast ScNhx1p,a prevacuole/vacuolar Na+/H+ antiporter,plays an important role in regulating pH to control trafficking out of the endosome.Evidence begins to accumulate that plant NHX antiporters might function in regulating membrane trafficking in plants.

  3. Small Computationally Complete Symport/Antiport P Systems

    OpenAIRE

    Csuhaj Varjú, Erzsébet; Margenstern, Maurice; Vaszil, György; Verlan, Sergey; Research Group on Natural Computing (Universidad de Sevilla) (Coordinador)

    2006-01-01

    It is known that P systems with symport/antiport rules simulate the register machines, i.e., they are computationally complete. Hence, due to the existence of universal register machines, there exist computationally complete subclasses of symport/antiport P systems with a number of rules limited by a constant. However, there was no estimation of this number in the literature. In this article, we first give a simple estimation of this constant, and then we show that the number c...

  4. 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*

    OpenAIRE

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

    2010-01-01

    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 resista...

  5. Molecular Characterization of the Na+/H+-Antiporter NhaA from Salmonella Typhimurium

    OpenAIRE

    Lentes, Christopher J.; Mir, Syed H.; Boehm, Marc; Ganea, Constanta; Fendler, Klaus; Hunte, Carola

    2014-01-01

    Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respect...

  6. Effects of thyroid hormone on the neonatal renal cortical Na+/H+ antiporter

    OpenAIRE

    Baum, Michel; Dwarakanath, Vangipuram; Alpern, Robert J; Moe, Orson W.

    1998-01-01

    The neonatal proximal tubule has a lower rate of bicarbonate absorption than that of adults. This is due, in part, to a lower rate of apical membrane Na+/H+ antiporter activity. The purpose of these studies was to examine if thyroid hormone could be a factor in the maturational increase in Na+/H+ antiporter activity. Hypothyroid (0.01% propylthiouracil in drinking water starting at day 14 gestation and throughout the postnatal period), euthyroid, and hyperthyroid (intraperitoneal triiodothyro...

  7. Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella Typhimurium

    OpenAIRE

    Lentes, Christopher J.; Syed H Mir; Boehm, Marc; Ganea, Constanta; Fendler, Klaus; Hunte, Carola

    2014-01-01

    Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respect...

  8. Maturation of the Na+/H+ antiporter (NHE3) in the proximal tubule of the hypothyroid adrenalectomized rat

    OpenAIRE

    Gupta, Neena; Dwarakanath, Vangipuram; Baum, Michel

    2004-01-01

    In previous studies examining the role of glucocorticoids and thyroid hormone on the maturation of the Na+/H+ antiporter (NHE3), we found attenuation in the maturational increase in proximal tubule apical Na+/H+ antiporter activity but no change in NHE3 mRNA abundance in either glucocorticoid-deficient or hypothyroid rats. In addition, prevention of the maturational increase in either hormone failed to totally prevent the maturational increase in Na+/H+ antiporter activity. We hypothesized th...

  9. EVIDENCE OF DIFFERENTIAL PH REGULATION OF THE ARABIDOPSIS VACUOLAR CA2+/H+ ANTIPORTERS CAX1 AND CAX2

    Science.gov (United States)

    The Arabidopsis Ca(2+)/H(+) antiporters cation exchanger (CAX) 1 and 2 utilise an electrochemical gradient to transport Ca(2+) into the vacuole to help mediate Ca(2+) homeostasis. Previous whole plant studies indicate that activity of Ca(2+)/H(+) antiporters is regulated by pH. However, the pH regul...

  10. pHluorin enables insights into the transport mechanism of antiporter Mdr1: R215 is critical for drug/H+ antiport.

    Science.gov (United States)

    Redhu, Archana Kumari; Khandelwal, Nitesh Kumar; Banerjee, Atanu; Moreno, Alexis; Falson, Pierre; Prasad, Rajendra

    2016-10-01

    Multidrug resistance 1 (MDR1) is a member of the major facilitator superfamily that contributes to MDR of Candida albicans This antiporter belongs to the drug/H(+) antiporter 1 family, pairing the downhill gradient of protons to drug extrusion. Hence, drug efflux from cytosol to extracellular space and the parallel import of H(+) towards cytosol are inextricably linked processes. For monitoring the drug/H(+) antiporter activity of Mdr1p, we developed a new system, exploiting a GFP variant pHluorin, which changes its fluorescence properties with pH. This enabled us to measure the cytosolic pH correlated to drug efflux. Since protonation of charged residues is a key step in proton movement, we explored the role of all charged residues of the 12 transmembrane segments (TMSs) of Mdr1p in drug/H(+) transport by mutational analysis. This revealed that the conserved residue R(215), positioned close to the C-terminal end of TMS-4, is critical for drug/H(+) antiport, allowing protonation over a range of pH, in contrast with its H(215) or K(215) variants that failed to transport drugs at basic pH. Mutation of other residues of TMS-4 highlights the role of this TMS in drug transport, as confirmed by in silico modelling of Mdr1p and docking of drugs. The model points to the importance of R(215) in proton transport, suggesting that it may adopt two main conformations, one oriented towards the extracellular face and the other towards the centre of Mdr1p. Together, our results not only establish a new system for monitoring drug/H(+) transport, but also unveil a positively charged residue critical to Mdr1p function.

  11. Whole-genome identiifcation and expression analysis of K+eflfux antiporter (KEA) and Na+/H+antiporter (NHX) families under abiotic stress in soybean

    Institute of Scientific and Technical Information of China (English)

    CHEN Hua-tao; CHEN Xin; WU Bing-yue; YUAN Xing-xing; ZHANG Hong-mei; CUI Xiao-yan; LIU Xiao-qing

    2015-01-01

    Sodium toxicity and potassium insufifcient are important factors affecting the growth and development of soybean in saline soil. As the capacity of plants to maintain a high cytosolic, K+/Na+ratio is the key determinant of tolerance under salt stress. The aims of the present study were to identify and analyse expression patterns of the soybean K+eflfux antiporter (KEA) gene and Na+/H+ antiporter (NHX) gene family, and to explore their roles under abiotic stress. As a result, 12 soybean GmKEAs genes and 10 soybean GmNHXs genes were identiifed and analyzed from soybean genome. Interestingly, the novel soybean KEA gene Glyma16g32821 which encodes 11 transmembrane domains were extremely up-regulated and remained high level until 48 h in root after the excessive potassium treatment and lack of potassium treatment, respectively. The novel soybean NHX gene Glyma09g02130 which encodes 10 transmembrane domains were extremely up-regulated and remained high level until 48 h in root with NaCl stress. Imaging of subcel ular locations of the two new Glyma16g32821-GFP and Glyma09g02130-GFP fusion proteins indicated al plasma membrane localizations of the two novel soybean genes. The 3D structures indicated that the two soybean novel proteins Glyma09g02130 (NHX) and Glyma16g32821 (KEA) al belong to the cation/hydrogen antiporter family.

  12. Structural basis of Na+-independent and cooperative substrate/product antiport in CaiT

    NARCIS (Netherlands)

    Schulze, Sabrina; Köster, Stefan; Geldmacher, Ulrike; Terwisscha van Scheltinga, Anke C.; Kühlbrandt, Werner

    2010-01-01

    Transport of solutes across biological membranes is performed by specialized secondary transport proteins in the lipid bilayer, and is essential for life. Here we report the structures of the sodium-independent carnitine/butyrobetaine antiporter CaiT from Proteus mirabilis (PmCaiT) at 2.3-Å and from

  13. Antiporter Gene from Hordum brevisubulatum (Trin.) Link and Its Overexpression in Transgenic Tobaccos

    Institute of Scientific and Technical Information of China (English)

    Shi-You Lü; Yu-Xiang JING; Shi-Hua SHEN; Hua-Yan ZHAO; Lan-Qing MA; Xiang-Juan ZHOU; Qing REN; Yan-Fang LI

    2005-01-01

    A vacuolar Na+/H+ antiporter cDNA gene was successfully isolated from Hordeum brevisubulatum (Trin.) Link using the rapid amplification of cDNA ends (RACE) method. The gene was named HbNHX1 and was found to consist of 1 916 bp encoding a predicted polypeptide of 540 amino acids with a conserved amiloride-binding domain. Phylogenetic tree analysis of the Na+/H+ antiporters showed that the HbNHX1gene shares 55.3%-74.8% similarity with the vacuolar-type Na+/H+ antiporters. Transgenic tobaccos that contain the HbNHX1 gene, integrated by forward insertion into the tobacco genome, were obtained via Agrobacterium tumerfaciens and characterized for the determination of the concentration of Na+ and K+ions, as well as proline, in the presence of 300 mmol/L NaCl. The T1 transgenic plants showed more tolerance to salt and drought than did wild-type plants. Our data suggest that overexpression of the HbNHX1 gene could improve the tolerance of transgenic tobaccos to salt and drought through the function of the vacuolar Na+/H+ antiporter.

  14. Sodium-Proton (Na(+)/H(+)) Antiporters: Properties and Roles in Health and Disease.

    Science.gov (United States)

    Padan, Etana; Landau, Meytal

    2016-01-01

    The transmembranal Na(+)/H(+) antiporters transport sodium (or several other monovalent cations) in exchange for H(+) across lipid bilayers in all kingdoms of life. They are critical in pH homeostasis of the cytoplasm and/or organelles. A particularly notable example is the SLC9 gene family, which encodes Na(+)/H(+) exchangers (NHEs) in many species from prokaryotes to eukaryotes. In humans, these proteins are associated with the pathophysiology of various diseases. Yet, the most extensively studied Na(+)/H(+) antiporter is Ec-NhaA, the main Na(+)/H(+) antiporter of Escherichia coli.The crystal structure of down-regulated Ec-NhaA, determined at acidic pH, has provided the first structural insights into the antiport mechanism and pH regulation of an Na(+)/H(+) antiporter. It reveals a unique structural fold (called the NhaA fold) in which transmembrane segments (TMs) are organized in inverted-topology repeats, including two antiparallel unfolded regions that cross each other, forming a delicate electrostatic balance in the middle of the membrane. This unique structural fold (The NhaA fold) contributes to the cation binding site and facilitates the rapid conformational changes expected for Ec-NhaA. The NhaA fold has now been recognized to be shared by four Na(+)/H(+) antiporters (bacterial and archaeal) and a Na(+) symporter. Remarkably, no crystal structure of any of the human Na(+)/H(+) antiporters exists. Nevertheless, the Ec-NhaA crystal structure has enabled the structural modeling of NHE1, NHE9, and NHA2, three human plasmalemmal proteins that are members of the SLC9 family that are involved in human pathophysiology. Moreover, as outlined in this review, developments in the field, including cellular and biophysical methods that enable ion levels and fluxes to be measured in intact cells as well as in knockout mice, have led to striking advances in the identification and characterization of plasma membrane NHEs and NHA.Very little is known about the endomembrane

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

    Indian Academy of Sciences (India)

    Jia Ning Yu; Jian Huang; Zi Ning Wang; Jin Song Zhang; Shou Yi Chen

    2007-09-01

    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 suppressed the salt sensitivity of the mutant, which was deficient in vacuolar Na+/H+ antiporter, and caused partial recovery of growth of nhx1 in NaCl and LiCl media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.

  16. Hydrophilic C terminus of Salicornia europaea vacuolar Na+/H+ antiporter is necessary for its function

    Indian Academy of Sciences (India)

    Guangxia Wu; Gang Wang; Jing Ji; Xiaowei Tian; Hailing Gao; Qing Zhao; Jing Li; Yurong Wang

    2014-08-01

    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 hydrophilic C terminus of a vacuolar Na+/H+ antiporter gene from Salicornia europaea (SeNHX1) to generate its derivative, SeNHX1-C. Expression of SeNHX1 and SeNHX1-C in yeast mutant showed that SeNHX1 significantly improved the tolerance to NaCl; however, the expression of SeNHX1-C enormously decreased the tolerance to NaCl. Overall, these results suggest that the hydrophilic C-terminal region of SeNHX1 is required for Na+/H+ exchanging activity of SeNHX1.

  17. Intracellular NHX-Type Cation/H+ Antiporters inPlants

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Cells depend on the homeostatic maintenance of pHwithin specific cellular compartments to ensure optimalconditions for metabolic and enzymatic processes as wellas protein structure and function. In the animal secre-tory pathway, cells maintain distinct luminal pHs withinvarious compartments (Paroutis et al., 2004). Among themany molecular players that contribute to pH and ionhomeostasis in plants, Na+(K+)/H+ exchangers (also knownas NHX-type cation/H+ antiporters) appear to be particu-larly important for the regulation of a wide variety ofphysiological processes, including cell expansion, cellvolume regulation, osmotic adjustment, pH regulation,membrane trafficking, protein processing, and cellularstress responses (Pardo et al., 2006; Rodriguez-Rosaleset al., 2009; Bassil et al., 2012). In plants, NHX antiportersappeared early in evolution and are ubiquitously encodedmembers of the CPA1 cation/H+ antiporters subgroupthat belongs to the large family of monovalent cation/H+ transporters CPA (Brett et al., 2005). NHX antiport-ers are found, thus far, in all sequenced plant genomes(Bassil et al., 2012; Chanroj et al., 2012). In Arabidopsis,the NHX family consists of eight isoforms, six of whichare intracellular (AtNHXl-AtNHX6), located either to thevacuole (AtNHXl to AtNHX4) or endosomes (AtNHX5 andAtNHX6) and an additional two more divergent members(AtNHX7/SOSl and AtNHX8) at the plasma membrane(Bassil et al., 2012). Orthologous sequences in each of thethree classes (plasma membrane, vacuolar, or endosomal)appear in all sequenced genomes, suggesting that distinctfunctional NHX classes appeared early in evolution andmay have conserved roles that are compartment-specific(Bassil et al., 2012). Emerging new evidence highlightsthe importance of particular intracellular NHX antiport-ers in the regulation of vesicular and vacuolar pH andK+ homeostasis. Vacuolar NHXs are needed to maintainK+ homeostasis

  18. 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...

  19. The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp.

    OpenAIRE

    Kitada, M; Onda, K.; Horikoshi, K

    1989-01-01

    The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the sam...

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

    OpenAIRE

    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 ...

  1. 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▿ †

    OpenAIRE

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

    2008-01-01

    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 ...

  2. 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.

  3. 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

    2015-05-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

    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 /sup 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.

  5. Transforming a drug/H+ antiporter into a polyamine importer by a single mutation.

    Science.gov (United States)

    Brill, Shlomo; Falk, Ofir Sade; Schuldiner, Shimon

    2012-10-16

    EmrE, a multidrug antiporter from Escherichia coli, has presented biochemists with unusual surprises. Here we describe the transformation of EmrE, a drug/H(+) antiporter to a polyamine importer by a single mutation. Antibiotic resistance in microorganisms may arise by mutations at certain chromosomal loci. To investigate this phenomenon, we used directed evolution of EmrE to assess the rate of development of novel specificities in existing multidrug transporters. Strikingly, when a library of random mutants of EmrE was screened for resistance to two major antibacterial drugs--norfloxacin, a fluoroquinolone, and erythromycin, a macrolide--proteins with single mutations were found capable of conferring resistance. The mutation conferring erythromycin resistance resulted from substitution of a fully conserved and essential tryptophan residue to glycine, and, as expected, this protein lost its ability to recognize and transport the classical EmrE substrates. However, this protein functions now as an electrochemical potential driven importer of a new set of substrates: aliphatic polyamines. This mutant provides a unique paradigm to understand the function and evolution of distinct modes of transport.

  6. Proton-Coupled Organic Cation Antiporter Contributes to the Hepatic Uptake of Matrine.

    Science.gov (United States)

    Wu, Chunyong; Sun, Xiaomin; Feng, Chao; Liu, Xiaoying; Wang, Hufang; Feng, Fang; Zhang, Junying

    2016-03-01

    Matrine is the major bioactive alkaloid found in certain Sophora plants and has been used for the treatment of liver diseases and protection of liver function. The aim of this study was to investigate the human liver uptake mechanism of matrine by using HepG2 cells as the in vitro model. Matrine was transported into HepG2 cells in a time- and temperature-dependent manner. The cellular uptake was saturable and was significantly reduced by the metabolic inhibitors, such as sodium azide and rotenone. Furthermore, the uptake of matrine was found to be regulated by a protonophore (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) and pH, indicating that this influx transporter may be a proton-coupled antiporter. The uptake of matrine was sensitive to inhibition by the cationic drugs including pyrilamine, quinidine, verapamil, amantadine, diphenhydramine, and cimetidine but insensitive to other typical substrates or inhibitors of well-known organic cation transport systems. The present study reveals that, for the first time, in HepG2 cells, the existence of a proton-coupled organic cation antiporter that contributes substantially to the hepatic uptake of matrine.

  7. 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. PMID:36390

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

    International Nuclear Information System (INIS)

    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 32Pi 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

  9. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    Science.gov (United States)

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  10. 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.

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

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    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.

  12. Functional characterization of a plasma membrane Na+/H+ antiporter from alkali grass (Puccinellia tenuiflora).

    Science.gov (United States)

    Wang, Xin; Yang, Ru; Wang, Baichen; Liu, Guifeng; Yang, Chuanping; Cheng, Yuxiang

    2011-10-01

    We have cloned a Na(+)/H(+) antiporter gene (GenBank accession no EF440291, PtNHA1) from Puccinellia tenuiflora (so-called alkali grass in Chinese) roots under NaCl salt stress. Its cDNA is 3775 bp and contains a 3414 bp open reading frame. The amino acid sequences of PtNHA1 show high identities with a putative plasma membrane Na(+)/H(+) antiporter from wheat. PtNHA1 was predicted to contain 11 hypothetical transmembrane domains in the N-terminal part and to localize in the plasma membrane. Genomic DNA gel blot analysis shows that PtNHA1 is a single-copy gene in the alkali grass genome. PtNHA1 is highly expressed in leaves, roots and shoots by RNA gel blot analysis. Furthermore, PtNHA1 gene expression of alkali grass was clearly up-regulated by NaCl salt stress. Overexpression of PtNHA1 in Arabidopsis resulted in enhanced tolerance of transgenic plants to NaCl stress. The ion contents analysis shows that, compared with the wild-type (WT), less Na(+) and more K(+) were accumulated in transgenic plants under NaCl stress. The results indicate that PtNHA1 play an important role in NaCl salt stress. Additionally, compared with the WT, total activities of ascorbate peroxidase (APX) and catalase (CAT), two key reactive oxygen species (ROS) detoxifying enzymes were high in transgenic plants under salt stress, respectively. The transcript levels of two APX genes (Apx1, s/mApx) and two CAT genes (Cat1, Cat2) in transgenic plants were higher than those in WT. This suggests that overexpression of PtNHA1 results in enhanced ROS-scavenging enzymes of transgenic plants under NaCl salt stress.

  13. 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.

  14. A yeast expression system for functional and pharmacological studies of the malaria parasite Ca2+/H+ antiporter

    Directory of Open Access Journals (Sweden)

    Salcedo-Sora J

    2012-08-01

    Full Text Available Abstract Background Calcium (Ca2+ signalling is fundamental for host cell invasion, motility, in vivo synchronicity and sexual differentiation of the malaria parasite. Consequently, cytoplasmic free Ca2+ is tightly regulated through the co-ordinated action of primary and secondary Ca2+ transporters. Identifying selective inhibitors of Ca2+ transporters is key towards understanding their physiological role as well as having therapeutic potential, therefore screening systems to facilitate the search for potential inhibitors are a priority. Here, the methodology for the expression of a Calcium membrane transporter that can be scaled to high throughputs in yeast is presented. Methods The Plasmodium falciparum Ca2+/H+ antiporter (PfCHA was expressed in the yeast Saccharomyces cerevisiae and its activity monitored by the bioluminescence from apoaequorin triggered by divalent cations, such as calcium, magnesium and manganese. Results Bioluminescence assays demonstrated that PfCHA effectively suppressed induced cytoplasmic peaks of Ca2+, Mg2+ and Mn2+ in yeast mutants lacking the homologue yeast antiporter Vcx1p. In the scalable format of 96-well culture plates pharmacological assays with a cation antiporter inhibitor allowed the measurement of inhibition of the Ca2+ transport activity of PfCHA conveniently translated to the familiar concept of fractional inhibitory concentrations. Furthermore, the cytolocalization of this antiporter in the yeast cells showed that whilst PfCHA seems to locate to the mitochondrion of P. falciparum, in yeast PfCHA is sorted to the vacuole. This facilitates the real-time Ca2+-loading assays for further functional and pharmacological studies. Discussion The functional expression of PfCHA in S. cerevisiae and luminescence-based detection of cytoplasmic cations as presented here offer a tractable system that facilitates functional and pharmacological studies in a high-throughput format. PfCHA is shown to behave as a divalent

  15. Mechanism of pH-dependent activation of the sodium-proton antiporter NhaA

    Science.gov (United States)

    Huang, Yandong; Chen, Wei; Dotson, David L.; Beckstein, Oliver; Shen, Jana

    2016-10-01

    Escherichia coli NhaA is a prototype sodium-proton antiporter, which has been extensively characterized by X-ray crystallography, biochemical and biophysical experiments. However, the identities of proton carriers and details of pH-regulated mechanism remain controversial. Here we report constant pH molecular dynamics data, which reveal that NhaA activation involves a net charge switch of a pH sensor at the entrance of the cytoplasmic funnel and opening of a hydrophobic gate at the end of the funnel. The latter is triggered by charging of Asp164, the first proton carrier. The second proton carrier Lys300 forms a salt bridge with Asp163 in the inactive state, and releases a proton when a sodium ion binds Asp163. These data reconcile current models and illustrate the power of state-of-the-art molecular dynamics simulations in providing atomic details of proton-coupled transport across membrane which is challenging to elucidate by experimental techniques.

  16. Simulation of a bounded symport/antiport P system with Brane calculi.

    Science.gov (United States)

    Vitale, Antonio; Mauri, Giancarlo; Zandron, Claudio

    2008-03-01

    Membrane systems (also called P systems) and Brane calculi have been recently introduced as formal models inspired by the structure and the functioning of living cells, but having in mind different goals. The aim of Membrane systems was the formal investigation of the computational nature and power of various features of the cell, while Brane calculi aims to define a model capable of a faithful and intuitive representation of various biological processes. The common background of the two formalisms and the recent growing of interests in applying P systems in Systems Biology have raised the natural question of bridging this two research areas. The present paper goes in this direction, as it presents a direct simulation of a variant of P systems by means of Brane calculi. In particular, we consider a Brane calculus based on three operations called Mate/Bud/Drip, and we show how to use such system to simulate Simple symport/antiport P systems, a variant of P systems purely based on communication of objects. As an example, a simplified sodium-potassium pump modeled in Simple SA is encoded in Mate/Bud/Drip Brane calculus. PMID:17889992

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

    International Nuclear Information System (INIS)

    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 [14C]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

  18. Membrane topology of aspartate:alanine antiporter AspT from Comamonas testosteroni.

    Science.gov (United States)

    Fujiki, Takashi; Nanatani, Kei; Nishitani, Kei; Yagi, Kyoko; Ohnishi, Fumito; Yoneyama, Hiroshi; Uchida, Takafumi; Nakajima, Tasuku; Abea, Keietsu

    2007-01-01

    We cloned the aspT gene encoding the L-aspartate:L-alanine antiporter AspTCt in Comamonas testosteroni genomic DNA. Analysis of the nucleotide sequence revealed that C. testosteroni has an asp operon containing aspT upstream of the l-aspartate 4-decarboxylase gene, and that the gene order of the asp operon of C. testosteroni is the inverse of that of Tetragenococcus halophilus. We used proteoliposomes to confirm the transport processes of AspTCt. To elucidate the two-dimensional structure of AspTCt, we analysed its membrane topology by means of alkaline phosphatase (PhoA) and beta-lactamase (BlaM) fusion methods. The fusion analyses revealed that AspTCt has seven transmembrane segments (TMs), a large cytoplasmic loop containing approximately 200 amino acid residues between TM4 and TM5, a cytoplasmic N-terminus, and a periplasmic C-terminus. These results suggest that the orientation of the N-terminus of AspTCt differs from that of tetragenococcal AspT, even though these two AspT orthologues catalyse the same transport reactions. PMID:17158863

  19. Evidence for nickel/proton antiport activity at the tonoplast of the hyperaccumulator plant Alyssum lesbiacum.

    Science.gov (United States)

    Ingle, R A; Fricker, M D; Smith, J A C

    2008-11-01

    The mechanism of nickel uptake into vacuoles isolated from leaf tissue of Alyssum lesbiacum was investigated to help understand the ability of this species to hyperaccumulate Ni. An imaging system was designed to monitor Ni uptake by single vacuoles using the metal-sensitive fluorescent dye, Newport Green. Nickel uptake into isolated vacuoles from leaf tissue of A. lesbiacum was enhanced by the presence of Mg/ATP, presumably via energisation of the vacuolar H(+)-ATPase (V-ATPase). This ATP-stimulated Ni uptake was abolished by bafilomycin (a diagnostic inhibitor of the V-ATPase) and by dissipation of the transmembrane pH difference with an uncoupler. These observations are consistent with Ni(2+)/nH(+) antiport activity at the tonoplast driven by a proton electrochemical gradient established by the V-ATPase, which would provide a mechanism for secondary active transport of Ni(2+) into the vacuole. This study provides insights into the molecular basis of Ni tolerance in Alyssum, and may aid in the identification of genes involved in Ni hyperaccumulation. PMID:18950432

  20. 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.

  1. Effects of NaCl and Iso-Osmotic Polyethylene Glycol Stress on Na+/H+Antiport Activity of Three Malus species with Different Salt Tolerance

    Institute of Scientific and Technical Information of China (English)

    YANG Hong-bing; DONG Chun-hai; XU Xue-feng; WANG Yi; HAN Zhen-hai

    2014-01-01

    Salt stress contains osmotic and ionic stress, while iso-osmotic polyethylene glycol (PEG) has only osmotic stress. This study aimed to compare the different effects on the activity of H+-ATPase, proton pump and Na+/H+antiport in Malus seedlings between osmotic and ionic stress. Species of salt tolerant Malus zumi, middle salt tolerant Malus xiaojinensis and salt sensitive Malus baccata were used as experimental materials. Malus seedlings were treated with NaCl and iso-osmotic PEG stress. The activity of H+-ATPase, proton pump and Na+/H+antiport of plasmolemma and tonoplast in Malus seedlings were obviously increased under salt stress, and those in salt-tolerant species increased more. Under the same NaCl concentration, the activity of H+-ATPase, proton pump and Na+/H+antiport of plasmolemma and tonoplast in salt-tolerant species were all obviously higher than those in salt-sensitive one. Higher Na+/H+antiport activity of plasmolemma and tonoplast in salt-tolerant species could help to extrude and compartmentalize sodium in roots under salt stress. The ascent rate of activity of H+-ATPase, proton pump and Na+/H+antiport in Malus seedlings under the three salt concentration stress was all obviously higher than that under the iso-osmotic PEG stress. It indicated that the sodium ion effect had more stimulation on the activity of H+-ATPase, proton pump and Na+/H+antiport in salt-tolerant species, and salt-tolerant species has higher capability of sodium extrusion and compartmentalization in roots and is therefore more salt tolerant.

  2. 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.

  3. System xc⁻ cystine/glutamate antiporter: an update on molecular pharmacology and roles within the CNS.

    Science.gov (United States)

    Bridges, Richard J; Natale, Nicholas R; Patel, Sarjubhai A

    2012-01-01

    System x(c)(-) is an amino acid antiporter that typically mediates the exchange of extracellular l-cystine and intracellular L-glutamate across the cellular plasma membrane. Studied in a variety of cell types, the import of L-cystine through this transporter is critical to glutathione production and oxidative protection. The exchange-mediated export of L-glutamate takes on added significance within the CNS, as it represents a non-vesicular route of release through which this excitatory neurotransmitter can participate in either neuronal signalling or excitotoxic pathology. When both the import of L-cystine and the export of L-glutamate are taken into consideration, system x(c)(-) has now been linked to a wide range of CNS functions, including oxidative protection, the operation of the blood-brain barrier, neurotransmitter release, synaptic organization, viral pathology, drug addiction, chemosensitivity and chemoresistance, and brain tumour growth. The ability to selectively manipulate system x(c)(-), delineate its function, probe its structure and evaluate it as a therapeutic target is closely linked to understanding its pharmacology and the subsequent development of selective inhibitors and substrates. Towards that goal, this review will examine the current status of our understanding of system x(c)(-) pharmacology and the structure-activity relationships that have guided the development of an initial pharmacophore model, including the presence of lipophilic domains adjacent to the substrate binding site. A special emphasis is placed on the roles of system x(c)(-) within the CNS, as it is these actions that are among the most exciting as potential long-range therapeutic targets.

  4. NhaA antiporter functions using 10 helices, and an additional 2 contribute to assembly/stability.

    Science.gov (United States)

    Padan, Etana; Danieli, Tsafi; Keren, Yael; Alkoby, Dudu; Masrati, Gal; Haliloglu, Turkan; Ben-Tal, Nir; Rimon, Abraham

    2015-10-13

    The Escherichia coli Na(+)/H(+) antiporter (Ec-NhaA) is the best-characterized of all pH-regulated Na(+)/H(+) exchangers that control cellular Na(+) and H(+) homeostasis. Ec-NhaA has 12 helices, 2 of which (VI and VII) are absent from other antiporters that share the Ec-NhaA structural fold. This α-hairpin is located in the dimer interface of the Ec-NhaA homodimer together with a β-sheet. Here we examine computationally and experimentally the role of the α-hairpin in the stability, dimerization, transport, and pH regulation of Ec-NhaA. Evolutionary analysis (ConSurf) indicates that the VI-VII helical hairpin is much less conserved than the remaining transmembrane region. Moreover, normal mode analysis also shows that intact NhaA and a variant, deleted of the α-hairpin, share similar dynamics, suggesting that the structure may be dispensable. Thus, two truncated Ec-NhaA mutants were constructed, one deleted of the α-hairpin and another also lacking the β-sheet. The mutants were studied at physiological pH in the membrane and in detergent micelles. The findings demonstrate that the truncated mutants retain significant activity and regulatory properties but are defective in the assembly/stability of the Ec-NhaA dimer. PMID:26417087

  5. Envelope K+/H+ Antiporters AtKEA1 and AtKEA2 Function in Plastid Development.

    Science.gov (United States)

    Aranda-Sicilia, María Nieves; Aboukila, Ali; Armbruster, Ute; Cagnac, Olivier; Schumann, Tobias; Kunz, Hans-Henning; Jahns, Peter; Rodríguez-Rosales, María Pilar; Sze, Heven; Venema, Kees

    2016-09-01

    It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K(+)/H(+) antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation. PMID:27443603

  6. Functional validation of a novel isoform of Na+/H+ antiporter from Pennisetum glaucum for enhancing salinity tolerance in rice

    Indian Academy of Sciences (India)

    Dheeraj Verma; Sneh L Singla-Pareek; Divya Rajagopal; M K Reddy; S K Sopory

    2007-04-01

    Salt stress is an environmental factor that severely impairs plant growth and productivity. 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 when overexpressed in Brassica juncea. Here, we report the functional validation of this antiporter in crop plant rice. Overexpression of PgNHX1 conferred high level of salinity tolerance in rice. 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 for growing in high saline areas.

  7. Change of Cystine/Glutamate Antiporter Expression in Ethanol-Dependent Rats

    Directory of Open Access Journals (Sweden)

    Alessandra Tiziana Peana

    2014-10-01

    Full Text Available Background: Some drugs of abuse down regulate the expression of cystine/glutamate (xCT antiporter in the nucleus accumbens (Acb after extinction or withdrawal. The altered level of xCT exchanger in Acb, a structure involved in ethanol reinforcement, may contribute to the pathological glutamatergic signalling, linked to addiction. We hypothesised that the expression of xCT may be changed in Acb and whole brain also in non-dependent (occasional drinkers, ethanol-dependent rats, as well as, during ethanol withdrawal.Methods: Wistar rats were made ethanol-dependent by chronic exposure to an alcoholic milk beverage (from 2.4 to 7.2% v/v ethanol. Ethanol non-dependent rats were exposed to a similar, but non-alcoholic liquid diet and self-administered ethanol (10% twice a week. Withdrawal in ethanol-dependent rats was studied at 12 hours after the last ethanol-enriched diet exposure. Immediately after the measurement of somatic signs of withdrawal, Western blot analysis with a polyclonal antibody against xCT was carried out in a naïve control group, non-dependent and ethanol-dependent rats as well as withdrawal rats, in order to study the level of xCT expression in Acb and whole brain. Results. Non-dependent rats self-administered an average dose of 1.21±0.02 g/kg per session (30 min. Daily ethanol consumption during chronic exposure to the alcoholic beverage ranged from 6.30±0.16 to 13.99±0.66 g/kg. Ethanol dependent rats after suspension of the ethanol-enriched diet have shown significant somatic signs of withdrawal. Western blotting analysis of Acb lysates revealed that xCT was over expressed in ethanol-dependent rats whereas in whole brain preparations xCT was over expressed in both non-dependent and ethanol-dependent rats compared to control group. On the contrary, xCT expression during withdrawal was down regulated in Acb and restored to control level in whole brain preparations. Conclusions: The changes of xCT expression in both Acb and

  8. NA+/H+ ANTIPORT ACTIVITY IN TONOPLAST VESICLES FROM ROOTS OF THE SALT-TOLERANT PLANTAGO-MARITIMA AND THE SALT-SENSITIVE PLANTAGO-MEDIA

    NARCIS (Netherlands)

    STAAL, M; MAATHUIS, FJM; ELZENGA, JTM; OVERBEEK, JHM; PRINS, HBA

    1991-01-01

    Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na+ ions. A salt-sensitive (Plantago media L.) and a salt-tolerant (P. maritima L.) species were compared with respect to Na+/H+ antiport activities at the tonoplast. After exposure

  9. Effect of salt stress on the expression of NHX-type Na+/H+antiporters in Populus euphratica and P. pruinosa calli

    Institute of Scientific and Technical Information of China (English)

    LiHua Hu; YuXia Wu

    2014-01-01

    Populus euphratica and Populus pruinosa, sister species in the Turanga Section (Salicaceae), growing in semi-arid saline areas are known for their high salinity tolerance. In this study, by combining growth level with Na+and K+contents, the expression level of vacuolar Na+/H+antiporters was investigated for NaCl-induced changes in P. euphratica and P. pru-inosa calli. Compared to P. euphratica, P. pruinosa calli grew well in 200 mM NaCl stress from 14 to 21 days. Increasing the stressed time caused an increase in Na+ content concomitant with a decrease in K+ content in P. euphratica calli, whereas, with the presence of 200 mM NaCl, K+content has a less increase in 14 and 21 days than in 7 days which was detected in P. pruinosa calli. The transcript levels of six genes coding for NHX-type Na+/H+ antiporters suggest that vacuolar NHX1-NHX6 antiporters play important roles in responding to salt stress in P. pruinosa. Our data suggest that there exists a higher salt tolerance for P. pruinosa than P. euphratica at the cellular level, Na+avoidance or accumulation is observed in cellular compartments, and that expression of NHX antiporters is linked to the accumulator phenotype.

  10. Efficient solutions to hard computational problems by P systems with symport/antiport rules and membrane division.

    Science.gov (United States)

    Song, Bosheng; Pérez-Jiménez, Mario J; Pan, Linqiang

    2015-04-01

    P systems are computing models inspired by some basic features of biological membranes. In this work, membrane division, which provides a way to obtain an exponential workspace in linear time, is introduced into (cell-like) P systems with communication (symport/antiport) rules, where objects are never modified but they just change their places. The computational efficiency of this kind of P systems is studied. Specifically, we present a (uniform) linear time solution to the NP-complete problem, Subset Sum by using division rules for elementary membranes and communication rules of length at most 3. We further prove that such P system allowing division rules for non-elementary membranes can efficiently solve the PSPACE-complete problem, QSAT in a uniform way.

  11. Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic Halomonas sp. Y2.

    Science.gov (United States)

    Cui, Yanbing; Cheng, Bin; Meng, Yiwei; Li, Chunfang; Yin, Huijia; Xu, Ping; Yang, Chunyu

    2016-09-01

    Na(+)/H(+) antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na(+)/H(+) antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na(+) and Li(+) translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0-8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na(+) and Li(+), while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues. PMID:27315164

  12. 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

    2011-08-19

    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-aspartate(1-) with L-alanine(0). Although physiological functions of AspT were well studied, L-aspartate(1-):L-alanine(0) 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 His(6)-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K(m) = 0.35 ± 0.03 mm for L-aspartate, K(m) = 0.098 ± 0 mm for D-aspartate, K(m) = 26 ± 2 mm for L-alanine, K(m) = 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

  13. 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

  14. 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. PMID:24139955

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

    OpenAIRE

    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 cla...

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

    Science.gov (United States)

    Zahrádka, Jaromír; Sychrová, Hana

    2012-06-01

    Saccharomyces cerevisiae extrudes K(+) cations even when potassium is only present in scarce amounts in the environment. Lost potassium is taken up by the Trk1 and Trk2 uptake systems. If the Trk transporters are absent or nonfunctional, the efflux of potassium is significantly diminished. A series of experiments with strains lacking various combinations of potassium efflux and uptake systems revealed that all three potassium-exporting systems the Nha1 antiporter, Ena ATPase and Tok1 channel contribute to potassium homeostasis and are active upon potassium limitation in wild-type cells. In trk1Δ trk2Δ mutants, the potassium efflux via potassium exporters Nha1 and Ena1 is diminished and can be restored either by the expression of TRK1 or deletion of TOK1. In both cases, the relative hyperpolarization of trk1Δ trk2Δ cells is decreased. Thus, it is the plasma-membrane potential which serves as the common mechanism regulating the activity of K(+) exporting systems. There is a continuous uptake and efflux of potassium in yeast cells to regulate their membrane potential and thereby other physiological parameters, and the cells are able to quickly and efficiently compensate for a malfunction of potassium transport in one direction by diminishing the transport in the other direction.

  17. Interaction of lanthanide cations and uranyl ion with the calcium/proton antiport system in Mycobacterium phlei.

    Science.gov (United States)

    Agarwal, N; Kalra, V K

    1983-01-19

    Uranyl ions (UO2+(2)) and lanthanide cations (La3+, Nd3+, Sm3+, Eu3+, Tb3+ and Dy3+) at 100-200 microM concentration inhibited active transport of Ca2+, mediated by respiratory linked substrates as well as by ATP hydrolysis, without affecting respiration and membrane-bound ATPase activity, in inside-out membrane vesicles of Mycobacterium phlei. The extent of inhibition in the uptake of Ca2+, mediated by ATP hydrolysis, increased with increase in ionic radii of these cations. Lanthanide cations did not dissipate the formation of a proton gradient, as measured by determining the effect either on the uptake of [14C]methylamine or energy-linked quenching of the fluorescence of 9-aminoacridine. However, uranyl ion (UO2+(2+)) caused reversal of the energy-linked quenching of 9-aminoacridine. UO2+(2)) concentration yielding 50% of Vmax (S0.5) was approx. 15 microM. Kinetic studies revealed that inhibition in the uptake of Ca2+ was competitive with UO2+(2) while non-competitive with rare-earth metals. It is proposed that inhibition in the uptake of Ca2+ by uranyl ion occurs as a result of UO2+(2) transport into the interior of vesicles in exchange for protons, while lanthanide cations are not being transported but affect the binding of Ca2+ to the membrane, presumably to the Ca2+/H+ antiporter. PMID:6838872

  18. Vacuolar Transport of the Medicinal Alkaloids from Catharanthus roseus Is Mediated by a Proton-Driven Antiport1[W

    Science.gov (United States)

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-01-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3′,4′-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4+ and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H+ gradient preestablished across the tonoplast by either vacuolar H+-ATPase or vacuolar H+-pyrophosphatase. The initial rates of H+ gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H+ antiport system and not by an ion-trap mechanism or ABC transporters. PMID:23686419

  19. Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

    Science.gov (United States)

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-07-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters. PMID:23686419

  20. Sulfasalazine attenuates ACL transection and medial menisectomy-induced cartilage destruction by inhibition of cystine/glutamate antiporter.

    Science.gov (United States)

    Tsai, Wei-Yuan; Tsai, Ru-Yin; Liu, Chih-Chung; Wu, Jia-Lin; Wong, Chih-Shung

    2016-04-01

    We had previously demonstrated that excitatory amino acid glutamate plays a role in the progression and severity of knee osteoarthritis (OA), and early hyaluronic acid injection attenuates the OA progression by attenuation of knee joint glutamate level, which was also related to the cystine/glutamate antiporter system X (system XC-) expression. System XC- uptakes cystine into chondrocytes for glutathione (GSH) synthesis, but the role of system XC- in OA is rarely addressed. Sulfasalazine (SSZ) is a system XC- inhibitor; SSZ was applied intra-articularly to study the function of system XC- in the development of OA in rats subjected to anterior cruciate ligament transection and medial meniscectomy (ACLT + MMx). Moerover, the system XC- activator N-acetylcysteine (NAC) was also applied to verify the role of system XC-. The intra-articular injection of SSZ significantly attenuated knee swelling and cartilage destruction in the knees of ACLT + MMx rats and this effect was blocked by NAC. The results showed that inhibition of system XC- function can attenuate ACLT + MMx-induced cartilage destruction. In the present study, system XC- inhibitor SSZ was shown to reduce glutamate content in synovial fluid and GSH in chondrocytes. It was also showed SSZ could attenuate ACLT + MMx-induced cartilage destruction, and treatment of NAC reversed the protective effect of SSZ.

  1. 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. PMID:17660287

  2. 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.

  3. Isolation and characterisation of transport-defective substrate-binding mutants of the tetracycline antiporter TetA(B).

    Science.gov (United States)

    Wright, David J; Tate, Christopher G

    2015-10-01

    The tetracycline antiporter TetA(B) is a member of the Major Facilitator Superfamily which confers tetracycline resistance to cells by coupling the efflux of tetracycline to the influx of protons down their chemical potential gradient. Although it is a medically important transporter, its structure has yet to be determined. One possibility for why this has proven difficult is that the transporter may be conformationally heterogeneous in the purified state. To overcome this, we developed two strategies to rapidly identify TetA(B) mutants that were transport-defective and that could still bind tetracycline. Up to 9 amino acid residues could be deleted from the loop between transmembrane α-helices 6 and 7 with only a slight decrease in affinity of tetracycline binding as measured by isothermal titration calorimetry, although the mutant was transport-defective. Scanning mutagenesis where all the residues between 2 and 389 were mutated to either valine, alanine or glycine (VAG scan) identified 15 mutants that were significantly impaired in tetracycline transport. Of these mutants, 12 showed no evidence of tetracycline binding by isothermal titration calorimetry performed on the purified transporters. In contrast, the mutants G44V and G346V bound tetracycline 4-5 fold more weakly than TetA(B), with Kds of 28 μM and 36 μM, respectively, whereas the mutant R70G bound tetracycline 3-fold more strongly (Kd 2.1 μM). Systematic mutagenesis is thus an effective strategy for isolating transporter mutants that may be conformationally constrained and which represent attractive targets for crystallisation and structure determination. PMID:26143388

  4. Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli.

    OpenAIRE

    Gerchman, Y.; Olami, Y; Rimon, A.; Taglicht, D; Schuldiner, S; Padan, E

    1993-01-01

    The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R. In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed. By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype ...

  5. Salt tolerance conferred by overexpression of Arabidopsis vacuolar Na(+)/H (+) antiporter gene AtNHX1 in common buckwheat (Fagopyrum esculentum).

    Science.gov (United States)

    Chen, Li-Hong; Zhang, Bo; Xu, Zi-Qin

    2008-02-01

    Agriculture productivity is severely affected by soil salinity. One possible mechanism by which plants could survive salt stress is to compartmentalize sodium ions away from the cytosol. In the present work, transgenic buckwheat plants overexpressing AtNHX1, a vacuolar Na(+)/H(+) antiporter gene from Arabidopsis thaliana, were regenerated after transformation with Agrobacterium tumefaciens. These plants were able to grow, flower and accumulate more rutin in the presence of 200 mmol/l sodium chloride. Moreover, the content of important nutrients in buckwheat was not affected by the high salinity of the soil. These results demonstrated the potential value of these transgenic plants for agriculture use in saline soil.

  6. 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.

  7. Isolation and characterization of plasma membrane Na+/H+ antiporter (SOS1 gene during salinity stress in kallar grass (Leptochloa fusca

    Directory of Open Access Journals (Sweden)

    Banafsheh Taherinia

    2015-03-01

    Full Text Available Background: Leptochloa fusca is a halophyte plant which is highly tolerant to saline and sodic soils and water. Moreover, L. fusca is an attractive model plant to study the mechanism of salt tolerance mainly due to its characteristics as a typical euhalophyte, having both accumulating and excreting salt properties. Soil salinity adversely affects plant growth, 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 of plants. Material and Methods: Using conserved sequences of SOS1, the coding sequence of plasma membrane Na+/H+ antiporter (SOS1 in kallar grass was partially isolated and its expression profile during salinity stress was investigated. Results: The aa (amino acid sequence of the isolated region of LfSOS1 possesses the maximum identity up to 96% of its orthologue in Distichlis spicata. The results of semi-quantitative RT-PCR revealed that salinization was affected SOS1 transcript level positively. The expression of LfSOS1 in leaves of kallar grass progressively increased under all salinity levels compared to control. Conclusions: The results suggest that LfSOS1 may play an essential role in the salt tolerance of L. fusca and may be useful for improving salt tolerance in other crop species.

  8. Ectopic Expression of a Bacterium NhaD-type Na+/H+Antiporter Leads to Increased Tolerance to Combined Salt/Alkali Stresses

    Institute of Scientific and Technical Information of China (English)

    Nai-Qin Zhong; Li-Bo Han; Xiao-Min Wu; Li-Li Wang; Fang Wang; Yan-He Ma; Gui-Xian Xia

    2012-01-01

    AaNhaD,a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica,encodes a Na+/H+antiporter crucial for the bacterium's resistance to salt/alkali stresses.However,it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses.To investigate the use of extremophile genetic resources in higher plants,transgenic tobacco BY-2 cells and plants harboring AaNhaD were generated and their stress tolerance was evaluated.Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner.Compared to wild-type controls,the transgenic cells exhibited increased Na+ concentrations and pH levels in the vacuoles.Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts.Similar to the transgenic BY-2 cells,AaNhaD-overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil.These results indicate that AaNhaD functions as a pH-dependent tonoplast Na+/H+ antiporter in plant cells,thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.

  9. 植物Cation/H+反向转运蛋白研究进展%Characteristics of Cation/H+ Antiporters in Plants

    Institute of Scientific and Technical Information of China (English)

    单喆; 张欣欣; 高野哲夫; 柳参奎

    2012-01-01

    CAX是一种通过质子梯度产生的能量运输协调再分配钙离子(Ca2+)等阳离子的转运蛋白,是Ca2+/Cation antiporter (CaCA)大家族的一个分化枝.植物CAXs属于CAX三大类的Ⅰ型CAX.大部分植物CAXs有11个跨膜区(TM)和5个典型的功能域,即N-端自抑制区域(NRR)、C-端功能区域、Ca2+功能域(CaD)、C功能域和D功能域.其中NRR存在于大部分CAX中,调节CAX的功能.以下综述了近年来国内外对CAX类蛋白的研究成果与进展,涉及到CAX家族的命名,亚家族的分类,CAX组织表达及亚细胞定位,特别是CAX的转运活性等研究.加强对CAX的研究对调节植物生长、提高农作物养分吸收和减轻土壤中污染物等有重要作用.%Cation/H+ antiporters (CAXs) are a group of proteins that efflux Ca2+ and other cations to maintain optimal ionic concentrations in cells, which are energized by the proton gradient. It is one of the five families that constitute the Ca2/cation antiporter (CaCA) superfamily. Plant CAXs is the type I of three major categories. Plant CAXs are characterized by 11 transmembrane (TM) domains and 5 regulatory domains-that is N-terminal autoinhi-bitory domains (NRR), C-terminal domains, Ca2+ domain (CaD), C domain and D domain, and NRR exists in most CAXs to regulate the function of CAX. In this paper, the research progress in the classification, location, structure and function of CAXs in plants is reviewed, especially in the transporter activity of CAXs. The studies of CAXs play an important role in regulating plant growth, increasing crop nutrient uptake and reducing pollutants in soil.

  10. Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

    Science.gov (United States)

    Kanno, Hitomi; Kawakami, Zenji; Mizoguchi, Kazushige; Ikarashi, Yasushi; Kase, Yoshio

    2014-01-01

    Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook. PMID:25551766

  11. Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

    Directory of Open Access Journals (Sweden)

    Hitomi Kanno

    Full Text Available Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells. Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

  12. Yokukansan, a kampo medicine, protects PC12 cells from glutamate-induced death by augmenting gene expression of cystine/glutamate antiporter system Xc-.

    Science.gov (United States)

    Kanno, Hitomi; Kawakami, Zenji; Mizoguchi, Kazushige; Ikarashi, Yasushi; Kase, Yoshio

    2014-01-01

    Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc-, which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc- subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

  13. 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, J K; Balderas, M A; Versalovic, J

    2014-03-01

    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 tumor necrosis factor (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 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 contribute to microbiome-mediated immunomodulation. PMID:24488273

  14. 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.

  15. 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.

  16. 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.

    Science.gov (United States)

    Sperling, Eva; Górecki, Kamil; Drakenberg, Torbjörn; Hägerhäll, Cecilia

    2016-01-01

    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. PMID:27391676

  17. Piperaquine and Lumefantrine resistance in Plasmodium berghei ANKA associated with increased expression of Ca2+/H+ antiporter and glutathione associated enzymes.

    Science.gov (United States)

    Kiboi, Daniel; Irungu, Beatrice; Orwa, Jennifer; Kamau, Luna; Ochola-Oyier, Lynette Isabella; Ngángá, Joseph; Nzila, Alexis

    2014-12-01

    We investigated the mechanisms of resistance of two antimalarial drugs piperaquine (PQ) and lumefantrine (LM) using the rodent parasite Plasmodium berghei as a surrogate of the human parasite, Plasmodium falciparum. We analyzed the whole coding sequence of Plasmodium berghei chloroquine resistance transporter (Pbcrt) and Plasmodium berghei multidrug resistance gene 1(Pbmdr-1) for polymorphisms. These genes are associated with quinoline resistance in Plasmodium falciparum. No polymorphic changes were detected in the coding sequences of Pbcrt and Pbmdr1 or in the mRNA transcript levels of Pbmdr1. However, our data demonstrated that PQ and LM resistance is achieved by multiple mechanisms that include elevated mRNA transcript levels of V-type H(+) pumping pyrophosphatase (vp2), Ca(2+)/H(+) antiporter (vcx1), gamma glutamylcysteine synthetase (ggcs) and glutathione-S-transferase (gst) genes, mechanisms also known to contribute to chloroquine resistance in P. falciparum and rodent malaria parasites. The increase in ggcs and gst transcript levels was accompanied by high glutathione (GSH) levels and elevated activity of glutathione-S-transferase (GST) enzyme. Taken together, these results demonstrate that Pbcrt and Pbmdr1 are not associated with PQ and LM resistance in P. berghei ANKA, while vp2, vcx1, ggcs and gst may mediate resistance directly or modulate functional mutations in other unknown genes. PMID:25448357

  18. 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.

  19. Hydrogen sulfide is involved in maintaining ion homeostasis via regulating plasma membrane Na+/H+ antiporter system in the hydrogen peroxide-dependent manner in salt-stress Arabidopsis thaliana root.

    Science.gov (United States)

    Li, Jisheng; Jia, Honglei; Wang, Jue; Cao, Qianhua; Wen, Zichao

    2014-07-01

    Hydrogen sulfide (H2S) and hydrogen peroxide (H2O2) function as the signaling molecules in plants responding to salt stresses. The present study presents a signaling network involving H2S and H2O2 in salt resistance pathway of the Arabidopsis root. Arabidopsis roots were sensitive to 100 mM NaCl treatment, which displayed a great increase in electrolyte leakage (EL) and Na(+)/K(+) ratio under salt stress. The treatment of H2S donors sodium hydrosulfide (NaHS) enhanced the salt tolerance by maintaining a lower Na(+)/K(+) ratio. In addition, the inhibition of root growth under salt stress was removed by H2S. Further studies indicated that H2O2 was involved in H2S-induced salt tolerance pathway. H2S induced the production of the endogenous H2O2 via regulating the activities of glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) NADPH oxidase, with the treatment with dimethylthiourea (DMTU, an ROS scavenger), diphenylene iodonium (DPI, a PM NADPH oxidase inhibitor), or glycerol (G6PDH inhibitor) removing the effect of H2S. Treatment with amiloride (an inhibitor of PM Na(+)/H(+) antiporter) and vanadate (an inhibitor of PM H(+)-ATPase) also inhibited the activity of H2S on Na(+)/K(+) ratio. Through an analysis of quantitative real-time polymerase chain reaction and Western blot, we found that H2S promoted the genes expression and the phosphorylation level of PM H(+)-ATPase and Na(+)/H(+) antiporter protein level. However, when the endogenous H2O2 level was inhibited by DPI or DMTU, the effect of H2S on the PM Na(+)/H(+) antiporter system was removed. Taken together, H2S maintains ion homeostasis in the H2O2-dependent manner in salt-stress Arabidopsis root.

  20. 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

  1. High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

    Science.gov (United States)

    Thomas, Ajit G; Sattler, Rita; Tendyke, Karen; Loiacono, Kara A; Hansen, Hans; Sahni, Vishal; Hashizume, Yutaka; Rojas, Camilo; Slusher, Barbara S

    2015-01-01

    The cystine-glutamate antiporter (system xc-) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc- expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc- in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc- is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc- inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc-. Human glioma cells were chosen based on their high system xc- activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of

  2. Thinking outside the cleft to understand synaptic activity: contribution of the cystine-glutamate antiporter (System xc-) to normal and pathological glutamatergic signaling.

    Science.gov (United States)

    Bridges, Richard; Lutgen, Victoria; Lobner, Doug; Baker, David A

    2012-07-01

    System x(c)(-) represents an intriguing target in attempts to understand the pathological states of the central nervous system. Also called a cystine-glutamate antiporter, system x(c)(-) typically functions by exchanging one molecule of extracellular cystine for one molecule of intracellular glutamate. Nonvesicular glutamate released during cystine-glutamate exchange activates extrasynaptic glutamate receptors in a manner that shapes synaptic activity and plasticity. These findings contribute to the intriguing possibility that extracellular glutamate is regulated by a complex network of release and reuptake mechanisms, many of which are unique to glutamate and rarely depicted in models of excitatory signaling. Because system x(c)(-) is often expressed on non-neuronal cells, the study of cystine-glutamate exchange may advance the emerging viewpoint that glia are active contributors to information processing in the brain. It is noteworthy that system x(c)(-) is at the interface between excitatory signaling and oxidative stress, because the uptake of cystine that results from cystine-glutamate exchange is critical in maintaining the levels of glutathione, a critical antioxidant. As a result of these dual functions, system x(c)(-) has been implicated in a wide array of central nervous system diseases ranging from addiction to neurodegenerative disorders to schizophrenia. In the current review, we briefly discuss the major cellular components that regulate glutamate homeostasis, including glutamate release by system x(c)(-). This is followed by an in-depth discussion of system x(c)(-) as it relates to glutamate release, cystine transport, and glutathione synthesis. Finally, the role of system x(c)(-) is surveyed across a number of psychiatric and neurodegenerative disorders.

  3. The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+ resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA).

    OpenAIRE

    Ivey, D M; Guffanti, A A; Shen, Z.; Kudyan, N; Krulwich, T A

    1992-01-01

    A 5.6-kb fragment of alkaliphilic Bacillus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989). The plasmid, designated pJB22, contained two genes that apparently encode transposition functions and two genes th...

  4. 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▿

    OpenAIRE

    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 substrat...

  5. Bioinformatics Analysis of 7 Plants' K+/H+ Antiporters%7种植物K+/H+逆向转运蛋白的生物信息学分析

    Institute of Scientific and Technical Information of China (English)

    韩蕾; 宋志忠; 王莉; 苏彦华

    2011-01-01

    K+/H+ antiporting is employed by almost all living organisms as an important mechanism of ion homeostasis. This machinery is composed of a multi-gene family known as KEA (K efflux antiporter). However, except a couple of reports from E. coli, the functionalities of most KEAs and their physiological significances remain largely unclear. In this report, homologous comparison of KEAs from seven plant species which had been accomplished in full genome sequencing had revealed members of the KEA family were highly conserved along evolution. Further analyses engaged the KEAs of the model plant A rabidopsis thaliana in regard of their transmembrane structure, putative functional hallmarks as well as predicted subcellular localizations. The results indicated that AtKEAs were membrane proteins and contained 10 to 12 transmembrane domains (TM), on which locating multiple serine phosphorylation sites to the N-termini of the TMs. Morever, KEAs harbor structurally, a putative K+/H+ exchange motif as well as related regulation components, which was in support of a functional deduction as K+/H+ antiporters. Results from microarrays indicated that expression of AtKEA genes varied in tissues/organs and developmental stages, suggesting a temporal and spatial pattern of their physiological roles. This report would be expected to provide useful inspiration toward discovery of the functionality of plant K+/H+ antiport system.%K+/H+逆向转运是普遍存在于几乎所有生物体内的重要离子平衡机制之一.该机制主要由多基因家族KEA (K efflux antiporter)介导.然而,长期以来对KEA的功能特征和生理意义的认识,除了在大肠杆菌中有零星的报道之外,绝大部分尚为空白.本文通过生物信息学分析,比较了7个植物物种KEA的同源和进化关系,发现KEA家族中的部分成员在物种进化过程中具有极高的保守性.以模式植物拟南芥为例,进一步研究了KEA的蛋白质跨膜结构、关键结构域和亚细

  6. Key point of Na+-H+ antiporter to transport sodium in osteoclast%破骨细胞钠氢转运蛋白关键位点研究

    Institute of Scientific and Technical Information of China (English)

    黄晓斌; 仲蕾蕾

    2012-01-01

    目的 在酿酒酵母中异源表达人破骨细胞分化成熟潜在因子钠氢转运蛋白2,对其关键氨基酸保守位点进行突变分析,鉴定其作为盐离子载体转运Na+的功能.方法 采用突变试剂盒依次把此蛋白的D278和D279 2个位点的天冬氨酸中1个氨基酸突变成半胱氨酸,得到2个不同位点突变的定点突变体;构建酵母双基因表达载体,通过电穿孔的方式转入到酵母菌株中;Western印迹检测2个定点突变基因在酵母菌株的表达;通过NaCl选择压力显示2个突变体在高盐环境中的相互作用,观察菌株的抗盐生长表型.结果 成功获得适合转化酵母的多个载体;在固体和液体培养基中,经半乳糖诱导后,各个载体均可使酵母异源表达钠氢转运蛋白2;生长曲线显示2个突变体可相互作用,部分恢复酵母菌株的抗盐能力.结论 钠氢转运蛋白2通过关键位点D278和D279 2,以形成二聚体的方式在细胞中发挥着钠离子转运功能.%Objective To express heterologously Na*-H+ antiporter 2 that potentially benefits the differentiation and maturation of the osteoclast in Saccharomyces cerevisiae, directly mutate the conservative key amino acid, and identify its function in transporting Na* as a salt transporter. Methods In turn mutating was carried out to make one of the two Asp of the antiporter into Cys by mutation kit. After two different mutants were achieved, two gene yeast vector was constructed to express them simultaneously in Saccharomyces cerevisiae. The obtained vectors were electroporated into yeast. The express of the two antiporter mutants in the strain was detected by Western blotting. Their interaction of the two mutants was observed by the growth curve of BW31a in high salty concentration medium. Results Several vectors which can be transformed into yeast was successfully constructed. Each vector expressed the Na*-H* antiporter 2 in the yeast after induction by galac-tose in the

  7. 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 (3)H-flucytosine and, to a moderate extent, of (3)H-clotrimazole, consistent with a direct role in antifungal drug efflux. Interestingly, no effect of CgAQR1 deletion could be found on the intracellular accumulation of (14)C-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

  8. The dual role of Candida glabrata Drug:H+ Antiporter CgAqr1 (ORF CAGL0J09944g in antifungal drug and acetic acid resistance

    Directory of Open Access Journals (Sweden)

    Catarina eCosta

    2013-06-01

    Full Text Available 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 GFP 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

  9. Cloning of Na+/H+ an Antiporter Gene from Bruguiera gymnorrhiza (L.) LAM%木榄质膜型Na+/H+逆向转运蛋白的基因克隆与序列分析

    Institute of Scientific and Technical Information of China (English)

    郭庆水; 于伟; 徐立新; 袁潜华

    2012-01-01

    Based on the homologous sequences from Populus trichocarpa, Populus euphratica, Ricinus communis, Solarium lycopersicum, Thellungiella halophila, PCR primers were designed to amplify the SOS1 DNA fragment using cDNA prepared from Bruguiera gymnorrhiza, A full length cDNA of SOS1 gene in Bruguiera gymnorrhiza (BgSOSl) was cloned using RACE. The data showed that the BgSOSl is 3 703 bp in length, including an open reading frame of 3 462 bp encoding a predicted polypeptide of 1 154 amino acids with 12 transmembrane domains. The sequence showed high homologies (70%) with Na7H+ antiporter genes from Ricinus communis; Populus trichocarpa, Ricinus communis, Populus euphratica, Vitis vinifera and other plants.%根据已发表的胡杨、毛白杨、蓖麻、番杏、番茄、盐角草等逆向转运蛋白基因的保守区设计兼并引物, 通过RT-PCR和RACE技术,从木榄(Bruguiera gymnorrhiza)中克隆出Na+/H+逆转运蛋白的cDNA全长序列,长度为3 703 bp,其中,开放阅读框3 462 bp,编码1 154个氨基酸,含12个跨膜区.序列同源性分析表明,该氨基酸序列与毛果杨、蓖麻、胡杨、葡萄等Na+/H+逆向转运蛋白氨基酸序列具有很高的同源性(70%以上),表明该序列确属Na+/H+质膜型逆向转运蛋白的家族基因.

  10. 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

  11. 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

  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. Cloning and Characterization of Na+/H+ Antiporter Gene (nhaA) from Pseudomonas sp.cn4902%假单胞菌Na+/H+逆向转运蛋白基因nhaA的克隆与鉴定

    Institute of Scientific and Technical Information of China (English)

    刘广发; 曾活水; 陈启伟; 高亚辉

    2005-01-01

    According to the sequences of the gene nhaA coding for Na+/H+ antiporter,a structural gene was cloned from Pseudomonas sp.cn4902 by PCR reaction with a set of primers.It was 1 089 bp in length and codes for 362 amino acids sharing homology with the gene nhaA of E.coli K12 as high as 97.0%.It was inserted into plasmid pBV220 to form a high level expression reconstruction plasmid pBVA.So an overexpression 41 kD protein band could be found in the lane of transformant harbored with pBVA after SDS-PAGE electrophoresis.The detection of growth curve showed that the biomass of the transformant was 2.3 times over that of the control in the medium containing 1.0 mol/L NaCl.It was found that Na+ concentration in cytoplasm of the transformant was low to 60.4% of the control by the detection of atomic absorption spectrum.Evidence of SDS-PAGE electrophoresis of membrane proteins also showed that the NhaA was located in membrane.Purified NhaA was harvested and digested by FXa proteinase.The sequence of eight amino acids in N termination of NhaA protein was entirely identical with the polypeptide deduced from the nhaA gene.Then ten strains of transformant were continuously cultivated for 18 generations under 42 ℃ hot shock condition,all of their reconstructed plasmids were lost with the result that salt-tolerant-level went back to the original standard.In summary,all the experiments proved that the cloned gene is nhaA gene.The gene has been accepted in GenBank by the accession number AY643494.%根据3种生物的Na+/H+逆向转运蛋白基因(nhaA)的两端序列设计引物,利用PCR从假单胞菌(Pseudomonas sp.cn4902)中克隆得到一结构基因.该基因长1 089 bp,编码362个氨基酸,与E.coli K12的nhaA基因的同源性高达97.0%.将该结构基因与pBV220构建成重组载体pBVA.SDS-PAGE电泳表明:含pBVA的转化子产生较高浓度的分子量约为41 kD的蛋白,与预期相符.在含NaCl 1.0 mol/L的培养基中生长达到平衡期时,转化

  14. 泌盐植物长叶红砂质膜 Na +/H +逆向转运蛋白基因(RtSOS1)全长 cDNA 的克隆及序列分析%Cloning and Sequence Analysis of the Plasma Membrane Na +/H +Antiporter cDNA in Recretohalophyte Reaumuria trigyna Maxim

    Institute of Scientific and Technical Information of China (English)

    党振华; 郑琳琳; 冯智; 王迎春

    2013-01-01

      Reaumuria trigyna Maxim.is an endangered small shrub with the features of a recretohalophyte .This species is endemic to the Eastern Alxa Western Ordos area and developed distinctive strategies to adapt to the semi -desert and salty soil environment .A full-length cDNA of the plasma Na+/H+antiporter (RtSOS1) was isolated from this species by using RT-PCR and RACE technologies.The 3 829 bp sequence comprised a 3 438 bp open reading frame,encoding an 1 145 amino acids protein with the molecular weight of 126.76 kDa.Bioinformatics analyze re-veals that RtSOS1 composed of 11 transmembrane domains within its N terminal portion ,and a hydrophilic cytoplas-mic tail with the length approximately 700 amino acids in its C-terminal portion.In the C-terminal region,the phos-phorylation domain and the auto -inhibited domain are found.The Homology comparison and phylogenetic analysis showed that RtSOS1 is related to plasma membrane Na+/H+antiporter in other plant species.%  长叶红砂为内蒙古东阿拉善-西鄂尔多斯地区特有珍稀泌盐,强旱生小灌木,对盐渍荒漠环境具有极强适应性。利用 RT-PCR 和 RACE 技术从该植物中分离出质膜 Na+/H+逆向转运蛋白基因(RtSOS1),该 cDNA 全长为3829 bp,开放阅读框为3438 bp,编码一个含1145个氨基酸的蛋白质,推测分子量为126.76 kDa。氨基酸序列的生物信息学分析推测,该蛋白 N 端含有11个跨膜结构域,C 端为一个长约700个氨基酸的亲水性尾,具有磷酸化和自我抑制结构域。同源性比对和系统发育分析证实,RtSOS1与其他植物的质膜 Na+/H+逆向转运蛋白亲缘关系较近。

  15. ‘富士’苹果Ca2+/H+反向转运蛋白基因生物信息学及表达模式分析%Bioinformatics and Expression Analysis of Ca2+/H+ Antiporter Gene Family in ‘Fuji’ Apple

    Institute of Scientific and Technical Information of China (English)

    程玉豆; 冯云霄; 关军锋

    2015-01-01

    本研究借助生物信息学分析从苹果基因组数据库中获得了6个含全长编码序列的Ca2+/H+反向转运蛋白(Ca2+/H+ antiporter, CAX)基因,分别命名为MdCAX1、MdCAX2、MdCAX3、MdCAX4、MdCAX5和MdCAX6。基因和蛋白结构特性分析结果显示:6个CAX在基因结构和蛋白特性等方面存在差异; MdCAXs隶属于I-A型和I-B 2个亚型,均含有N-端自我抑制区(N-terminal regulatory region, NRR)、CaD功能域、C-端功能域、C功能域和D功能域。荧光定量PCR分析表明,MdCAX1在‘富士’苹果叶片中表达量表现为基部叶片>中部叶片>顶端叶片,在果皮和果肉中的表达量低于叶片;MdCAX2在果皮和果肉中表达量高于叶片;MdCAX3在叶片和果实中的表达模式与MdCAX1相似,但在叶片中的表达量低于MdCAX1;MdCAX4在各被检测样本中表达量无明显变化;MdCAX5和MdCAX6在果皮和果肉组织中的表达量随发育进程呈逐渐升高趋势。叶片中Ca2+含量在不同叶位叶片中差异显著,果皮和果肉中Ca2+含量随着果实发育均呈下降趋势。上述研究结果表明:6个MdCAXs基因在基因结构、蛋白特性以及表达模式上存在差异, MdCAX1和MdCAX3基因表达与‘富士’苹果叶片和果实Ca2+含量变化密切相关。%Six Ca2+/H+ antiporter(CAX) genes which contained full-length coding sequences were obtained from the apple genome database by bioinformatics analysis in this work, and designated asMdCAX1, MdCAX2, Md-CAX3, MdCAX4, MdCAX5 and MdCAX6respectively. The studies of gene and protein characteristics indicated that the gene formations and protein characteristics of the sixMdCAXs were different. The MdCAXs were clus-tered into two groups: I-A type and I-B type, and all of the 6 CAXs contained N-terminal regulatory region (NRR), CaD domain, C-terminal domain, C domain and D domain. The results of real-time PCR showed that the expres-sion level ofMdCAX1presented base leaf> middle leaf > top leaf, and

  16. Cloning and Expression to Salt Stress of Na +/H +Antiporter Gene (MnNHX1) in Mulberry Tree%桑树Na+/H+逆向转运蛋白基因(MnNHX1)的克隆与耐盐力表达

    Institute of Scientific and Technical Information of China (English)

    边晨凯; 龙定沛; 刘雪琴; 魏从进; 龚加红; 赵爱春

    2015-01-01

    ;连续浇灌含高浓度 NaCl 营养液的转基因拟南芥生长状态更为优良。【结论】MnNHX1为优良的植物耐盐基因,在桑树中为组成型表达,并受NaCl胁迫诱导,表现出组织特异性。过量表达 MnNHX1的拟南芥耐盐能力显著提高,生存在盐胁迫环境中,依然具有良好的生长和发育能力。%Objective] To study the function of Na + /H +antiporter ( NHX) in vacuolar membrane from mulberry tree Morus notabilis,and to explore the mechanism of salt tolerance in mulberry,and to provide an excellent candidate gene for the screening of plant resistance gene engineering. [Method]In this study,a Na + /H +antiporter gene named as MnNHX1 was identified based on the M. notabilis genomic database and other homologous sequences. The MnNHX1 was cloned using the cDNA from M. notabilis leaves as template. The analysis of the primary structure and functional domains from MnNHX1 was completed by the bioinformatics analysis. The phylogenetic tree was generated to analyse the relationships between mulberry NHX1 and other species. Quantitative PCR was conducted to analyse the expression profiles of mulberry NHX1 in different tissues of M. multicaulis‘Husang No. 32’and treatment time under NaCl stress. The overexpression vector was constructed and transformed into Arabidopsis thaliana. The seed germination rate,the growth of roots and the survival rate of seedlings of the transgenic A. thaliana were analyzed under NaCl stress. Furthermore,the transgenic A. thaliana was continuously irrigated with the nutrient solution containing high concentration of NaCl to study the functional effects of MnNHX1 gene in the transgenic A. thaliana. [Result]We cloned a Na + /H + antiporter gene designated as MnNHX1(GenBank accession No. KJ720637). The open reading frame (ORF) of MnNHX1 is 1 644 bp and encodes a protein of 547 amino acid with a Na + /H + exchange pump. At the upstream of this pump,there are some domains such as inhibitors amiloride binding sites

  17. Malolactic Fermentation : Electrogenic Malate Uptake and Malate/Lactate Antiport Generate Metabolic Energy

    NARCIS (Netherlands)

    Poolman, Bert; Molenaar, Douwe; Smid, Eddy J.; Ubbink, Trees; Abee, Tjakko; Renault, Pierre P.; Konings, Wil N.

    1991-01-01

    The mechanism of metabolic energy production by malolactic fermentation in Lactococcus lactis has been investigated. In the presence of L-malate, a proton motive force composed of a membrane potential and pH gradient is generated which has about the same magnitude as the proton motive force generate

  18. Natrium/Protonen-Antiporter und mechanosensitive Kanäle von Halomonas elongata

    OpenAIRE

    Kurz, M.

    2003-01-01

    Die Arbeit gibt einen Einblick in die Antwort der halophilen Eubakterien Halomonas elongata, Marinococcus halophilus und Lake Bogoria Isolat 25B1 auf einen Wechsel von osmotischen Verhältnissen und Salinität. Physiologisch wurde gezeigt, dass H.elongata und L.B.I.25B1 mechanosensitive Kanäle besitzen. M.halophilus verfügt nicht über diesen Mechanismus. Während der Arbeiten an dieser Problemstellung wurde ein Verfahren zum Nachweis cytotoxischer Verbindungen weiterentwickelt. Die Funktio...

  19. Membrane topology of the electrogenic aspartate-alanine antiporter AspT of Tetragenococcus halophilus.

    Science.gov (United States)

    Nanatani, Kei; Ohonishi, Fumito; Yoneyama, Hiroshi; Nakajima, Tasuku; Abe, Keietsu

    2005-03-01

    AspT is an electrogenic aspartate:alanine exchange protein that represents the vectorial component of a proton-motive metabolic cycle found in some strains of Tetragenococcus halophilus. AspT is the sole member of a new family, the Aspartate: Alanine Exchanger (AAE) family, in secondary transporters, according to the computational classification proposed by Saier et al. (http://www.biology.ucsd.edu/~msaier/transport/). We analyzed the topology of AspT biochemically, by using fusion methods in combination with alkaline phosphatase or beta-lactamase. These results suggested that AspT has a unique topology; 8 TMS, a large cytoplasmic loop (183 amino acids) between TMS5 and TMS6, and N- and C-termini that both face the periplasm. These results demonstrated a unique 2D-structure of AspT as the novel AAE family. PMID:15670744

  20. Zebrafish ("Danio rerio") endomembrane antiporter similar to a yeast cation/H(+) transporter is required for neural crest development

    Science.gov (United States)

    CAtion/H (+) eXchangers (CAXs) are integral membrane proteins that transport Ca (2+) or other cations by exchange with protons. While several yeast and plant CAX proteins have been characterized, no functional analysis of a vertebrate CAX homologue has yet been reported. In this study, we further ch...

  1. 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

    1993-01-01

    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 addit

  2. Generation of a Proton Motive Force by Histidine Decarboxylation and Electrogenic Histidine/Histamine Antiport in Lactobacillus buchneri

    OpenAIRE

    Molenaar, Douwe; Bosscher, Jaap S.; Brink, Bart ten; Arnold J M Driessen; Konings, Wil N.

    1993-01-01

    Lactobaciflus 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 (Δψ), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histi...

  3. Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

    OpenAIRE

    Molenaar, D; Bosscher, J S; ten Brink, B.; Driessen, A J; Konings, W N

    1993-01-01

    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 (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate tha...

  4. Generation of a Proton Motive Force by Histidine Decarboxylation and Electrogenic Histidine/Histamine Antiport in Lactobacillus buchneri

    NARCIS (Netherlands)

    Molenaar, Douwe; Bosscher, Jaap S.; Brink, Bart ten; Driessen, Arnold J.M.; Konings, Wil N.

    1993-01-01

    Lactobaciflus 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 (Δψ), inside negative, upon addition of

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

    OpenAIRE

    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 deve...

  6. 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.

  7. Apical Na+/H+ antiporter and glycolysis-dependent H+-ATPase regulate intracellular pH in the rabbit S3 proximal tubule.

    OpenAIRE

    Kurtz, I

    1987-01-01

    The apical transport processes responsible for proton secretion were studied in the isolated perfused rabbit S3 proximal tubule. Intracellular pH (pHi) was measured with the pH dye, 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady state pHi in S3 tubules in nominally HCO3(-)-free solutions was 7.08 +/- 0.03. Removal of Na+ (lumen) caused a decrease in pHi of 0.34 +/- 0.06 pH/min. The decrease in pHi was inhibited 62% by 1 mM amiloride (lumen) and was unaffected by 50 microM 4,4'-diisoth...

  8. Isolation and characterization of plasma membrane Na+/H+ antiporter (SOS1) gene during salinity stress in kallar grass (Leptochloa fusca)

    OpenAIRE

    Banafsheh Taherinia; Hamid Reza Kavousi; Sara Dehghan

    2015-01-01

    Background: Leptochloa fusca is a halophyte plant which is highly tolerant to saline and sodic soils and water. Moreover, L. fusca is an attractive model plant to study the mechanism of salt tolerance mainly due to its characteristics as a typical euhalophyte, having both accumulating and excreting salt properties. Soil salinity adversely affects plant growth, development and disturbs intracellular ion homeostasis resulting cellular toxicity. The Salt Overly Sensitive 1 (SOS1) gene encodes a ...

  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. NCBI nr-aa BLAST: CBRC-TTRU-01-1371 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1371 ref|ZP_01070173.1| putative Na+/H+ antiporter [Campylobacter jeju...ni subsp. jejuni 260.94] emb|CAI38704.1| putative Na+/H+ antiporter [Campylobacter jejuni] gb|EAQ58317.1| pu...tative Na+/H+ antiporter [Campylobacter jejuni subsp. jejuni 260.94] ZP_01070173.1 0.40 27% ...

  11. 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% ...

  12. NCBI nr-aa BLAST: CBRC-DMEL-03-0067 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-03-0067 ref|YP_966641.1| Na+/H+ antiporter NhaC [Desulfovibrio vulgaris s...ubsp. vulgaris DP4] gb|ABM28214.1| Na+/H+ antiporter NhaC [Desulfovibrio vulgaris subsp. vulgaris DP4] YP_966641.1 0.029 28% ...

  13. NCBI nr-aa BLAST: CBRC-DSIM-03-0068 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DSIM-03-0068 ref|YP_966641.1| Na+/H+ antiporter NhaC [Desulfovibrio vulgaris s...ubsp. vulgaris DP4] gb|ABM28214.1| Na+/H+ antiporter NhaC [Desulfovibrio vulgaris subsp. vulgaris DP4] YP_966641.1 0.010 27% ...

  14. 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% ...

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

    Lifescience Database Archive (English)

    Full Text Available CBRC-CREM-01-1301 ref|YP_131006.1| putative Na+/H+ antiporter [Photobacterium profund...um SS9] emb|CAG21204.1| putative Na+/H+ antiporter [Photobacterium profundum SS9] YP_131006.1 8e-80 51% ...

  16. 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% ...

  17. 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 upsalien...sis RM3195] gb|EAL53227.1| NA+/H+ antiporter (napA) [Campylobacter upsaliensis RM3195] ZP_00371235.1 0.025 22% ...

  18. NCBI nr-aa BLAST: CBRC-LAFR-01-1203 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-LAFR-01-1203 ref|YP_791111.1| putative Na+/H+ antiporter [Pseudomonas aerugino...sa UCBPP-PA14] gb|ABJ11311.1| putative Na+/H+ antiporter [Pseudomonas aeruginosa UCBPP-PA14] YP_791111.1 3.9 29% ...

  19. 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% ...

  20. NCBI nr-aa BLAST: CBRC-CBRE-01-0086 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-CBRE-01-0086 gb|AAM42931.1| Na+:H+ antiporter [Xanthomonas campestris pv. campest...ris str. ATCC 33913] gb|AAY50772.1| Na+:H+ antiporter [Xanthomonas campestris pv. campestris str. 8004] AAM42931.1 0.42 31% ...

  1. 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% ...

  2. NCBI nr-aa BLAST: CBRC-TTRU-01-1218 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TTRU-01-1218 ref|YP_001250401.1| amino acid antiporter [Legionella pneumophila... str. Corby] gb|ABQ55055.1| amino acid antiporter [Legionella pneumophila str. Corby] YP_001250401.1 0.010 23% ...

  3. AcEST: DK962528 [AcEST

    Lifescience Database Archive (English)

    Full Text Available -tartrate/succinate antiporter OS=Escherichia coli O6:K15:H31 (strain 536 / UPEC)...OUT L-tartrate/succinate antiporter OS=Escherichia coli (strain UTI89 / UPEC) GN=ttdT PE=2 SV=1 Length = 487

  4. AcEST: DK963290 [AcEST

    Lifescience Database Archive (English)

    Full Text Available SWAVSGFSNSVIWLIFAAFMFGTGY 116 >sp|Q0TD43|TTDT_ECOL5 L-tartrate/succinate antiporter OS=Escherichia coli O6:K15:H31 (strain 536 / UPEC...tartrate/succinate antiporter OS=Escherichia coli (strain UTI89 / UPEC) GN=ttdT PE=2 SV=1 Length = 487 Score

  5. AcEST: DK946142 [AcEST

    Lifescience Database Archive (English)

    Full Text Available P-GAVVAMVGISIIAIL 68 >sp|Q0TD43|TTDT_ECOL5 L-tartrate/succinate antiporter OS=Escherichia coli O6:K15:H31 (strain 536 / UPEC...PIKDVLVPSSQPPRPKVTLKSSECLGHN 216 >sp|Q1R6R8|TTDT_ECOUT L-tartrate/succinate antiporter OS=Escherichia coli (strain UTI89 / UPEC

  6. AcEST: DK953813 [AcEST

    Lifescience Database Archive (English)

    Full Text Available sp|Q0TD43|TTDT_ECOL5 L-tartrate/succinate antiporter OS=Escherichia coli O6:K15:H31 (strain 536 / UPEC...AFMF 112 >sp|Q1R6R8|TTDT_ECOUT L-tartrate/succinate antiporter OS=Escherichia coli (strain UTI89 / UPEC) GN=

  7. NCBI nr-aa BLAST: CBRC-DYAK-03-0008 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-03-0008 ref|YP_536514.1| Na+/H+ antiporter [Lactobacillus salivarius subsp. saliva...rius UCC118] gb|ABE00431.1| Na+/H+ antiporter [Lactobacillus salivarius subsp. salivarius UCC118] YP_536514.1 0.80 24% ...

  8. NCBI nr-aa BLAST: CBRC-DMEL-03-0008 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-03-0008 ref|YP_536514.1| Na+/H+ antiporter [Lactobacillus salivarius subsp. saliva...rius UCC118] gb|ABE00431.1| Na+/H+ antiporter [Lactobacillus salivarius subsp. salivarius UCC118] YP_536514.1 1.4 22% ...

  9. NCBI nr-aa BLAST: CBRC-MDOM-02-0142 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-MDOM-02-0142 ref|YP_687482.1| putative Na(+)/H(+) antiporter [uncultured methanogenic... archaeon RC-I] emb|CAJ38156.1| putative Na(+)/H(+) antiporter [uncultured methanogenic archaeon RC-I] YP_687482.1 0.75 25% ...

  10. Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles.

    Science.gov (United States)

    Dedukhova, V I; Mokhova, E N; Skulachev, V P; Starkov, A A; Arrigoni-Martelli, E; Bobyleva, V A

    1991-12-16

    The effect of ATP/ADP-antiporter inhibitors on palmitate-induced uncoupling was studied in heart muscle mitochondria and inside-out submitochondrial particles. In both systems palmitate is found to decrease the respiration-generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non-penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate-induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl-CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP-antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation. PMID:1765167

  11. Fatty acid circuit as a physiological mechanism of uncoupling of oxidative phosphorylation.

    Science.gov (United States)

    Skulachev, V P

    1991-12-01

    Free fatty acids, natural uncouplers of oxidative phosphorylation, are shown to differ from artificial ones in that they fail to increase conductance of phospholipid bilayers which are permeable for the protonated form of fatty acids but impermeable for their anionic form. Recent studies have revealed that uncoupling by fatty acids in mitochondria is mediated by the ATP/ADP antiporter and, in brown fat, by thermogenin which is structurally very similar to the antiporter. It is suggested that both the ATP/ADP antiporter and thermogenin facilitate translocation of the fatty anions through the mitochondrial membrane. PMID:1756853

  12. 新疆盐生植物猪毛菜逆向运输蛋白基因SaNHX1 3'-RACE的克隆及序列分析%Clone and Sequence Analysis of 3'RACE of Antiporter Protein Gene SaNHX1 in Xinjiang Halophytes Plant Salsola collina Pall.

    Institute of Scientific and Technical Information of China (English)

    张雨良; 罗淑萍; 杨峰山; 魏岩; 袁辉; 郭长奎

    2008-01-01

    以新疆盐生植物猪毛菜( Salsola collina Pall.)为材料提取总RNA,根据 NHX1家族同源序列保守区设计1对简并引物,进行RT-PCR扩增,得到猪毛菜逆向运输蛋白基因cDNA中间一段619 bp序列.再用快速扩增cDNA 3'末端(3' RACE)方法,得到约1 000 bp 3'末端序列.将两段序列进行拼接,得到猪毛菜长为1 585 bp(GenBank登陆号为EU072932)的逆向运输蛋白基因(SaNHX1) cDNA序列,编码370个氨基酸,其编码序列、氨基酸序列与同科耐盐植物盐角草相应序列同源性为85%和87%,研究为进一步克隆猪毛菜NHX1全基因序列和研究其功能打下基础.

  13. 疏叶骆驼刺耐盐基因AsNHX的3′-RACE克隆及序列分析%Clone and Sequence Analysis of 3′-RACE of Na+/H+Antiporter Gene AsNHX1 of Alhagi sparsifolia

    Institute of Scientific and Technical Information of China (English)

    努尔凯麦尔·木拉提; 帕尔哈提·阿布都克日木; 王希东

    2015-01-01

    通过Trizol法提取骆驼刺叶片总RNA的方法,反转录合成cDNA,PCR扩增得到一条600-800bp的DNA条带,与报道的AspNHX1基因的核心序列基本相符,经3′-RACE扩增的DNA片段并连接到载体上进行测序.测序结果显示cDNA长度为1484bp,编码470个氨基酸,与猪毛菜SaNHX1 (Salsola affinis)、梭梭HaNHX1 (Haloxylon ammodendron)、盐爪爪 Kf NHX1(Kalidium foliatum)的核苷酸序列同源性高达96%、89%、89%,氨基酸序列同源性达到97% 、93%和90%.生物信息学分析显示AsNHX蛋白的分子式为C4578H7674N1484O1907S291,蛋白的分子量为123347.5,理论等电点pI为5.01,不稳定参数为46.41,是一个富含疏水性氨基酸的不太稳定蛋白,理论上有35个苏氨酸的磷酸化位点,属于NHX1家族.说明己经在骆驼刺叶片中成功的克隆到Na+/H+逆向转运蛋白基因cDNA序列的部分片段,为下一步克隆骆驼刺叶片Na+/H+逆向转运蛋白基因的cDNA全序列提供了可能.%Alhagi sparsifolia is a very good biological resistance material with the characteristics of cold resistance, drought resistance, salt resistance and wind characteristics. In order to study on the core sequence of the Alhagi sparsifolia's NHX1 gene, Extracted the total RNA from the Alhagi sparsifolia's leaves used Trizol kit method, synthesis the cDNA,and amplified by PCR,gained a DNA belt about 600bp to 800bp,which conforms to the core sequence of the NHX1 gene that had published.Amplified and sequenced the core As-NHX by 3′-RACE. The se-quence result shows the length of the As-NHX is 1484bp,encode 470 amino acid. The nucleotide sequence ho-mology is 96%,89%and 89%,The amino acid sequence homology is 97%, 93%and 90%by compared the se-quence with NHX1 between SaNHX1(Salsola affinis),HaNHX1(Haloxylon ammodendron)and Kf NHX1(Kalidi-um foliatum)reported in NCBI. The result analyses with bioinformatics shows the formula of As-NHX protein is C4578H7674N1484O1907S291,Mr is 123347.5,the isoelectric point (pI)is 5.01,it is a kind of unstable protein with rich hydrophobic amino acids,35 Threonine phosphorylation sites. We had cloned the fragment of NHX from Alhagi sparsifolia leaf and lay a foundation for the molecular biology research works of the resistance aspects on Alhagi sparsifolia.

  14. NCBI nr-aa BLAST: CBRC-MDOM-06-0025 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available tiporter, putative [Candida dubliniensis CD36] emb|CAX42824.1| drug resistance protein, putative; drug:H+ antiporter, putative [Candida dubliniensis CD36] XP_002419235.1 0.058 28% ...

  15. NCBI nr-aa BLAST: CBRC-MDOM-03-0035 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available tiporter, putative [Candida dubliniensis CD36] emb|CAX42824.1| drug resistance protein, putative; drug:H+ antiporter, putative [Candida dubliniensis CD36] XP_002419235.1 0.50 26% ...

  16. NCBI nr-aa BLAST: CBRC-MDOM-08-0087 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available tiporter, putative [Candida dubliniensis CD36] emb|CAX42824.1| drug resistance protein, putative; drug:H+ antiporter, putative [Candida dubliniensis CD36] XP_002419235.1 0.009 28% ...

  17. Characterization of the Intracellular Glutamate Decarboxylase System: Analysis of Its Function, Transcription, and Role in the Acid Resistance of Various Strains of Listeria monocytogenes

    OpenAIRE

    Karatzas, Kimon-Andreas G.; Suur, Laura; O'Byrne, Conor P.

    2012-01-01

    The glutamate decarboxylase (GAD) system is important for the acid resistance of Listeria monocytogenes. We previously showed that under acidic conditions, glutamate (Glt)/γ-aminobutyrate (GABA) antiport is impaired in minimal media but not in rich ones, like brain heart infusion. Here we demonstrate that this behavior is more complex and it is subject to strain and medium variation. Despite the impaired Glt/GABA antiport, cells accumulate intracellular GABA (GABAi) as a standard response aga...

  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; Henriksen, Kim; Sørensen, Mette Guldmann;

    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. Enhanced tolerance to NaC1 and LiC1 stresses by over-expressing Caragana korshinskii sodium/proton exchange 1 (CkNHX1) and the hydrophilic C terminus is required for the activity of CkNHX1 in Atsos3-1 mutant and yeast

    Science.gov (United States)

    Sodium/proton exchangers (NHX antiporters) play important roles in plant responses to salt stress. Previous research showed that hydrophilic C-terminal region of Arabidopsis AtNHX1 negatively regulates the Na+/H+ transporting activity. In this study, CkNHX1 were isolated from Caragana korshinskii,...

  20. InterProScan Result: CA946045 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CA946045 CA946045_6_ORF2 F2879795FABF2ACC PFAM PF00999 Na_H_Exchanger NA ? IPR006153 unintegrated Biologica...l Process: cation transport (GO:0006812)|Molecular Function: solute:hydrogen antipor...ter activity (GO:0015299)|Cellular Component: integral to membrane (GO:0016021)|Biological Process: transmembrane transport (GO:0055085) ...

  1. InterProScan Result: CA946045 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available CA946045 CA946045_6_ORF2 F2879795FABF2ACC PFAM PF00999 Na_H_Exchanger 6.7e-19 T IPR...006153 Cation/H+ exchanger Biological Process: cation transport (GO:0006812)|Molecular Function: solute:hydr...ogen antiporter activity (GO:0015299)|Cellular Component: integral to membrane (GO:0016021)|Biological Process: transmembrane transport (GO:0055085) ...

  2. 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.

    1994-01-01

    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 nitr

  3. Dependence of chlorophyll P700 redox transients during the induction period on the transmembrane distribution of protons in chloroplasts of pea leaves

    NARCIS (Netherlands)

    Bulychev, A. A.; Cherkashin, A. A.; Rubin, A. B.

    2010-01-01

    Differential absorbance measurements and fluorometry were applied to examine the impact of dicyclohexylcarbodiimide (DCCD, an inhibitor of H(+) conductance in thylakoid membranes) and nigericin (a K(+)/H(+) antiporter) on photoinduced redox state transients of chlorophyll P700 and the induction curv

  4. AcEST: DK960764 [AcEST

    Lifescience Database Archive (English)

    Full Text Available ntiporter OS=Escherichia coli O6:K15:H31 (strain 536 / UPEC) GN=ttdT PE=2 SV=1 Length = 487 Score = 62.4 bit... 119 >sp|Q1R6R8|TTDT_ECOUT L-tartrate/succinate antiporter OS=Escherichia coli (strain UTI89 / UPEC

  5. AcEST: DK949251 [AcEST

    Lifescience Database Archive (English)

    Full Text Available tiporter OS=Escherichia coli (strain UTI89 / UPEC) GN=ttdT PE=2 SV=1 Length = 487 Score = 111 bits (277), Ex...artrate/succinate antiporter OS=Escherichia coli O6:K15:H31 (strain 536 / UPEC) GN=ttdT PE=2 SV=1 Length = 4

  6. AcEST: DK954795 [AcEST

    Lifescience Database Archive (English)

    Full Text Available OS=Escherichia coli O6:K15:H31 (strain 536 / UPEC) GN=ttdT PE=2 SV=1 Length = 487 Score = 67.4 bits (163), E...T L-tartrate/succinate antiporter OS=Escherichia coli (strain UTI89 / UPEC) GN=tt

  7. Multidrug transporters in lactic acid bacteria

    NARCIS (Netherlands)

    Mazurkiewicz, P; Sakamoto, K; Poelarends, GJ; Konings, WN

    2005-01-01

    Gram-positive lactic acid bacteria possess several Multi-Drug Resistance systems (MDRs) that excrete out of the cell a wide variety of mainly cationic lipophilic cytotoxic compounds as well as many clinically relevant antibiotics. These MDRs are either proton/drug antiporters belonging to the major

  8. Multidrug transporters and antibiotic resistance in Lactococcus lactis

    NARCIS (Netherlands)

    Poelarends, GJ; Mazurkiewicz, P; Konings, WN

    2002-01-01

    The Gram-positive bacterium Lactococcus lactis produces two distinct multidrug transporters, designated LmrA and LmrP, that both confer resistance to a wide variety of cationic lipophilic cytotoxic compounds as well as to many clinically relevant antibiotics. While LmrP is a proton/drug antiporter t

  9. Compositional asynchronous membrane systems

    Institute of Scientific and Technical Information of China (English)

    Cosmin Bonchis; Cornel Izbasa; Gabriel Ciobanu

    2007-01-01

    This paper presents an algorithmic way of building complex membrane systems by coupling elementary membranes. Its application seems particularly valuable in the case of asynchronous membrane systems, since the resulting membrane system remains asynchronous. The composition method is based on a handshake mechanism implemented by using antiport rules and promoters.

  10. AcEST: BP918940 [AcEST

    Lifescience Database Archive (English)

    Full Text Available containing protein 1 OS=Homo sap... 33 1.1 sp|A8FHE3|NHAK_BACP2 Sodium, potassium, lithium and rubidium/H(+....LKEINKMADHWETAPRHMMRLDI 1804 >sp|A8FHE3|NHAK_BACP2 Sodium, potassium, lithium and rubidium/H(+) antiporter O

  11. 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%.

  12. Model Construction and Analysis of Respiration in Halobacterium salinarum.

    Directory of Open Access Journals (Sweden)

    Cherryl O Talaue

    Full Text Available The archaeon Halobacterium salinarum can produce energy using three different processes, namely photosynthesis, oxidative phosphorylation and fermentation of arginine, and is thus a model organism in bioenergetics. Compared to its bacteriorhodopsin-driven photosynthesis, less attention has been devoted to modeling its respiratory pathway. We created a system of ordinary differential equations that models its oxidative phosphorylation. The model consists of the electron transport chain, the ATP synthase, the potassium uniport and the sodium-proton antiport. By fitting the model parameters to experimental data, we show that the model can explain data on proton motive force generation, ATP production, and the charge balancing of ions between the sodium-proton antiporter and the potassium uniport. We performed sensitivity analysis of the model parameters to determine how the model will respond to perturbations in parameter values. The model and the parameters we derived provide a resource that can be used for analytical studies of the bioenergetics of H. salinarum.

  13. CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

    OpenAIRE

    Reed, Anita A.C.; Loh, Nellie Y.; TERRYN, Sara; Lippiat, Jonathan D.; Partridge, Chris; Galvanovskis, Juris; Williams, Sian E; Jouret, François; Wu, Fiona T. F.; Courtoy, Pierre J.; Nesbit, M Andrew; Rorsman, Patrik; Devuyst, Olivier; Ashcroft, Frances M.; Thakker, Rajesh V

    2010-01-01

    Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function a...

  14. Resistance Index of Penicillin-Resistant Bacteria to Various Physicochemical Agents

    OpenAIRE

    M Kazemi; Kasra Kermanshahi, R.; Heshmat Dehkordi, E.; F. Payami; Behjati, M

    2012-01-01

    Widespread use of various antimicrobial agents resulted in the emergence of bacterial resistance. Mechanisms like direct efflux, formation, and sequestration of metals and drugs in complexes and antiporter pumps are some examples. This investigation aims to investigate the resistance pattern of penicillin-resistant bacterial strains to some physicochemical agents. Sensitivity/resistance pattern of common bacterial strains to antimicrobial agents were evaluated by disk diffusion assay. Broth a...

  15. THE ENERGETIC FUNCTIONS OF PLANT MITOCHONDRIA UNDER STRESS

    OpenAIRE

    Grabelnych O.I.

    2005-01-01

    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(P)H 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.

  16. Molecular Mechanism of Inhibition of the Mitochondrial Carnitine/Acylcarnitine Transporter by Omeprazole Revealed by Proteoliposome Assay, Mutagenesis and Bioinformatics

    OpenAIRE

    Annamaria Tonazzi; Ivano Eberini; Cesare Indiveri

    2013-01-01

    The effect of omeprazole on the mitochondrial carnitine/acylcarnitine transporter has been studied in proteoliposomes. Externally added omeprazole inhibited the carnitine/carnitine antiport catalysed by the transporter. The inhibition was partially reversed by DTE indicating that it was caused by the covalent reaction of omeprazole with Cys residue(s). Inhibition of the C-less mutant transporter indicated also the occurrence of an alternative non-covalent mechanism. The IC50 of the inhibition...

  17. Respiratory complex I: A dual relation with H(+) and Na(+)?

    Science.gov (United States)

    Castro, Paulo J; Silva, Andreia F; Marreiros, Bruno C; Batista, Ana P; Pereira, Manuela M

    2016-07-01

    Respiratory complex I couples NADH:quinone oxidoreduction to ion translocation across the membrane, contributing to the buildup of the transmembrane difference of electrochemical potential. H(+) is well recognized to be the coupling ion of this system but some studies suggested that this role could be also performed by Na(+). We have previously observed NADH-driven Na(+) transport opposite to H(+) translocation by menaquinone-reducing complexes I, which indicated a Na(+)/H(+) antiporter activity in these systems. Such activity was also observed for the ubiquinone-reducing mitochondrial complex I in its deactive form. The relation of Na(+) with complex I may not be surprising since the enzyme has three subunits structurally homologous to bona fide Na(+)/H(+) antiporters and translocation of H(+) and Na(+) ions has been described for members of most types of ion pumps and transporters. Moreover, no clearly distinguishable motifs for the binding of H(+) or Na(+) have been recognized yet. We noticed that in menaquinone-reducing complexes I, less energy is available for ion translocation, compared to ubiquinone-reducing complexes I. Therefore, we hypothesized that menaquinone-reducing complexes I perform Na(+)/H(+) antiporter activity in order to achieve the stoichiometry of 4H(+)/2e(-). In agreement, the organisms that use ubiquinone, a high potential quinone, would have kept such Na(+)/H(+) antiporter activity, only operative under determined conditions. This would imply a physiological role(s) of complex I besides a simple "coupling" of a redox reaction and ion transport, which could account for the sophistication of this enzyme. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt. PMID:26711319

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

    International Nuclear Information System (INIS)

    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 [35S]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

  19. 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. PMID:24813633

  20. 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.

  1. The permeability transition pore as a Ca2+ release channel: New answers to an old question

    OpenAIRE

    Bernardi, Paolo; von Stockum, Sophia

    2012-01-01

    Mitochondria possess a sophisticated array of Ca2+ transport systems reflecting their key role in physiological Ca2+ homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca2+ uptake, the ruthenium red (RR)-sensitive mitochondrial Ca2+ uniporter (MCU); and one main mechanism for Ca2+ release, the RR-insensitive 3Na+–Ca2+ antiporter. An additional mechanism for Ca2+ release is provided by a Na+ and RR-insensitive re...

  2. Mdt(A), a New Efflux Protein Conferring Multiple Antibiotic Resistance in Lactococcus lactis and Escherichia coli

    OpenAIRE

    Perreten, Vincent; Schwarz, Franziska V.; Teuber, Michael; Levy, Stuart B.

    2001-01-01

    The mdt(A) gene, previously designated mef214, from Lactococcus lactis subsp. lactis plasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lin...

  3. Microwave accelerated synthesis of isoxazole hydrazide inhibitors of the system xc- transporter: Initial homology model.

    Science.gov (United States)

    Matti, Afnan A; Mirzaei, Joseph; Rudolph, John; Smith, Stephen A; Newell, Jayme L; Patel, Sarjubhai A; Braden, Michael R; Bridges, Richard J; Natale, Nicholas R

    2013-11-01

    Microwave accelerated reaction system (MARS) technology provided a good method to obtain selective and open isoxazole ligands that bind to and inhibit the Sxc- antiporter. The MARS provided numerous advantages, including: shorter time, better yield and higher purity of the product. Of the newly synthesized series of isoxazoles the salicyl hydrazide 6 exhibited the highest level of inhibitory activity in the transport assay. A homology model has been developed to summarize the SAR results to date, and provide a working hypothesis for future studies.

  4. Interdependence of two NarK domains in a fused nitrate/nitrite transporter.

    Science.gov (United States)

    Goddard, Alan D; Moir, James W B; Richardson, David J; Ferguson, Stuart J

    2008-11-01

    Nitrate uptake is essential for various bacterial processes and combines with nitrite export to form the usual initial steps of denitrification, a process that reduces nitrate to dinitrogen gas. Although many bacterial species contain NarK-like transporters that are proposed to function as either nitrate/proton symporters or nitrate/nitrite antiporters based on sequence homology, these transporters remain, in general, poorly characterized. Several bacteria appear to contain a transporter that is a fusion of two NarK-like proteins, although the significance of this arrangement remains elusive. We demonstrate that NarK from Paracoccus denitrificans is expressed as a fusion of two NarK-like transporters. NarK1 and NarK2 are separately capable of supporting anaerobic denitrifying growth but with growth defects that are partially mitigated by coexpression of the two domains. NarK1 appears to be a nitrate/proton symporter with high affinity for nitrate and NarK2 a nitrate/nitrite antiporter with lower affinity for nitrate. Each transporter requires two conserved arginine residues for activity. A transporter consisting of inactivated NarK1 fused to active NarK2 has a dramatically increased affinity for nitrate compared with NarK2 alone, implying a functional interaction between the two domains. A potential model for nitrate and nitrite transport in P. denitrificans is proposed. PMID:18823285

  5. Protective effects of cariporide on endothelial dysfunction induced by high glucose

    Institute of Scientific and Technical Information of China (English)

    Shuang-xi WANG; Xiao-ming XIONG; Tao SONG; Li-ying LIU

    2005-01-01

    Aim: To explore the effects of cariporide, a selective sodium-hydrogen antiporter inhibitor, on endothelial dysfunction induced by high glucose. Methods: Acetylcholine (ACh)-induced endothelium-dependent relaxation (EDR), sodium nitroprusside (SNP)-induced endothelium-independent relaxation and biochemical parameters including malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) were measured in rat isolated aorta. Results: A 6-h incubation of aortic rings with high glucose (44 mmol/L) resulted in a significant inhibition of EDR, but had no effects on endothelium-independent relaxation. After the 6-h incubation of aortic rings in the co-presence of cariporide (0.01, 0.1, and 1 μmol/L) with high glucose, cariporide prevented the inhibition of EDR caused by high glucose in concentration-dependent manners. Similarly, high glucose decreased SOD activity and contents of NO, and increased MDA concentration in aortic tissue. Cariporide (1 μmol/L) significantly resisted the decrease of NO content and SOD activity, and elevation of MDA concentration caused by high glucose in aortic tissues. Mannitol (44 mmol/L) or cariporide (1 μmol/L) alone had no effect on EDR, endothelium-independent relaxation and biochemical parameters.Conclusion: Cariporide significantly prevented endothelial dysfunction induced by high glucose. The mechanisms of endothelial dysfunction induced by high glucose may involve the activation of sodium-hydrogen antiporter and the generation of oxygen-free radicals, but it is not related to the change of osmolarity.

  6. A flow cytometric method for measurement of intracellular chloride concentration in lymphocytes using the halide-specific probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ).

    Science.gov (United States)

    Pilas, B; Durack, G

    1997-08-01

    A flow cytometry method using the halide-specific fluorescent dye, 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ), has been developed to measure intracellular chloride concentration in single cells. Collisions with chloride quench the fluorescence of SPQ, making it possible to relate the measured fluorescence intensity to chloride concentration with a Stern-Volmer equation. To demonstrate the method, porcine lymphocytes were loaded in vitro, using a hypotonic method, with 5 mM SPQ. Fluorescence excitation was provided by a UV laser and the fluorescence emission intensity at 485 nm was recorded. Calibration was performed by using 7 microM nigericin (a K/H antiporter) and 10 microM tributyltin (a Cl/OH antiporter) to equilibrate the concentrations of intracellular and extracellular chloride. Calibration measurements were made for chloride concentrations between 0 mM and 140 mM. The calibration produced a Stern-Volmer quenching constant of 16.2 M(-1) which was used to relate measured cell fluorescence to intracellular chloride concentration. The intracellular chloride concentration for fresh porcine lymphocytes was determined to be 56.2 +/- 3.3 mM. Stable loading of cells with 5 mM SPQ was accomplished in 15 minutes, leakage of SPQ from the cells was minimal, and over 95% of the cells remained viable after loading. PMID:9266752

  7. The permeability transition pore as a Ca2+ release channel: New answers to an old question

    Science.gov (United States)

    Bernardi, Paolo; von Stockum, Sophia

    2012-01-01

    Mitochondria possess a sophisticated array of Ca2+ transport systems reflecting their key role in physiological Ca2+ homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca2+ uptake, the ruthenium red (RR)-sensitive mitochondrial Ca2+ uniporter (MCU); and one main mechanism for Ca2+ release, the RR-insensitive 3Na+–Ca2+ antiporter. An additional mechanism for Ca2+ release is provided by a Na+ and RR-insensitive release mechanism, the putative 3H+–Ca2+ antiporter. A potential kinetic imbalance is present, however, because the Vmax of the MCU is of the order of 1400 nmol Ca2+ mg−1 protein min−1 while the combined Vmax of the efflux pathways is about 20 nmol Ca2+ mg−1 protein min−1. This arrangement exposes mitochondria to the hazards of Ca2+ overload when the rate of Ca2+ uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca2+]. In this short review we discuss the hypothesis that transient opening of the Ca2+-dependent permeability transition pore may provide mitocondria with a fast Ca2+ release channel preventing Ca2+ overload. We also address the relevance of a mitochondrial Ca2+ release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals. PMID:22513364

  8. The permeability transition pore as a Ca(2+) release channel: new answers to an old question.

    Science.gov (United States)

    Bernardi, Paolo; von Stockum, Sophia

    2012-07-01

    Mitochondria possess a sophisticated array of Ca(2+) transport systems reflecting their key role in physiological Ca(2+) homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca(2+) uptake, the ruthenium red (RR)-sensitive mitochondrial Ca(2+) uniporter (MCU); and one main mechanism for Ca(2+) release, the RR-insensitive 3Na(+)-Ca(2+) antiporter. An additional mechanism for Ca(2+) release is provided by a Na(+) and RR-insensitive release mechanism, the putative 3H(+)-Ca(2+) antiporter. A potential kinetic imbalance is present, however, because the V(max) of the MCU is of the order of 1400nmol Ca(2+)mg(-1) proteinmin(-1) while the combined V(max) of the efflux pathways is about 20nmol Ca(2+)mg(-1) proteinmin(-1). This arrangement exposes mitochondria to the hazards of Ca(2+) overload when the rate of Ca(2+) uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca(2+)]. In this short review we discuss the hypothesis that transient opening of the Ca(2+)-dependent permeability transition pore may provide mitocondria with a fast Ca(2+) release channel preventing Ca(2+) overload. We also address the relevance of a mitochondrial Ca(2+) release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals. PMID:22513364

  9. Unveiling the Mechanism of Arginine Transport through AdiC with Molecular Dynamics Simulations: The Guiding Role of Aromatic Residues

    Science.gov (United States)

    Krammer, Eva-Maria; Ghaddar, Kassem; André, Bruno

    2016-01-01

    Commensal and pathogenic enteric bacteria have developed several systems to adapt to proton leakage into the cytoplasm resulting from extreme acidic conditions. One such system involves arginine uptake followed by export of the decarboxylated product agmatine, carried out by the arginine/agmatine antiporter (AdiC), which thus works as a virtual proton pump. Here, using classical and targeted molecular dynamics, we investigated at the atomic level the mechanism of arginine transport through AdiC of E. coli. Overall, our MD simulation data clearly demonstrate that global rearrangements of several transmembrane segments are necessary but not sufficient for achieving transitions between structural states along the arginine translocation pathway. In particular, local structural changes, namely rotameric conversions of two aromatic residues, are needed to regulate access to both the outward- and inward-facing states. Our simulations have also enabled identification of a few residues, overwhelmingly aromatic, which are essential to guiding arginine in the course of its translocation. Most of them belong to gating elements whose coordinated motions contribute to the alternating access mechanism. Their conservation in all known E. coli acid resistance antiporters suggests that the transport mechanisms of these systems share common features. Last but not least, knowledge of the functional properties of AdiC can advance our understanding of the members of the amino acid-carbocation-polyamine superfamily, notably in eukaryotic cells. PMID:27482712

  10. Fatty acids as natural uncouplers preventing generation of O2.- and H2O2 by mitochondria in the resting state.

    Science.gov (United States)

    Korshunov, S S; Korkina, O V; Ruuge, E K; Skulachev, V P; Starkov, A A

    1998-09-18

    Both natural (laurate) and artificial (m-chlorocarbonylcyanide phenylhydrazone; CCCP) uncouplers strongly inhibit O2.- and H2O2 formation by rat heart mitochondria oxidizing succinate. Carboxyatractylate, an ATP/ADP antiporter inhibitor, abolishes the laurate inhibition, the CCCP inhibition being unaffected. Atractylate partially releases the inhibition by laurate and decelerates the releasing effect of carboxyatractylate. GDP is much less effective than carboxyatractylate in releasing the laurate inhibition of reactive oxygen species (ROS) formation. Micromolar laurate concentrations arresting the ROS formation cause strong inhibition of reverse electron transfer from succinate to NAD+, whereas State 4 respiration and the transmembrane electric potential difference (delta psi) level are affected only slightly. It is suggested that (i) free fatty acids operate as natural 'mild uncouplers' preventing the transmembrane electrochemical H+ potential difference (delta muH+) from being above a threshold critical for ROS formation by complex I and, to a lesser degree, by complex III of the respiratory chain, and (ii) it is the ATP/ADP-antiporter, rather than uncoupling protein 2, that is mainly involved in this antioxidant mechanism of heart muscle mitochondria. PMID:9762912

  11. Differential hypersaline stress response in Zygosaccharomyces rouxii complex yeasts: a physiological and transcriptional study.

    Science.gov (United States)

    Solieri, Lisa; Vezzani, Veronica; Cassanelli, Stefano; Dakal, Tikam Chand; Pazzini, Jacopo; Giudici, Paolo

    2016-09-01

    The Zygosaccharomyces rouxii complex comprises three distinct lineages of halotolerant yeasts relevant in food processing and spoilage, such as Z. sapae, Z. rouxii and a mosaic group of allodiploid strains. They manifest plastic genome architecture (variation in karyotype, ploidy level and Na(+)/H(+) antiporter-encoding gene copy number), and exhibit diverse tolerances to salt concentrations. Here, we investigated accumulation of compatible osmolytes and transcriptional regulation of Na(+)/H(+) antiporter-encoding ZrSOD genes during salt exposure in strains representative for the lineages, namely Z. sapae ABT301(T) (low salt tolerant), Z. rouxii CBS 732(T) (middle salt tolerant) and allodiploid strain ATCC 42981 (high salt tolerant). Growth curve modelling in 2 M NaCl-containing media supplemented with or without yeast extract as nitrogen source indicates that moderate salt tolerance of CBS 732(T) mainly depends on nitrogen availability rather than intrinsic inhibitory effects of salt. All the strains produce glycerol and not mannitol under salt stress and use two different glycerol balance strategies. ATCC 42981 produces comparatively more glycerol than Z. sapae and Z. rouxii under standard growth conditions and better retains it intracellularly under salt injuries. Conversely, Z. sapae and Z. rouxii enhance glycerol production under salt stress and intracellularly retain glycerol less efficiently than ATCC 42981. Expression analysis shows that, in diploid Z. sapae and allodiploid ATCC 42981, transcription of gene variants ZrSOD2-22/ZrSOD2 and ZrSOD22 is constitutive and salt unresponsive. PMID:27493145

  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. Lactate transport by rainbow trout white muscle: kinetic characteristics and sensitivity to inhibitors.

    Science.gov (United States)

    Wang, Y; Wright, P M; Heigenhauser, G J; Wood, C M

    1997-05-01

    This study used an isolated-perfused tail-trunk preparation of rainbow trout to examine the uptake and release of lactate (Lac) and metabolic protons (delta H+M) in resting and exercised fish white muscle. In exercised muscle, L(+)-Lac efflux was inhibited (approximately 40%) by 5 mM alpha-cyano-4-hydroxycinnamate (CIN), but not by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) or 0.1 mM amiloride. These results suggest that Lac release occurs through a Lac(-)-H- symport and the free diffusion of lactic acid (HLac) or Lac-, but not via the Lac-/HCO3(-)-Cl- antiporter. Lac efflux was accompanied by delta H+m influx in all treatments, and increased delta H+m influx occurred after SITS treatment. In resting muscle, Lac uptake rates were greater than Lac efflux rates in the postexercise preparation. L-Lac influx exhibited partial saturation kinetics, whereas D(-)-Lac influx was linearly related to its extracellular concentration (0-32 mM). At 16 mM extracellular L-Lac, with a negligible transmembrane L-HLac gradient and an outwardly directed not driving force on L-Lac-, CIN, and SITS reduced net L-Lac uptake by 75 and 45%, respectively. At 16 mM extracellular concentration, D-Lac influx was 64% of the net L-Lac influx. These results suggest that in trout muscle at 16 mM extracellular L-Lac, the Lac -H+ symport accounts for 30-36%, the Lac-/HCO3(-)-Cl- antiport for 39-45%, and diffusion for 19-25% of uptake, although the latter is probably overestimated and the former underestimated for methodological reasons. Net L-Lac efflux was not affected by extracellular D-Lac concentration and/or D-Lac influx, implying the existence of a concurrent L-Lac efflux during L-Lac influx. The D-Lac influx kinetics data indicated that the Lac-/HCO3 antiport was not saturable in the extracellular D-Lac concentration range of 0-32 mM. This study clearly demonstrates the involvement of carrier-mediated transport in transmembrane Lac movement in fish muscle and

  14. 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

    The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption...... 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......, the effect of valinomycin, inhibitor sensitivity, and the ion profile of the human osteoclast microsomes. The expression level of chloride channel 7 was increased in the human osteoclastic microsomes compared with whole osteoclasts. Acid influx was induced by 1.25 mM adenosine triphosphate. Further 1.1 μ...

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

    DEFF Research Database (Denmark)

    Henriksen, Kim; Sørensen, Mette G; Jensen, Vicki 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 currently not well understood. We used a battery of ion channel inhibitors, human osteoclasts, and their subcellular compartments to perform an unbiased analysis of the importance of the different ion transporters for acidification of the resorption lacuna in osteoclasts. CD14(+) monocytes from human...... peripheral blood were isolated, and mature osteoclasts were generated using RANKL and M-CSF. The human osteoclasts were (1) used for acridine orange assays for evaluation of lysosomal acidification, (2) used for bone resorption assays, (3) used for generation of osteoclasts membranes for acid influx...

  16. Comparative study of effects of magnesium and taurine on electrical parameters of natural and artificial membranes. VII. Effects on cellular and paracellular ionic transfer through isolated human amnion.

    Science.gov (United States)

    Bara, M; Guiet-Bara, A; Durlach, J

    1990-12-01

    The comparative effects of 2 mM magnesium and taurine on various components of the human transamniotic conductance, Gt, were observed. The use of both microelectrodes and metabolic inhibitors enables 10 components of Gt to be distinguished: six cellular components (Na-K ATPase, Na-H antiport, Na-K-2Cl cotransport and Na, K, Cl channels), one coupling component, and three paracellular components (Na, K, Cl). Mg increased all components of Gt while taurine only increased five of them (Na and K channels, coupling, Na and K paracellular conductance). A potentiometric effect of taurine on Mg2+ modified membrane, obtained on paracellular components, was not measured on cellular components. There was only a vicarious effect between Mg and taurine on the non-enzymatic cellular and paracellular transfer of Na and K.

  17. Salt tolerance conferred by over-expression of OsNHX1 gene in Poplar 84K

    Institute of Scientific and Technical Information of China (English)

    WANG Shuyao; CHEN Qijun; WANG Wenlong; WANG Xuechen; LU Mengzhu

    2005-01-01

    OsNHX1 gene (Na+/H+ antiporter gene of Oryza sativa L.) was introduced into Poplar 84K with Agrobacterium tumefaciens-mediated transformation. PCR, Southern and Northern blot analysis showed that OsNHX1 gene was incorporated successfully into the genome of Poplar 84K and expressed in these transgenic plants. Salt tolerance test showed that three lines of transgenic plants grew normally in the presence of 200 mmol/L NaCl, while the Na+ content in the leaves of the transgenic plants grown at 200 mmol/L NaCl was significantly higher than that in plants grown at 0 mmol/L NaCl. The osmotic potential in the transgenic plants with high salinity treatment was lower than that of control plants. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils.

  18. Does the intracellular ionic concentration or the cell water content (cell volume) determine the activity of TonEBP in NIH3T3 cells?

    DEFF Research Database (Denmark)

    Rødgaard, Tina; Schou, Kenneth; Friis, Martin Barfred;

    2008-01-01

    of the present investigation was to investigate whether cell shrinkage or high intracellular ionic concentration induced the activation of TonEBP. We designed a model system for isotonically shrinking cells over a prolonged period of time. Cells swelled in hypotonic medium and performed a regulatory...... volume decrease (RVD). Upon return to the original isotonic medium, cells shrank initially followed by a regulatory volume increase (RVI). To maintain cell shrinkage, the RVI process was inhibited as follows: Ethyl-isopropyl-amiloride (EIPA) inhibited the Na(+)/H(+) antiport, Bumetanide inhibited the Na......(+)/K(+)/2Cl(-) co-transporter, and Gadolinium inhibited shrinkage-activated Na(+) channels. Cells remained shrunken for at least 4 hours (isotonically shrunken cells). The activity of TonEBP was investigated with a Luciferase assay after isotonic shrinkage and after shrinkage in a high NaCl hypertonic...

  19. 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. PMID:23808339

  20. Identidades y alteridades en el Río de la Plata. Una visión histórica desde la banda oriental del "río mar"

    Directory of Open Access Journals (Sweden)

    Gerardo Caetano

    2015-06-01

    Full Text Available From a long-term historical perspective, this article studies the itineraries and core elements that have influenced the construction of the Uruguayan national identity, referred to particular dialectics kept with its regional alterities. Special emphasis is made on the roots and effects of the “antiporteñista”, or anti Buenos Aires, sign (frequently turn into plain “anti-argentinism” of Uruguayan nationalism in its most classical version. Under this perspective, the paper analyses the influence of some topics such as geopolitical changes within the Rio de la Plata basin; controversial trajectories of predominant accounts on the notion of “nation” in Uruguayan history, in its cosmopolitan profiles and in its regional alterities; and this issue’s liaison with the challenges for the construction of an “international Uruguay”. The article closes with a reflection that seeks to pinpoint some historical roots of the contemporary contentious agenda between Argentina and Uruguay.

  1. R76 in transmembrane domain 3 of the aspartate:alanine transporter AspT is involved in substrate transport.

    Science.gov (United States)

    Suzuki, Satomi; Nanatani, Kei; Abe, Keietsu

    2016-01-01

    The L-aspartate:L-alanine antiporter of Tetragenococcus halophilus (AspT) possesses an arginine residue (R76) within the GxxxG motif in the central part of transmembrane domain 3 (TM3)-a residue that has been estimated to transport function. In this study, we carried out amino acid substitutions of R76 and used proteoliposome reconstitution for analyzing the transport function of each substitution. Both l-aspartate and l-alanine transport assays showed that R76K has higher activity than the AspT-WT (R76), whereas R76D and R76E have lower activity than the AspT-WT. These results suggest that R76 is involved in AspT substrate transport. PMID:26849958

  2. Mdt(A), a New Efflux Protein Conferring Multiple Antibiotic Resistance in Lactococcus lactis and Escherichia coli

    Science.gov (United States)

    Perreten, Vincent; Schwarz, Franziska V.; Teuber, Michael; Levy, Stuart B.

    2001-01-01

    The mdt(A) gene, previously designated mef214, from Lactococcus lactis subsp. lactis plasmid pK214 encodes a protein [Mdt(A) (multiple drug transporter)] with 12 putative transmembrane segments (TMS) that contain typical motifs conserved among the efflux proteins of the major facilitator superfamily. However, it also has two C-motifs (conserved in the fifth TMS of the antiporters) and a putative ATP-binding site. Expression of the cloned mdt(A) gene decreased susceptibility to macrolides, lincosamides, streptogramins, and tetracyclines in L. lactis and Escherichia coli, but not in Enterococcus faecalis or in Staphylococcus aureus. Glucose-dependent efflux of erythromycin and tetracycline was demonstrated in L. lactis and in E. coli. PMID:11257023

  3. Membrane-Transport Systems for Sucrose in Relation to Whole-Plant Carbon Partitioning

    Institute of Scientific and Technical Information of China (English)

    Brian G. Ayre

    2011-01-01

    T Sucrose is the principal product of photosynthesis used for the distribution of assimilated carbon in plants. Transport mechanisms and efficiency influence photosynthetic productivity by relieving product inhibition and contribute to plant vigor by controlling source/sink relationships and biomass partitioning. Sucrose is synthesized in the cytoplasm and may move cell to cell through plasmodesmata or may cross membranes to be compartmentalized or exported to the apoplasm for uptake into adjacent cells. As a relatively large polar compound, sucrose requires proteins to facilitate efficient membrane transport. Transport across the tonoplast by facilitated diffusion, antiport with protons, and symport with protons have been proposed; for transport across plasma membranes, symport with protons and a mechanism resembling facilitated diffusion are evident. Despite decades of research, only symport with protons is well established at the molecular level. This review aims to integrate recent and older studies on sucrose flux across membranes with principles of whole-plant carbon partitioning.

  4. Chloride Channels: Often enigmatic, rarely predictable

    Science.gov (United States)

    Duran, Charity; Thompson, Christopher H.; Xiao, Qinghuan; Hartzell, Criss

    2010-01-01

    Until recently, anion (Cl−) channels have received considerably less attention than cation channels. One reason for this may be that many Cl− channels perform functions that might be considered cell biological, like fluid secretion and cell volume regulation, whereas cation channels have historically been associated with cellular excitability that typically happens more rapidly. In this review, we discuss the recent explosion of interest in Cl− channels with special emphasis on new and often surprising developments over the last 5 years. This is exemplified by the findings that more than half of the ClC family members are antiporters, and not channels as was previously thought, and that bestrophins, previously prime candidates for Ca2+-activated Cl− channels, have been supplanted by the newly discovered anoctamins and now hold a tenuous position in the Cl− channel world. PMID:19827947

  5. A Rigorous Theory of Remote Loading of Drugs into Liposomes: Transmembrane Potential and Induced pH-Gradient Loading and Leakage of Liposomes

    Science.gov (United States)

    Ceh; Lasic

    1997-01-01

    Many drugs are successfully loaded into preformed liposomes by using various gradients and transmembrane potential. Several experimental breakthroughs, however, have not been paralleled by theoretical understanding of the processes. Recently, we have developed a rigorous treatment of loading of weak acids and bases into liposomes. The model is based on equilibration of chemical potentials of permeable neutral species. Charged molecules are not allowed to permeate the membrane. Although this assumption is quite reasonable and experimental data fit the theoretical predictions rather well, we have extended the model of liposome loading. In the expanded model, terms which allow leakage of protons, buildup of the transmembrane pH gradient, an antiport exchange of various cations with protons, and leakage of other molecules from or into liposomes are added to the basic model.

  6. 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.

  7. Vibrio anguillarum Is Genetically and Phenotypically Unaffected by Long-Term Continuous Exposure to the Antibacterial Compound Tropodithietic Acid

    DEFF Research Database (Denmark)

    Rasmussen, Bastian Barker; Grotkjær, Torben; D'Alvise, Paul;

    2016-01-01

    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...... 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...

  8. Inhibition of xc⁻ transporter-mediated cystine uptake by sulfasalazine analogs.

    Science.gov (United States)

    Shukla, Krupa; Thomas, Ajit G; Ferraris, Dana V; Hin, Niyada; Sattler, Rita; Alt, Jesse; Rojas, Camilo; Slusher, Barbara S; Tsukamoto, Takashi

    2011-10-15

    A series of sulfasalazine analogs were synthesized and tested for their ability to block cystine-glutamate antiporter system xc⁻ using L-[(14)C]cystine as a substrate. Replacement of sulfasalazine's diazo group with an alkyne group led to an equally potent inhibitor, 2-hydroxy-5-((4-(N-pyridin-2-ylsulfamoyl)phenyl)ethynyl)benzoic acid 6. Our SAR studies also revealed that the carboxylate group of sulfasalazine is essential for its inhibitory activity while the phenolic hydroxyl group is dispensable. Truncated analogs lacking an N-pyridin-2-ylsulfamoyl moiety were less potent than sulfasalazine, but may serve as more tractable templates because of their low molecular weight by applying a variety of fragment growing approaches. Given that sulfasalazine is rapidly metabolized through cleavage of the diazo bond, these analogs may possess a more desirable pharmacological profile as system xc- blockers, in particular, for in vivo studies.

  9. One step beyond a ribosome: The ancient anaerobic core.

    Science.gov (United States)

    Sousa, Filipa L; Nelson-Sathi, Shijulal; Martin, William F

    2016-08-01

    Life arose in a world without oxygen and the first organisms were anaerobes. Here we investigate the gene repertoire of the prokaryote common ancestor, estimating which genes it contained and to which lineages of modern prokaryotes it was most similar in terms of gene content. Using a phylogenetic approach we found that among trees for all 8779 protein families shared between 134 archaea and 1847 bacterial genomes, only 1045 have sequences from at least two bacterial and two archaeal groups and retain the ancestral archaeal-bacterial split. Among those, the genes shared by anaerobes were identified as candidate genes for the prokaryote common ancestor, which lived in anaerobic environments. We find that these anaerobic prokaryote common ancestor genes are today most frequently distributed among methanogens and clostridia, strict anaerobes that live from low free energy changes near the thermodynamic limit of life. The anaerobic families encompass genes for bifunctional acetyl-CoA-synthase/CO-dehydrogenase, heterodisulfide reductase subunits C and A, ferredoxins, and several subunits of the Mrp-antiporter/hydrogenase family, in addition to numerous S-adenosyl methionine (SAM) dependent methyltransferases. The data indicate a major role for methyl groups in the metabolism of the prokaryote common ancestor. The data furthermore indicate that the prokaryote ancestor possessed a rotor stator ATP synthase, but lacked cytochromes and quinones as well as identifiable redox-dependent ion pumping complexes. The prokaryote ancestor did possess, however, an Mrp-type H(+)/Na(+) antiporter complex, capable of transducing geochemical pH gradients into biologically more stable Na(+)-gradients. The findings implicate a hydrothermal, autotrophic, and methyl-dependent origin of life. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:27150504

  10. Bioinformatic analyses of integral membrane transport proteins encoded within the genome of the planctomycetes species, Rhodopirellula baltica.

    Science.gov (United States)

    Paparoditis, Philipp; Västermark, Ake; Le, Andrew J; Fuerst, John A; Saier, Milton H

    2014-01-01

    Rhodopirellula baltica (R. baltica) is a Planctomycete, known to have intracellular membranes. Because of its unusual cell structure and ecological significance, we have conducted comprehensive analyses of its transmembrane transport proteins. The complete proteome of R. baltica was screened against the Transporter Classification Database (TCDB) to identify recognizable integral membrane transport proteins. 342 proteins were identified with a high degree of confidence, and these fell into several different classes. R. baltica encodes in its genome channels (12%), secondary carriers (33%), and primary active transport proteins (41%) in addition to classes represented in smaller numbers. Relative to most non-marine bacteria, R. baltica possesses a larger number of sodium-dependent symporters but fewer proton-dependent symporters, and it has dimethylsulfoxide (DMSO) and trimethyl-amine-oxide (TMAO) reductases, consistent with its Na(+)-rich marine environment. R. baltica also possesses a Na(+)-translocating NADH:quinone dehydrogenase (Na(+)-NDH), a Na(+) efflux decarboxylase, two Na(+)-exporting ABC pumps, two Na(+)-translocating F-type ATPases, two Na(+):H(+) antiporters and two K(+):H(+) antiporters. Flagellar motility probably depends on the sodium electrochemical gradient. Surprisingly, R. baltica also has a complete set of H(+)-translocating electron transport complexes similar to those present in α-proteobacteria and eukaryotic mitochondria. The transport proteins identified proved to be typical of the bacterial domain with little or no indication of the presence of eukaryotic-type transporters. However, novel functionally uncharacterized multispanning membrane proteins were identified, some of which are found only in Rhodopirellula species, but others of which are widely distributed in bacteria. The analyses lead to predictions regarding the physiology, ecology and evolution of R. baltica. PMID:23969110

  11. Studies on the overexpression of the soybean GmNHX1 in Lotus corniculatus: The reduced Na+ level is the basis of the increased salt tolerance

    Institute of Scientific and Technical Information of China (English)

    SUN Yanxiang; WANG Dan; BAI Yanling; WANG Ningning; WANG Yong

    2006-01-01

    The full length cDNA coding for a novel vacuolar Na+/H+ antiporter (GmNHX1) was cloned from soybean and determined to consist of 2591 bp with a 5(-untranslated region of 464 bp, an open reading frame (ORF) of 1641 bp, and a 3(-untrans- lated region of 486 bp. The deduced protein sequence contains 546 aa with the typical characters of the vacuolar Na+/H+ antiporters, and shares high similarity with that of AtNHX1, OsNHX1 and AgNHX1. The soybean genome showed a single copy of the GmNHX1 gene. Northern blot analysis demonstrated that the expression of the GmNHX1 was tissue-spe- cific, increased by ABA treatment, NaCl, KCl, LiCl and dehydration stress, and lower in leaves but higher in roots and hypocotyls of salt-tolerant than salt-sensitive cultivars. The GmNHX1 was overexpressed under the control of a tandem cauliflower mosaic virus (CaMV) 35S promoter in the model leguminous plant Lotus corniculatus L. and conferred salt-tolerance of the transgenic plants. Measurements of Na+ and K+ contents in both roots and shoots demonstrated that the plantlets of GmNHX1- overexpressing lines had lower Na+ and K+ content, and higher K+/Na+ ratio than the controlled lines that were transformed with the empty vector, which indicates that the salt-tolerance conferred by GmNHX1 is closely related with decreased accumulation of Na+ in the transgenic plants.

  12. Membrane transporters for the special amino acid glutamine: Structure/function relationships and relevance to human health.

    Science.gov (United States)

    Pochini, Lorena; Scalise, Mariafrancesca; Galluccio, Michele; Indiveri, Cesare

    2014-08-01

    Glutamine together with glucose is essential for body’s homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na+ and H+. Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7 and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5 and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologues. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.

  13. A sodium requirement for growth, solute transport, and pH homeostasis in Bacillus firmus RAB.

    Science.gov (United States)

    Krulwich, T A; Guffanti, A A; Bornstein, R F; Hoffstein, J

    1982-02-25

    Activity of a Na+/H+ antiporter has been suggested to be critically involved in pH homeostasis in obligately alkalophilic bacteria (Krulwich, K. A., Mandel, K. G., Bornstein, R. F., and Guffanti, A. A. (1979) Biochem. Biophys. Res. Commun. 91, 58-62) and in Escherichia coli (Zilberstein, D., Padan, E., and Schuldiner, S. (1980) FEBS Lett. 116, 177-180). A concern with respect to these proposals has been the failure of either Bacillus alcalophilus or E. coli to exhibit a requirement for added Na+ for growth. Thus, it became of interest to examine Na+-coupled porter functions in obligately alkalophilic Bacillus firmus RAB, a species that exhibits an absolute requirement for added Na+ for growth at pH 10.5. In a comparative study using membrane vesicles from B. alcalophilus and B. firmus RAB it was found that both the Na+/H+ antiporter and the Na+/alpha-aminoisobutyric acid symporter from the "Na+-requiring" species had much lower apparent affinities for Na+ than corresponding porters from B. alcalophilus. At high concentrations of Na+, the porters from the two species were functionally similar. These findings support the argument that the absence of a growth requirement for added Na+ may reflect an ability of at least some bacteria to effectively utilize and recycle the available levels of Na+ that contaminate all media, rather than reflect true Na+ independence. Studies with a nonalkalophilic derivative of B. firmus RAB confirmed earlier findings with B. alcalophilus of a pleiotropic loss of Na+ coupling to porters in nonalkalophilic mutants. PMID:7056750

  14. 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.

  15. A high-affinity Ca{sup 2+} pump, ECA1, from the endoplasmic reticulum is inhibited by cyclopiazonic acid but not by thapsigargin

    Energy Technology Data Exchange (ETDEWEB)

    Feng Liang; Sze, H. [Univ. of Maryland, College Park, MD (United States). Dept. of Cell Biology and Molecular Genetics

    1998-11-01

    To identify and characterize individual Ca{sup 2+} pumps, the authors have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca{sup 2+}-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca{sup 2+} pump and grows very poorly on Ca{sup 2+}-depleted medium. Membranes isolated from the mutant showed high H{sup +}/Ca{sup 2+}-antiport but no Ca{sup 2+}-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca{sup 2+}-depleted medium. {sup 45}Ca{sup 2+} pumping into vesicles from ECA1 transformants was detected after the H{sup +}/Ca{sup 2+}-antiport activity was eliminated with bafilomycin A{sub 1} and gramicidin D. The pump had a high affinity for Ca{sup 2+} (K{sub m} = 30 nM) and displayed two affinities for ATP. Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca{sup 2+}-ATPase, inhibited Ca{sup 2+} transport but thapsigargin did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca{sup 2+}-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca{sup 2+} and replenishing Ca{sup 2+} in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca{sup 2+} pumps from eukaryotes.

  16. Signal transducer and activator of transcription 3 and 5 regulate system Xc- and redox balance in human breast cancer cells.

    Science.gov (United States)

    Linher-Melville, Katja; Haftchenary, Sina; Gunning, Patrick; Singh, Gurmit

    2015-07-01

    System Xc- is a cystine/glutamate antiporter that contributes to the maintenance of cellular redox balance. The human xCT (SLC7A11) gene encodes the functional subunit of system Xc-. Transcription factors regulating antioxidant defense mechanisms including system Xc- are of therapeutic interest, especially given that aggressive breast cancer cells exhibit increased system Xc- function. This investigation provides evidence that xCT expression is regulated by STAT3 and/or STAT5A, functionally affecting the antiporter in human breast cancer cells. Computationally analyzing two kilobase pairs of the xCT promoter/5' flanking region identified a distal gamma-activated site (GAS) motif, with truncations significantly increasing luciferase reporter activity. Similar transcriptional increases were obtained after treating cells transiently transfected with the full-length xCT promoter construct with STAT3/5 pharmacological inhibitors. Knock-down of STAT3 or STAT5A with siRNAs produced similar results. However, GAS site mutation significantly reduced xCT transcriptional activity, suggesting that STATs may interact with other transcription factors at more proximal promoter sites. STAT3 and STAT5A were bound to the xCT promoter in MDA-MB-231 cells, and binding was disrupted by pre-treatment with STAT inhibitors. Pharmacologically suppressing STAT3/5 activation significantly increased xCT mRNA and protein levels, as well as cystine uptake, glutamate release, and total levels of intracellular glutathione. Our data suggest that STAT proteins negatively regulate basal xCT expression. Blocking STAT3/5-mediated signaling induces an adaptive, compensatory mechanism to protect breast cancer cells from stress, including reactive oxygen species, by up-regulating xCT expression and the function of system Xc-. We propose that targeting system Xc- together with STAT3/5 inhibitors may heighten therapeutic anti-cancer effects.

  17. Main path and byways: non-vesicular glutamate release by system xc(-) as an important modifier of glutamatergic neurotransmission.

    Science.gov (United States)

    Massie, Ann; Boillée, Séverine; Hewett, Sandra; Knackstedt, Lori; Lewerenz, Jan

    2015-12-01

    System xc(-) is a cystine/glutamate antiporter that exchanges extracellular cystine for intracellular glutamate. Cystine is intracellularly reduced to cysteine, a building block of GSH. As such, system xc(-) can regulate the antioxidant capacity of cells. Moreover, in several brain regions, system xc(-) is the major source of extracellular glutamate. As such this antiporter is able to fulfill key physiological functions in the CNS, while evidence indicates it also plays a role in certain brain pathologies. Since the transcription of xCT, the specific subunit of system xc(-), is enhanced by the presence of reactive oxygen species and inflammatory cytokines, system xc(-) could be involved in toxic extracellular glutamate release in neurological disorders that are associated with increased oxidative stress and neuroinflammation. System xc(-) has also been reported to contribute to the invasiveness of brain tumors and, as a source of extracellular glutamate, could participate in the induction of peritumoral seizures. Two independent reviews (Pharmacol. Rev. 64, 2012, 780; Antioxid. Redox Signal. 18, 2013, 522), approached from a different perspective, have recently been published on the functions of system xc(-) in the CNS. In this review, we highlight novel achievements and insights covering the regulation of system xc(-) as well as its involvement in emotional behavior, cognition, addiction, neurological disorders and glioblastomas, acquired in the past few years. System xc(-) constitutes an important source of extrasynaptic glutamate in the brain. By modulating the tone of extrasynaptic metabotropic or ionotropic glutamate receptors, it affects excitatory neurotransmission, the threshold for overexcitation and excitotoxicity and, as a consequence, behavior. This review describes the current knowledge of how system xc(-) is regulated and involved in physiological as well as pathophysiological brain functioning.

  18. 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

  19. Comparative Study on Regeneration and Genetic Transformation between Puna Chicory and Commander Chicory%2种菊苣再生体系及遗传转化效率的比较

    Institute of Scientific and Technical Information of China (English)

    赵龙; 陈丹丹; 梁明祥; 郑青松; 王长海; 刘兆普

    2012-01-01

    Effects of culture medium composition on callus induction, shoot regeneration and root formation from cotyledon explants in two kinds of chicory(Puna chicory and Commander chicory) were evaluated to optimize the plant regeneration system. The Aeluropus littoralis Na+/H+ antiporter (AINHX) gene, which encodes a Na+/H+ antiporter, was introduced into chicory to evaluate their genetic transformation efficiency. The results showed that the callus induction and shoot regeneration varied from chicory genotypes. The optimum medium for Puna chicory and Commander chicory were MS+1. 5 mg/L 6-BA + 0. 2 mg/L IBA and MS+1. 0 mg/L 6-BA + 0. 5 mg/L NAA,respectively. The optimum medium for Puna and Commander chicory root formation was 1/2MS+0. 1 mg/L NAA. The insertion of AINHX gene into chicory genome was confirmed by PCR. The genetic transformation efficiency of Puna chicory and Commander chicory were 10. 0% and 13. 3% respectively.%以普那菊苣和将军菊苣子叶为材料,通过植物组织培养的方法,探讨了不同激素浓度配比对二者愈伤组织诱导、芽分化以及根再生的影响,并通过农杆菌介导法将编码獐茅液泡膜Na+/H+逆向转运蛋白基因(AlNHX)导入菊苣中,比较普那菊苣和将军菊苣的遗传转化效率.结果表明:不同基因型的菊苣愈伤组织诱导和芽分化条件不同,普那菊苣最佳培养基为MS+1.5 mg/L6-BA+0.2 mg/L IBA;将军菊苣最佳培养基为MS+ 1.0 mg/L 6-BA+0.5 mg/L NAA;二者最佳生根培养基均为1/2MS+0.1mg/L NAA.获得的抗性芽经PCR检测,初步证实AINHX已插入到菊苣基因组中,且普那菊苣转化效率为10.0%,将军菊苣转化效率为13.3%.

  20. B content and Si/C ratios from cultured diatoms (Thalassiosira pseudonana and Thalassiosira weissflogii): Relationship to seawater pH and diatom carbon acquisition

    Science.gov (United States)

    Mejía, Luz María; Isensee, Kirsten; Méndez-Vicente, Ana; Pisonero, Jorge; Shimizu, Nobumichi; González, Cristina; Monteleone, Brian; Stoll, Heather

    2013-12-01

    Despite the importance of diatoms in regulating climate and the existence of large opal-containing sediments in key air-ocean exchange areas, most geochemical proxy records are based on carbonates. Among them, Boron (B) content and isotopic composition have been widely used to reconstruct pH from foraminifera and coral fossils. We assessed the possibility of a pH/CO2 seawater concentration control on B content in diatom opal to determine whether or not frustule B concentrations could be used as a pH proxy or to clarify algae physiological responses to acidifying pH. We cultured two well-studied diatom species, Thalassiosira pseudonana and Thalassiosira weissflogii at varying pH conditions and determined Si and C quotas. Frustule B content was measured by both laser-ablation inductively coupled mass spectrometry (LA-ICPMS) and secondary ion mass spectrometry (SIMS/ion probe). For both species, frustules grown at higher pH have higher B contents and higher Si requirements per fixed C. If this trend is representative of diatom silicification in a future more acidic ocean, it could contribute to changes in the efficiency of diatom ballasting and C export, as well as changes in the contribution of diatoms relative to other phytoplankton groups in Si-limited regions. If B enters the cell through the same transporter employed for HCO3- uptake, an increased HCO3- requirement with decreasing CO2 concentrations (higher pH), and higher B(OH)4/HCO3- ratios would explain the observed increase in frustule B content with increasing pH. The mechanism of B transport from the site of uptake to the site of silica deposition is unknown, but may occur via silicon transport vesicles, in which B(OH)4- may be imported for B detoxification and/or as part of a pH regulation strategy either though Na-dependent B(OH)4-/Cl- antiport or B(OH)4-/H+ antiport. B deposition in the silica matrix may occur via substitution of a B(OH)4- for a negatively charged SiO- formed during silicification. With

  1. Transcriptional Regulation of Arabidopsis in Response to Salt Stress

    Institute of Scientific and Technical Information of China (English)

    Zhulong Chan

    2012-01-01

    Salt stress is a major factor limiting agricultural productivity worldwide.Adaptations to salt stress include avoidance by reduced sodium uptake,sequestration of toxic sodium ions away from the cytoplasm,or production of compatible solutes or osmoprotectants to reduce molecular disruption.Approaches to engineer salt stress resistance have included regulation of ion transport through introduction of Na+/H+ antiporter; synthesis of compatible solutes; or the introduction of transcription factors regulating expression of stress-responsive genes.On the other hand,naturally occurring variation among wild-type populations of plants also can be used to understand plant adaptive responses to their environments.In this study,we compared phenotypic and transcriptomic effects of constitutive expression of genes intended to confer salt stress tolerance by three different mechanisms:a transcription factor,CBF3/DREB1a; a metabolic gene,M6PR,for mannitol biosynthesis; and the Na+/H+ antiporter,SOS1.In the absence of salt,M6PR and SOS1 lines performed comparably with wild type; CBF3 lines exhibited dwarfing as reported previously.All three transgenes conferred fitness advantage when subjected to 100 mmol/L NaCI in the growth chamber.CBF3 and M6PR affected transcription of numerous abiotic stress-related genes as measured by Affymetrix microarray analysis.M6PR additionally modified expression of biotic stress and oxidative stress genes.Transcriptional effects of SOS1 were smaller and primarily limited to redox-related genes.In addition,we compared natural variations in salt tolerance between Ler and Sha ecotypes based on their responses to salt treatments and the results indicated that Ler was salt-sensitive,but Sha,which obtained a truncated RAS1 protein,was salt-tolerant.Transcriptome analysis revealed that many genes involved in secondary metabolism,photosynthesis,and protein synthesis were mainly down-regulated by salinity effects,while transposable element genes,microRNA and

  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. 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

  4. 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.

  5. Projection structure of the secondary citrate/sodium symporter CitS at 6 Å resolution by electron crystallography.

    Science.gov (United States)

    Kebbel, Fabian; Kurz, Mareike; Grütter, Markus G; Stahlberg, Henning

    2012-04-20

    CitS from Klebsiella pneumoniae acts as a secondary symporter of citrate and sodium ions across the inner membrane of the host. The protein is the best characterized member of the 2-hydroxycarboxylate transporter family, while no experimental structural information at sub-nanometer resolution is available on this class of membrane proteins. Here, we applied electron crystallography to two-dimensional crystals of CitS. Carbon-film-adsorbed tubular two-dimensional crystals were studied by cryo-electron microscopy, producing the 6-Å-resolution projection structure of the membrane-embedded protein. In the p22(1)2(1)-symmetrized projection map, the predicted dimeric structure is clearly visible. Each monomeric unit can tentatively be interpreted as being composed of 11 transmembrane α-helices. In projection, CitS shows a high degree of structural similarity to NhaP1, the Na(+)/H(+) antiporter of Methanococcus jannaschii. We discuss possible locations for the dimer interface and models for the helical arrangements and domain organizations of the symporter based on existing models.

  6. Identification and validation of selected universal stress protein domain containing drought-responsive genes in pigeonpea (Cajanus cajan L.

    Directory of Open Access Journals (Sweden)

    Pallavi eSinha

    2016-01-01

    Full Text Available Pigeonpea is a resilient crop, which is relatively more drought tolerant than many other legume crops. To understand the molecular mechanisms of this unique feature of pigeonpea, 51 genes were selected using the Hidden Markov Models those codes for proteins having close similarity to universal stress protein domain. Validation of these genes was conducted on three pigeonpea genotypes (ICPL 151, ICPL 8755 and ICPL 227 having different levels of drought tolerance. Gene expression analysis using qRT-PCR revealed 6, 8 and 18 genes to be ≥2 fold differentially expressed in ICPL 151, ICPL 8755 and ICPL 227, respectively. A total of 10 differentially expressed genes showed ≥2 fold up-regulation in the more drought tolerant genotype. Of these, four genes each encoded proteins for plant U-box and universal stress protein A- (uspA like, while one gene encoded for cation/H(+ antiporter protein and one uncharacterized protein. Genes C.cajan_29830 and C.cajan_33874 belonging to uspA, were found significantly expressed in all the three genotypes with ≥2 fold expression variations. Expression profiling of these two genes on the four other legume crops revealed their specific role in pigeonpea. Therefore, these genes seem to be promising candidates for conferring drought tolerance specifically to pigeonpea.

  7. Identification and Validation of Selected Universal Stress Protein Domain Containing Drought-Responsive Genes in Pigeonpea (Cajanus cajan L.).

    Science.gov (United States)

    Sinha, Pallavi; Pazhamala, Lekha T; Singh, Vikas K; Saxena, Rachit K; Krishnamurthy, L; Azam, Sarwar; Khan, Aamir W; Varshney, Rajeev K

    2015-01-01

    Pigeonpea is a resilient crop, which is relatively more drought tolerant than many other legume crops. To understand the molecular mechanisms of this unique feature of pigeonpea, 51 genes were selected using the Hidden Markov Models (HMM) those codes for proteins having close similarity to universal stress protein domain. Validation of these genes was conducted on three pigeonpea genotypes (ICPL 151, ICPL 8755, and ICPL 227) having different levels of drought tolerance. Gene expression analysis using qRT-PCR revealed 6, 8, and 18 genes to be ≥2-fold differentially expressed in ICPL 151, ICPL 8755, and ICPL 227, respectively. A total of 10 differentially expressed genes showed ≥2-fold up-regulation in the more drought tolerant genotype, which encoded four different classes of proteins. These include plant U-box protein (four genes), universal stress protein A-like protein (four genes), cation/H(+) antiporter protein (one gene) and an uncharacterized protein (one gene). Genes C.cajan_29830 and C.cajan_33874 belonging to uspA, were found significantly expressed in all the three genotypes with ≥2-fold expression variations. Expression profiling of these two genes on the four other legume crops revealed their specific role in pigeonpea. Therefore, these genes seem to be promising candidates for conferring drought tolerance specifically to pigeonpea. PMID:26779199

  8. Lipid binding protein response to a bile acid library: a combined NMR and statistical approach.

    Science.gov (United States)

    Tomaselli, Simona; Pagano, Katiuscia; Boulton, Stephen; Zanzoni, Serena; Melacini, Giuseppe; Molinari, Henriette; Ragona, Laura

    2015-11-01

    Primary bile acids, differing in hydroxylation pattern, are synthesized from cholesterol in the liver and, once formed, can undergo extensive enzyme-catalysed glycine/taurine conjugation, giving rise to a complex mixture, the bile acid pool. Composition and concentration of the bile acid pool may be altered in diseases, posing a general question on the response of the carrier (bile acid binding protein) to the binding of ligands with different hydrophobic and steric profiles. A collection of NMR experiments (H/D exchange, HET-SOFAST, ePHOGSY NOESY/ROESY and (15) N relaxation measurements) was thus performed on apo and five different holo proteins, to monitor the binding pocket accessibility and dynamics. The ensemble of obtained data could be rationalized by a statistical approach, based on chemical shift covariance analysis, in terms of residue-specific correlations and collective protein response to ligand binding. The results indicate that the same residues are influenced by diverse chemical stresses: ligand binding always induces silencing of motions at the protein portal with a concomitant conformational rearrangement of a network of residues, located at the protein anti-portal region. This network of amino acids, which do not belong to the binding site, forms a contiguous surface, sensing the presence of the bound lipids, with a signalling role in switching protein-membrane interactions on and off.

  9. Analysis and update of the human solute carrier (SLC gene superfamily

    Directory of Open Access Journals (Sweden)

    He Lei

    2009-01-01

    Full Text Available Abstract The solute-carrier gene (SLC superfamily encodes membrane-bound transporters. The SLC superfamily comprises 55 gene families having at least 362 putatively functional protein-coding genes. The gene products include passive transporters, symporters and antiporters, located in all cellular and organelle membranes, except, perhaps, the nuclear membrane. Transport substrates include amino acids and oligopeptides, glucose and other sugars, inorganic cations and anions (H+, HCO3-, Cl-, Na+, K+, Ca2+, Mg2+, PO43-, HPO42-, H2PO4-, SO42-, C2O42-, OH-,CO32-, bile salts, carboxylate and other organic anions, acetyl coenzyme A, essential metals, biogenic amines, neurotransmitters, vitamins, fatty acids and lipids, nucleosides, ammonium, choline, thyroid hormone and urea. Contrary to gene nomenclature commonly assigned on the basis of evolutionary divergence http://www.genenames.org/, the SLC gene superfamily has been named based largely on transporter function by proteins having multiple transmembrane domains. Whereas all the transporters exist for endogenous substrates, it is likely that drugs, non-essential metals and many other environmental toxicants are able to 'hitch-hike' on one or another of these transporters, thereby enabling these moieties to enter (or leave the cell. Understanding and characterising the functions of these transporters is relevant to medicine, genetics, developmental biology, pharmacology and cancer chemotherapy.

  10. Proton Gradients and Proton-Dependent Transport Processes in the Chloroplast

    Science.gov (United States)

    Höhner, Ricarda; Aboukila, Ali; Kunz, Hans-Henning; Venema, Kees

    2016-01-01

    Proton gradients are fundamental to chloroplast function. Across thylakoid membranes, the light induced -proton gradient is essential for ATP synthesis. As a result of proton pumping into the thylakoid lumen, an alkaline stromal pH develops, which is required for full activation of pH-dependent Calvin Benson cycle enzymes. This implies that a pH gradient between the cytosol (pH 7) and the stroma (pH 8) is established upon illumination. To maintain this pH gradient chloroplasts actively extrude protons. More than 30 years ago it was already established that these proton fluxes are electrically counterbalanced by Mg2+, K+, or Cl- fluxes, but only recently the first transport systems that regulate the pH gradient were identified. Notably several (Na+,K+)/H+ antiporter systems where identified, that play a role in pH gradient regulation, ion homeostasis, osmoregulation, or coupling of secondary active transport. The established pH gradients are important to drive uptake of essential ions and solutes, but not many transporters involved have been identified to date. In this mini review we summarize the current status in the field and the open questions that need to be addressed in order to understand how pH gradients are maintained, how this is interconnected with other transport processes and what this means for chloroplast function. PMID:26973667

  11. Uptake of codeine into intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells.

    Science.gov (United States)

    Fischer, Wiebke; Bernhagen, Jennifer; Neubert, Reinhard H H; Brandsch, Matthias

    2010-09-11

    Orally administered codeine has to permeate both the intestinal and the blood-brain barrier in order to act as analgesic and cough suppressant. In this study we characterized the uptake of codeine at intestinal epithelial (Caco-2) and brain endothelial (RBE4) cells. At both cell types, uptake of [(3)H]codeine was independent of an inwardly directed Na(+) gradient. Uptake was, however, strongly stimulated by an outwardly directed H(+) gradient and inhibited by the protonophore FCCP. [(3)H]Codeine uptake into Caco-2 cells was strongly temperature dependent. In the presence of excess amounts of unlabeled codeine, the uptake was inhibited by up to 87% (Caco-2) or 94% (RBE4), respectively. Synthetic opioids and some non-opioid organic cations like propranolol, pyrilamine and quinidine potently inhibited [(3)H]codeine uptake. Several prototype substrates of known transporters for amino acids, neurotransmitters and organic cations were ineffective. Our data are consistent with a hypothetic saturable, H(+)-dependent (antiport) mechanism not yet identified on a molecular level. The pH dependence of codeine uptake and its intracellular accumulation can partially also be explained by a model comprising diffusional membrane permeation of unionized species of codeine followed by codeine sequestration into acidic vesicles and distribution into cellular lipids. PMID:20510359

  12. Recombination suppression at the dominant Rhg1/Rfs2 locus underlying soybean resistance to the cyst nematode.

    Science.gov (United States)

    Afzal, Ahmed J; Srour, Ali; Saini, Navinder; Hemmati, Naghmeh; El Shemy, Hany A; Lightfoot, David A

    2012-04-01

    Host resistance to "yellow dwarf" or "moonlight" disease cause by any population (Hg type) of Heterodera glycines I., the soybean cyst nematode (SCN), requires a functional allele at rhg1. The host resistance encoded appears to mimic an apoptotic response in the giant cells formed at the nematode feeding site about 24-48 h after nematode feeding commences. Little is known about how the host response to infection is mediated but a linked set of 3 genes has been identified within the rhg1 locus. This study aimed to identify the role of the genes within the locus that includes a receptor-like kinase (RLK), a laccase and an ion antiporter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles, gene-based markers, and a new Hg type 0 and new recombination events. A syntenic gene cluster on Lg B1 was found. The effectiveness of SNP probes from the RLK for distinguishing homolog sequence variants on LgB1 from alleles at the rhg1 locus on LgG was shown. The resistant allele of the rhg1 locus was shown to be dominant in NILs. None of the recombination events were within the cluster of the three candidate genes. Finally, rhg1 was shown to reduce the plant root development. A model for rhg1 as a dominant multi-gene resistance locus based on the developmental control was inferred. PMID:22200919

  13. Understanding Abiotic Stress Tolerance Mechanisms: Recent Studies on Stress Response in Rice

    Institute of Scientific and Technical Information of China (English)

    Ji-Ping Gao; Dai-Yin Chao; Hong-Xuan Lin

    2007-01-01

    Abiotic stress is the main factor negatively affecting crop growth and productivity worldwide. The advances in physiology, genetics, and molecular biology have greatly improved our understanding of plant responses to stresses. Rice plants are sensitive to various abiotic stresses. In this short review, we present recent progresses in adaptation of rice to salinity, water deficit and submergence. Many studies show that salt tolerance is tightly associated with the ability to maintain ion homeostasis under salinity. Na+ transporter SKC1 unloads NaMrom xylem, plasma membrane NaVHTantiporter SOS1 excludes sodium out of cytosol and tonoplast Na+/H+antiporter NHX1 sequesters Na+ into the vacuole. Silicon deposition in exodermis and endodermis of rice root reduces sodium transport through the apoplastic pathway. A number of transcription factors regulate stress-inducible gene expression that leads to initiating stress responses and establishing plant stress tolerance. Overexpression of some transcription factors, including DREB/CBF and MAC, enhances salt, drought, and cold tolerance in rice. A variant of one of ERF family genes, Sub1A-1, confers immersion tolerance to lowland rice. These findings and their exploitation will hold promise for engineering breeding to protect crop plants from certain abiotic stresses.

  14. 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.

  15. Identification of mRNA transcript and screening of amino acids in response to interaction of salinity and nitrate in aquatic fern Azolla caroliniana.

    Science.gov (United States)

    Tammam, A A; Mostafa, E M

    2012-06-01

    The mechanisms by which Azolla caroliniana respond to salt stress in absence and presence of nitrate is investigated. Screening of amino acid and differential display is used to compare overall differences in gene expression between salinity-stressed and unstressed Azolla caroliniana by quantitative reverse transcriptase polymerase chain reaction (RT-PC R). Results showed that under saline conditions, aspartic acid, glutamic acid, alanine and leucine were the amino acids found to be abundant in Azolla caroliniana, accounting for 11.26%, 8.66%, 9.43%, and 12.36%, respectively. Following salinity stress, a decrease in free glutamate concomitant with a parallel decrease in free proline was indeed evident. Interaction between nitrate and salinity stress increased proline content significantly. By screening a cDNA library, we have identified protein products by homology with known proteins. The RNA transcripts encoding protein influencing secondary metabolites and vacuolar Na+/H+ antiporter that facilitate the transport system. The databasematched under interaction of nitrate and 50 mM NaCl were associated with wall biosynthesis, disease resistance, metabolite transport and protein regulator, other gene for metabolism of steroids and secondary transport. Results obtained from this research could represent a key step in understanding the molecular mechanism of salt tolerance of Azolla caroliniana in the presence and absence of nitrate. PMID:22695523

  16. Proton-dependent coniferin transport, a common major transport event in differentiating xylem tissue of woody plants.

    Science.gov (United States)

    Tsuyama, Taku; Kawai, Ryo; Shitan, Nobukazu; Matoh, Toru; Sugiyama, Junji; Yoshinaga, Arata; Takabe, Keiji; Fujita, Minoru; Yazaki, Kazufumi

    2013-06-01

    Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms. PMID:23585651

  17. System x(c)(-) regulates microglia and macrophage glutamate excitotoxicity in vivo.

    Science.gov (United States)

    Kigerl, Kristina A; Ankeny, Daniel P; Garg, Sanjay K; Wei, Ping; Guan, Zhen; Lai, Wenmin; McTigue, Dana M; Banerjee, Ruma; Popovich, Phillip G

    2012-01-01

    It is widely believed that microglia and monocyte-derived macrophages (collectively referred to as central nervous system (CNS) macrophages) cause excitotoxicity in the diseased or injured CNS. This view has evolved mostly from in vitro studies showing that neurotoxic concentrations of glutamate are released from CNS macrophages stimulated with lipopolysaccharide (LPS), a potent inflammogen. We hypothesized that excitotoxic killing by CNS macrophages is more rigorously controlled in vivo, requiring both the activation of the glutamate/cystine antiporter (system x(c)(-)) and an increase in extracellular cystine, the substrate that drives glutamate release. Here, we show that non-traumatic microinjection of low-dose LPS into spinal cord gray matter activates CNS macrophages but without causing overt neuropathology. In contrast, neurotoxic inflammation occurs when LPS and cystine are co-injected. Simultaneous injection of NBQX, an antagonist of AMPA glutamate receptors, reduces the neurotoxic effects of LPS+cystine, implicating glutamate as a mediator of neuronal cell death in this model. Surprisingly, neither LPS nor LPS+cystine adversely affects survival of oligodendrocytes or oligodendrocyte progenitor cells. Ex vivo analyses show that redox balance in microglia and macrophages is controlled by induction of system x(c)(-) and that high GSH:GSSG ratios predict the neurotoxic potential of these cells. Together, these data indicate that modulation of redox balance in CNS macrophages, perhaps through regulating system x(c)(-), could be a novel approach for attenuating injurious neuroinflammatory cascades.

  18. L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia

    Institute of Scientific and Technical Information of China (English)

    Peilong Lu; Dan Ma; Yuling Chen; Yingying Guo; Guo-Qiang Chen; Haiteng Deng; Yigong Shi

    2013-01-01

    Bacteria,exemplified by enteropathogenic Escherichia coli (E.coli),,rely on elaborate acid resistance systems to survive acidic environment (such as the stomach).Comprehensive understanding of bacterial acid resistance is important for prevention and clinical treatment.In this study,we report a previously uncharacterized type of acid resistance system in E.coli that relies on L-glutamine (Gln),one of the most abundant food-borne free amino acids.Upon uptake into E.coli,Gln is converted to L-glutamate (Glu) by the acid-activated glutaminase YbaS,with concomitant release of gaseous ammonia.The free ammonia neutralizes proton,resulting in elevated intracellular pH under acidic environment.We show that YbaS and the amino acid antiporter GadC,which exchanges extracellular Gln with intracellular Glu,together constitute an acid resistance system that is sufficient for E.coli survival under extremely acidic environment.

  19. Barley Genes as Tools to Confer Abiotic Stress Tolerance in Crops.

    Science.gov (United States)

    Gürel, Filiz; Öztürk, Zahide N; Uçarlı, Cüneyt; Rosellini, Daniele

    2016-01-01

    Barley is one of the oldest cultivated crops in the world with a high adaptive capacity. The natural tolerance of barley to stress has led to increasing interest in identification of stress responsive genes through small/large-scale omics studies, comparative genomics, and overexpression of some of these genes by genetic transformation. Two major categories of proteins involved in stress tolerance are transcription factors (TFs) responsible from the re-programming of the metabolism in stress environment, and genes encoding Late Embryogenesis Abundant (LEA) proteins, antioxidant enzymes, osmolytes, and transporters. Constitutive overexpression of several barley TFs, such as C-repeat binding factors (HvCBF4), dehydration-responsive element-binding factors (HvDREB1), and WRKYs (HvWRKY38), in transgenic plants resulted in higher tolerance to drought and salinity, possibly by effectively altering the expression levels of stress tolerance genes due to their higher DNA binding affinity. Na(+)/H(+) antiporters, channel proteins, and lipid transporters can also be the strong candidates for engineering plants for tolerance to salinity and low temperatures. PMID:27536305

  20. Proteome scale census of major facilitator superfamily transporters in Trichoderma reesei using protein sequence and structure based classification enhanced ranking.

    Science.gov (United States)

    Chaudhary, Nitika; Kumari, Indu; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2016-07-01

    Trichoderma spp. have been acknowledged as potent bio-control agents against microbial pathogens and also as plant growth promoters. Various secondary metabolites are attributed for these beneficial activities. Major facilitator superfamily (MFS) includes the large proportion of efflux-pumps which are linked with membrane transport of these secondary metabolites. We have carried out a proteome-wide identification of MFS transporters using protein sequence and structure based hierarchical method in Trichoderma reesei. 448 proteins out of 9115 were detected to carry transmembrane helices. MFS specific intragenic gene duplication and its context with transport function have been presented. Finally, using homology based techniques, domains and motifs of MFS families have been identified and utilized to classify them. From query dataset of 448 transmembrane proteins, 148 proteins are identified as potential MFS transporters. Sugar porter, drug: H(+) antiporter-1, monocarboxylate porter and anion: cation symporter emerged as major MFS families with 51, 35, 17 and 11 members respectively. Representative protein tertiary structures of these families are homology modeled for structure-function analysis. This study may help to understand the molecular basis of secretion and transport of agriculturally valuable secondary metabolites produced by these bio-control fungal agents which may be exploited in future for enhancing its biotechnological applications in eco-friendly sustainable development.

  1. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

    Energy Technology Data Exchange (ETDEWEB)

    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  2. Sequencing, characterization, and gene expression analysis of the histidine decarboxylase gene cluster of Morganella morganii.

    Science.gov (United States)

    Ferrario, Chiara; Borgo, Francesca; de Las Rivas, Blanca; Muñoz, Rosario; Ricci, Giovanni; Fortina, Maria Grazia

    2014-03-01

    The histidine decarboxylase gene cluster of Morganella morganii DSM30146(T) was sequenced, and four open reading frames, named hdcT1, hdc, hdcT2, and hisRS were identified. Two putative histidine/histamine antiporters (hdcT1 and hdcT2) were located upstream and downstream the hdc gene, codifying a pyridoxal-P dependent histidine decarboxylase, and followed by hisRS gene encoding a histidyl-tRNA synthetase. This organization was comparable with the gene cluster of other known Gram negative bacteria, particularly with that of Klebsiella oxytoca. Recombinant Escherichia coli strains harboring plasmids carrying the M. morganii hdc gene were shown to overproduce histidine decarboxylase, after IPTG induction at 37 °C for 4 h. Quantitative RT-PCR experiments revealed the hdc and hisRS genes were highly induced under acidic and histidine-rich conditions. This work represents the first description and identification of the hdc-related genes in M. morganii. Results support the hypothesis that the histidine decarboxylation reaction in this prolific histamine producing species may play a role in acid survival. The knowledge of the role and the regulation of genes involved in histidine decarboxylation should improve the design of rational strategies to avoid toxic histamine production in foods.

  3. Urinary Dopamine as a Potential Index of the Transport Activity of Multidrug and Toxin Extrusion in the Kidney

    Science.gov (United States)

    Kajiwara, Moto; Ban, Tsuyoshi; Matsubara, Kazuo; Nakanishi, Yoichi; Masuda, Satohiro

    2016-01-01

    Dopamine is a cationic natriuretic catecholamine synthesized in proximal tubular cells (PTCs) of the kidney before secretion into the lumen, a key site of its action. However, the molecular mechanisms underlying dopamine secretion into the lumen remain unclear. Multidrug and toxin extrusion (MATE) is a H+/organic cation antiporter that is highly expressed in the brush border membrane of PTCs and mediates the efflux of organic cations, including metformin and cisplatin, from the epithelial cells into the urine. Therefore, we hypothesized that MATE mediates dopamine secretion, a cationic catecholamine, into the tubule lumen, thereby regulating natriuresis. Here, we show that [3H]dopamine uptake in human (h) MATE1-, hMATE-2K- and mouse (m) MATE-expressing cells exhibited saturable kinetics. Fluid retention and decreased urinary excretion of dopamine and Na+ were observed in Mate1-knockout mice compared to that in wild-type mice. Imatinib, a MATE inhibitor, inhibited [3H]dopamine uptake by hMATE1-, hMATE2-K- and mMATE1-expressing cells in a concentration-dependent manner. At clinically-relevant concentrations, imatinib inhibited [3H]dopamine uptake by hMATE1- and hMATE2-K-expressing cells. The urinary excretion of dopamine and Na+ decreased and fluid retention occurred in imatinib-treated mice. In conclusion, MATE transporters secrete renally-synthesized dopamine, and therefore, urinary dopamine has the potential to be an index of the MATE transporter activity. PMID:27483254

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

    Directory of Open Access Journals (Sweden)

    Sunita Bijarnia-Mahay

    2015-01-01

    Full Text Available 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 associated with hyperammonemia, who was diagnosed to be having citrin deficiency. Sequencing of the SLC25A13 gene revealed two novel mutations, a single base pair deletion, c. 650delT (p.Phe217SerfsFNx0133 in exon 7, and a missense mutation, c. 869T>C (p.Ile290Thr in exon 9. Confirmation of the diagnosis allowed establishment of the appropriate management. The latter is an essential pre-requisite for obtaining a good prognosis as well as for family counseling.

  5. Effects of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline on glutamate transporter 1 and cysteine/glutamate exchanger as well as ethanol drinking behavior in male, alcohol-preferring rats.

    Science.gov (United States)

    Aal-Aaboda, Munaf; Alhaddad, Hasan; Osowik, Francis; Nauli, Surya M; Sari, Youssef

    2015-06-01

    Alcohol consumption is largely associated with alterations in the extracellular glutamate concentrations in several brain reward regions. We recently showed that glutamate transporter 1 (GLT-1) is downregulated following chronic exposure to ethanol for 5 weeks in alcohol-preferring (P) rats and that upregulation of the GLT-1 levels in nucleus accumbens and prefrontal cortex results, in part, in attenuating ethanol consumption. Cystine glutamate antiporter (xCT) is also downregulated after chronic ethanol exposure in P rats, and its upregulation could be valuable in attenuating ethanol drinking. This study examines the effect of a synthetic compound, (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), on ethanol drinking and expressions of GLT-1 and xCT in the amygdala and the hippocampus of P rats. P rats were exposed to continuous free-choice access to water, 15% and 30% ethanol, and food for 5 weeks, after which they received treatments of MS-153 or vehicle for 5 days. The results show that MS-153 treatment significantly reduces ethanol consumption. It was revealed that GLT-1 and xCT expressions were downregulated in both the amygdala and the hippocampus of ethanol-vehicle-treated rats (ethanol-vehicle group) compared with water-control animals. MS-153 treatment upregulated GLT-1 and xCT expressions in these brain regions. These findings demonstrate an important role for MS-153 in these glutamate transporters for the attenuation of ethanol-drinking behavior.

  6. Exploring regulation genes involved in the expression of L-amino acid oxidase in Pseudoalteromonas sp. Rf-1.

    Directory of Open Access Journals (Sweden)

    Zhiliang Yu

    Full Text Available Bacterial L-amino acid oxidase (LAAO is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO.

  7. Cultivating the uncultured: growing the recalcitrant cluster-2 Frankia strains.

    Science.gov (United States)

    Gtari, Maher; Ghodhbane-Gtari, Faten; Nouioui, Imen; Ktari, Amir; Hezbri, Karima; Mimouni, Wajdi; Sbissi, Imed; Ayari, Amani; Yamanaka, Takashi; Normand, Philippe; Tisa, Louis S; Boudabous, Abdellatif

    2015-01-01

    The repeated failures reported in cultivating some microbial lineages are a major challenge in microbial ecology and probably linked, in the case of Frankia microsymbionts to atypical patterns of auxotrophy. Comparative genomics of the so far uncultured cluster-2 Candidatus Frankia datiscae Dg1, with cultivated Frankiae has revealed genome reduction, but no obvious physiological impairments. A direct physiological assay on nodule tissues from Coriaria myrtifolia infected with a closely-related strain permitted the identification of a requirement for alkaline conditions. A high pH growth medium permitted the recovery of a slow-growing actinobacterium. The strain obtained, called BMG5.1, has short hyphae, produced diazovesicles in nitrogen-free media, and fulfilled Koch's postulates by inducing effective nodules on axenically grown Coriaria spp. and Datisca glomerata. Analysis of the draft genome confirmed its close proximity to the Candidatus Frankia datiscae Dg1 genome with the absence of 38 genes (trehalose synthase, fumarylacetoacetase, etc) in BMG5.1 and the presence of 77 other genes (CRISPR, lanthionine synthase, glutathione synthetase, catalase, Na+/H+ antiporter, etc) not found in Dg1. A multi-gene phylogeny placed the two cluster-2 strains together at the root of the Frankia radiation. PMID:26287281

  8. Transcriptome profiling of TDC cluster deletion mutant of Enterococcus faecalis V583.

    Science.gov (United States)

    Perez, Marta; Ladero, Victor; Del Rio, Beatriz; Redruello, Begoña; de Jong, Anne; Kuipers, Oscar P; Kok, Jan; Martin, M Cruz; Fernandez, Maria; Alvarez, Miguel A

    2016-09-01

    The species Enterococcus faecalis is able to catabolise the amino acid tyrosine into the biogenic amine tyramine by the tyrosine decarboxilase (TDC) pathway Ladero et al. (2012) [1]. The TDC cluster comprises four genes: tyrS, an aminoacyl-tRNA synthetase-like gene; tdcA, which encodes the tyrosine decarboxylase; tyrP, a tyrosine/tyramine exchanger gene and nhaC-2, which encodes an Na(+)/H(+) antiporter and whose role in the tyramine biosynthesis remains unknown [2]. In E. faecalis V583 the last three genes are co-transcribed as a single polycistronic mRNA forming the catabolic operon, while tyrS is transcribed independently of the catabolic genes as a monocistronic mRNA [2]. The catabolic operon is transcriptionally induced by tyrosine and acidic pH. On the opposite, the tyrS expression is repressed by tyrosine concentrations [2]. In this work we report the transcriptional profiling of the TDC cluster deletion mutant (E. faecalis V583 ΔTDC) [2] compared to the wild-type strain, both grown in M17 medium supplemented with tyrosine. The transcriptional profile data of TDC cluster-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE77864. PMID:27408815

  9. 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.

  10. Targeting glia with N-Acetylcysteine modulates brain glutamate and behaviours relevant to neurodevelopmental disorders in C57BL/6J mice

    Directory of Open Access Journals (Sweden)

    Alice Marie Sybille Durieux

    2015-12-01

    Full Text Available An imbalance between excitatory (E glutamate and inhibitory (I GABA transmission may underlie neurodevelopmental conditions such as Autism Spectrum Disorder (ASD and schizophrenia. This may be direct, through alterations in synaptic genes, but there is increasing evidence for the importance of indirect modulation of E/I balance through glial mechanisms. Here we used C57BL/6J mice to test the hypothesis that striatal glutamate levels can be shifted by N-acetylcysteine (NAC, which acts at the cystine-glutamate antiporter of glial cells. Striatal glutamate was quantified in-vivo using proton magnetic resonance spectroscopy. The effect of NAC on behaviours relevant to ASD was examined in a separate cohort. NAC induced a time-dependent decrease in striatal glutamate, which recapitulated findings of lower striatal glutamate reported in ASD. NAC-treated animals were significantly less active and more anxious in the open field test; and NAC-treated females had significantly impaired prepulse inhibition of startle response. This at least partly mimics greater anxiety and impaired sensorimotor gating reported in neurodevelopmental disorders. Thus glial mechanisms regulate glutamate acutely and have functional consequences even in adulthood. Glial cells may be a potential drug target for the development of new therapies for neurodevelopmental disorders across the life-span.

  11. Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa.

    Science.gov (United States)

    Hernandez, D; Dias, F M; Rowe, J J

    1991-04-01

    Pseudomonas aeruginosa is an obligate respirer which can utilize nitrate as a terminal electron acceptor under anaerobic conditions (denitrification). Immediate, transient regulation of nitrate respiration is mediated by oxygen through the inhibition of nitrate uptake. In order to gain an understanding of the bioenergetics of nitrate transport and its regulation by oxygen, the effects of various metabolic inhibitors on the uptake process and on oxygen regulation were investigated. Nitrate uptake was stimulated by the protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, indicating that nitrate uptake is not strictly energized by, but may be affected by the proton motive force. Oxygen regulation of nitrate uptake might in part be through redox-sensitive thiol groups since N-ethylmaleimide at high concentrations decreased the rate of nitrate transport. Cells grown with tungstate (deficient in nitrate reductase activity) and azide-treated cells transported nitrate at significantly lower rates than untreated cells, indicating that physiological rates of nitrate transport are dependent on nitrate reduction. Furthermore, tungstate grown cells transported nitrate only in the presence of nitrite, lending support to the nitrate/nitrite antiport model for transport. Oxygen regulation of nitrate transport was relieved (10% that of typical anaerobic rates) by the cytochrome oxygen reductase inhibitors carbon monoxide and cyanide. PMID:1910283

  12. N-acetylcysteine decreases binge eating in a rodent model.

    Science.gov (United States)

    Hurley, M M; Resch, J M; Maunze, B; Frenkel, M M; Baker, D A; Choi, S

    2016-07-01

    Binge-eating behavior involves rapid consumption of highly palatable foods leading to increased weight gain. Feeding in binge disorders resembles other compulsive behaviors, many of which are responsive to N-acetylcysteine (NAC), which is a cysteine prodrug often used to promote non-vesicular glutamate release by a cystine-glutamate antiporter. To examine the potential for NAC to alter a form of compulsive eating, we examined the impact of NAC on binge eating in a rodent model. Specifically, we monitored consumption of standard chow and a high-fat, high carbohydrate western diet (WD) in a rodent limited-access binge paradigm. Before each session, rats received either a systemic or intraventricular injection of NAC. Both systemic and central administration of NAC resulted in significant reductions of binge eating the WD without decreasing standard chow consumption. The reduction in WD was not attributable to general malaise as NAC did not produce condition taste aversion. These results are consistent with the clinical evidence of NAC to reduce or reverse compulsive behaviors, such as, drug addiction, skin picking and hair pulling. PMID:26975440

  13. Identification and Characterization of hmr19 Gene Encoding a Multidrug Resistance Efflux Protein from Streptomyces hygroscopicus subsp.yingchengensis Strain 10-22

    Institute of Scientific and Technical Information of China (English)

    Lei QIN; Heng-An WANG; Zhong-Qin WU; Xiao-Feng ZHANG; Mei-Lei JIN; Zi-Xin DENG; Guo-Ping ZHAO

    2004-01-01

    The hmr19 gene was cloned from Streptomyces hygroscopicus subsp.yingchengensis strain 10-22,a bacterium strain producing agricultural antibiotics.Sequence similarity comparison indicates that hmr19gene may encode a predicted protein with 14 putative transmembrane α-helical spanners,belonging to the drug:H+ antiporter-2 family of the major facilitator superfamily.The expression ofhmr19 in the mycelium of strain 10-22 was detected by Western blotting analysis.Gene replacement technology was employed to construct an hmr19 disruption mutant.The growth inhibition test against different antibiotics indicated that the mutant strain was 5-20 fold more susceptible to tetracycline,vancomycin and mitomycin C than the parental wild type strain.The mutant took up tetracycline much faster and accumulated more antibiotics than the wild type strain 10-22.While with the addition of an energy uncoupler,carbonyl cyanide mchlorophenylhydrazone,the characteristics of the accumulation of [3H]tetracycline in these two strains were almost the same.It was thus concluded that hmr19 encoded a multidrug resistance efflux protein.

  14. 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. PMID:26284097

  15. V-ATPase, ScNhxlp and Yeast Vacuole Fusion

    Institute of Scientific and Technical Information of China (English)

    Quan-Sheng Qiu

    2012-01-01

    Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos.It is a central cellular reaction that plays important roles in signal transduction,protein sorting and subcellular compartmentation.Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summanzed in this article.It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhxlp are key components of the vacuole fusion machinery in yeast.Yeast ScNhxlp regulates vacuole fusion by controlling the luminal pH.V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast.Fission defects are epistatic to fusion defects.Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast,the fusion reaction does not need the transport activity but requires the physical presence of the proton pump.Vo,the membrane-integral sector of the V-ATPase,forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the Vo trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

  16. 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.

  17. 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'. PMID:7823046

  18. GmCLC1 Confers Enhanced Salt Tolerance through Regulating Chloride Accumulation in Soybean.

    Science.gov (United States)

    Wei, Peipei; Wang, Longchao; Liu, Ailin; Yu, Bingjun; Lam, Hon-Ming

    2016-01-01

    The family of chloride channel proteins that mediate Cl(-) transportation play vital roles in plant nutrient supply, cellular action potential and turgor pressure adjustment, stomatal movement, hormone signal recognition and transduction, Cl(-) homeostasis, and abiotic and biotic stress tolerance. The anionic toxicity, mainly caused by chloride ions (Cl(-)), on plants under salt stress remains poorly understood. In this work, we investigated the function of soybean Cl(-)/H(+) antiporter GmCLC1 under salt stress in transgenic Arabidopsis thaliana, soybean, and yeast. We found that GmCLC1 enhanced salt tolerance in transgenic A. thaliana by reducing the Cl(-) accumulation in shoots and hence released the negative impact of salt stress on plant growth. Overexpression of GmCLC1 in the hairy roots of soybean sequestered more Cl(-) in their roots and transferred less Cl(-) to their shoots, leading to lower relative electrolyte leakage values in the roots and leaves. When either the soybean GmCLC1 or the yeast chloride transporter gene, GEF1, was transformed into the yeast gef1 mutant, and then treated with different chloride salts (MnCl2, KCl, NaCl), enhanced survival rate was observed. The result indicates that GmCLC1 and GEF1 exerted similar effects on alleviating the stress of diverse chloride salts on the yeast gef1 mutant. Together, this work suggests a protective function of GmCLC1 under Cl(-) stress. PMID:27504114

  19. 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.

  20. Cadmium Induced Changes in Metabolic Function of Mitochondrial Isolated from Potato Tissue (Solanum tuberosum L.

    Directory of Open Access Journals (Sweden)

    Chagra Ali

    2009-01-01

    Full Text Available Problem statement: Cadmium is highly toxic at low concentrations, but the mechanism of its toxicity is still not understood particularly at the cellular and subcellular level. Approach: In this study we examined the effects of cadmium on the oxidophosphorylation properties of mitochondria isolated from potatoes. Results: Cadmium strongly disturbed the respiratory metabolism of mitochondria isolated especially in the transfer of electrons by cyanide pathway. Meanwhile, cadmium altered the composition of lipid fatty acids polar while inhibiting catalase activity, a key enzyme in the detoxification (antioxidant process. In addition, cadmium caused an increase in mitochondrial volume associated with strong inhibition of ATPase activity, which could be explained by a transport of the potassium ion stimulation at the origin of the massive influx of H+ by antiport through the K+/H+ leading to a decoupling (cut of mitochondrial oxidative phosphorylation. The swelling of mitochondria was accompanied by the rupture of the mitochondrial outer membrane and thus the release of Cytochrome C, which appears to be the initial phase of apoptosis. Conclusion: Following this study, it appeared that cadmium generates in potato the isolated mitochondria a concentration-dependent oxidative stress.

  1. 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).

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    Mukul Joshi

    Full Text Available 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.

  4. Sodic alkaline stress mitigation by interaction of nitric oxide and polyamines involves antioxidants and physiological strategies in Solanum lycopersicum.

    Science.gov (United States)

    Gong, Biao; Li, Xiu; Bloszies, Sean; Wen, Dan; Sun, Shasha; Wei, Min; Li, Yan; Yang, Fengjuan; Shi, Qinghua; Wang, Xiufeng

    2014-06-01

    Nitric oxide (NO) and polyamines (PAs) are two kinds of important signal in mediating plant tolerance to abiotic stress. In this study, we observed that both NO and PAs decreased alkaline stress in tomato plants, which may be a result of their role in regulating nutrient balance and reactive oxygen species (ROS), thereby protecting the photosynthetic system from damage. Further investigation indicated that NO and PAs induced accumulation of each other. Furthermore, the function of PAs could be removed by a NO scavenger, cPTIO. On the other hand, application of MGBG, a PA synthesis inhibitor, did little to abolish the function of NO. To further elucidate the mechanism by which NO and PAs alleviate alkaline stress, the expression of several genes associated with abiotic stress was analyzed by qRT-PCR. NO and PAs significantly upregulated ion transporters such as the plasma membrane Na(+)/H(+) antiporter (SlSOS1), vacuolar Na(+)/H(+) exchanger (SlNHX1 and SlNHX2), and Na(+) transporter and signal components including ROS, MAPK, and Ca(2+) signal pathways, as well as several transcription factors. All of these play important roles in plant adaptation to stress conditions.

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

    International Nuclear Information System (INIS)

    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® 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: ► We prepared oriented membranes based on a liquid crystalline columnar polyether. ► In this structure, the inner polyether chain could work as an ion channel. ► We obtained membranes by casting a chloroform solution in the presence of water. ► Membranes showed good proton permeability due to the presence of oriented channels.

  6. 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.

  7. MFS transporters of Candida species and their role in clinical drug resistance.

    Science.gov (United States)

    K Redhu, Archana; Shah, Abdul H; Prasad, Rajendra

    2016-06-01

    ABC (ATP-binding cassette) and MFS (major facilitator superfamily) exporters, belonging to two different superfamilies, are one of the most prominent contributors of multidrug resistance (MDR) in yeast. While the role of ABC efflux pump proteins in the development of MDR is well documented, the MFS transporters which are also implicated in clinical drug resistance have not received due attention. The MFS superfamily is the largest known family of secondary active membrane carriers, and MFS exporters are capable of transporting a host of substrates ranging from small molecules, including organic and inorganic ions, to complex biomolecules, such as peptide and lipid moieties. A few of the members of the drug/H(+) antiporter family of the MFS superfamily function as multidrug transporters and employ downhill transport of protons to efflux their respective substrates. This review focuses on the recent developments in MFS of Candida and highlights their role in drug transport by using the example of the relatively well characterized promiscuous Mdr1 efflux pump of the pathogenic yeast C. albicans.

  8. Novel role of a family of major facilitator transporters in biofilm development and virulence of Candida albicans.

    Science.gov (United States)

    Shah, Abdul Haseeb; Singh, Ashutosh; Dhamgaye, Sanjiveeni; Chauhan, Neeraj; Vandeputte, Patrick; Suneetha, Korivi Jyothiraj; Kaur, Rupinder; Mukherjee, Pranab K; Chandra, Jyotsna; Ghannoum, Mahmoud A; Sanglard, Dominique; Goswami, Shyamal K; Prasad, Rajendra

    2014-06-01

    The QDR (quinidine drug resistance) family of genes encodes transporters belonging to the MFS (major facilitator superfamily) of proteins. We show that QDR transporters, which are localized to the plasma membrane, do not play a role in drug transport. Hence, null mutants of QDR1, QDR2 and QDR3 display no alterations in susceptibility to azoles, polyenes, echinocandins, polyamines or quinolines, or to cell wall inhibitors and many other stresses. However, the deletion of QDR genes, individually or collectively, led to defects in biofilm architecture and thickness. Interestingly, QDR-lacking strains also displayed attenuated virulence, but the strongest effect was observed with qdr2∆, qdr3∆ and in qdr1/2/3∆ strains. Notably, the attenuated virulence and biofilm defects could be reversed upon reintegration of QDR genes. Transcripts profiling confirmed differential expression of many biofilm and virulence-related genes in the deletion strains as compared with wild-type Candida albicans cells. Furthermore, lipidomic analysis of QDR-deletion mutants suggests massive remodelling of lipids, which may affect cell signalling, leading to the defect in biofilm development and attenuation of virulence. In summary, the results of the present study show that QDR paralogues encoding MFS antiporters do not display conserved functional linkage as drug transporters and perform functions that significantly affect the virulence of C. albicans.

  9. The cmbT gene encodes a novel major facilitator multidrug resistance transporter in Lactococcus lactis.

    Science.gov (United States)

    Filipic, Brankica; Golic, Natasa; Jovcic, Branko; Tolinacki, Maja; Bay, Denice C; Turner, Raymond J; Antic-Stankovic, Jelena; Kojic, Milan; Topisirovic, Ljubisa

    2013-01-01

    Functional characterization of the multidrug resistance CmbT transporter was performed in Lactococcus lactis. The cmbT gene is predicted to encode an efflux protein homologous to the multidrug resistance major facilitator superfamily. The cmbT gene (1377 bp) was cloned and overexpressed in L. lactis NZ9000. Results from cell growth studies revealed that the CmbT protein has an effect on host cell resistance to lincomycin, cholate, sulbactam, ethidium bromide, Hoechst 33342, sulfadiazine, streptomycin, rifampicin, puromycin and sulfametoxazole. Moreover, in vivo transport assays showed that overexpressed CmbT-mediated extrusion of ethidium bromide and Hoechst 33342 was higher than in the control L. lactis NZ9000 strain. CmbT-mediated extrusion of Hoechst 33342 was inhibited by the ionophores nigericin and valinomycin known to dissipate proton motive force. This indicates that CmbT-mediated extrusion is based on a drug-proton antiport mechanism. Taking together results obtained in this study, it can be concluded that CmbT is a novel major facilitator multidrug resistance transporter candidate in L. lactis, with a possible signaling role in sulfur metabolism.

  10. Micron dimensioned cavity array supported lipid bilayers for the electrochemical investigation of ionophore activity.

    Science.gov (United States)

    Maher, Sean; Basit, Hajra; Forster, Robert J; Keyes, Tia E

    2016-12-01

    Microcavity supported lipid bilayers, MSLBs, were applied to an electrochemical investigation of ionophore mediated ion transport. The arrays comprise of a 1cm(2) gold electrode imprinted with an ordered array of uniform spherical-cap pores of 2.8μm diameter prepared by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aqueous buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by the micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coefficient of 12.58±1.28μm(2)s(-1) and anomalous exponent of 1.03±0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3days and electrochemical impedance spectroscopy of the membrane with and without ionic gradient over experimental windows of 6h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K(+) uniporter and Nigericin, a K(+)/H(+) antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance respectively. Significant increases in Valinomycin and Nigericin activity were observed, reflected in large decreases in membrane resistance when K(+) was present in the contacting buffer and in the presence of H(+) ionic gradient across the membrane respectively. PMID:27420132

  11. The uniqueness of the plant mitochondrial potassium channel

    Directory of Open Access Journals (Sweden)

    Donato Pastore

    2013-08-01

    Full Text Available The ATP-inhibited Plant Mitochondrial K+ Channel (PmitoKATPwas discovered about fifteen years ago in Durum WheatMitochondria (DWM. PmitoKATP catalyses the electrophoreticK+ uniport through the inner mitochondrial membrane;moreover, the co-operation between PmitoKATP and K+/H+antiporter allows such a great operation of a K+ cycle tocollapse mitochondrial membrane potential (ΔΨ and ΔpH, thusimpairing protonmotive force (Δp. A possible physiological roleof such ΔΨ control is the restriction of harmful reactive oxygenspecies (ROS production under environmental/oxidative stressconditions. Interestingly, DWM lacking Δp were found to benevertheless fully coupled and able to regularly accomplish ATPsynthesis; this unexpected behaviour makes necessary to recastin some way the classical chemiosmotic model. In the whole,PmitoKATP may oppose to large scale ROS production bylowering ΔΨ under environmental/oxidative stress, but, whenstress is moderate, this occurs without impairing ATP synthesisin a crucial moment for cell and mitochondrial bioenergetics.[BMB Reports 2013; 46(8: 391-397

  12. Molecular cloning of functional genes for high growth-temperature and salt tolerance of the basidiomycete Fomitopsis pinicola isolated in a mangrove forest in Micronesia.

    Science.gov (United States)

    Miyazaki, Yasumasa; Hiraide, Masakazu; Shibuya, Hajime

    2007-01-01

    Several functional genes encoding putative proteins, heat shock protein 70, sphingosine phosphate lyase, and Na+/H+ antiporter, were cloned from the basidiomycete Fomitopsis pinicola, a wood-rotting fungus isolated in the tropical mangrove forest of Pohnpei Island of the Federated States of Micronesia. The deduced amino acid sequences of the obtained genes involved in heat shock resistance, lipid synthesis, and salt tolerance showed diverse similarities to other homologous proteins. Molecular phylogenetic trees of these proteins suggested that encoded proteins of the cloned genes of F. pinicola differed remarkably from other homologs in various organisms, even fungal proteins. Putative candidates for other genes related to several cellular metabolisms were also amplified, implying the possible existence of those genes in F. pinicola. This is the first report of possibly functional genes derived from a basidiomycetous mushroom growing in tropical islands such as Micronesia. The genes found in this study might play important roles in the cellular survival of the basidiomycete F. pinicola under severe environmental conditions. PMID:17213639

  13. Micron dimensioned cavity array supported lipid bilayers for the electrochemical investigation of ionophore activity.

    Science.gov (United States)

    Maher, Sean; Basit, Hajra; Forster, Robert J; Keyes, Tia E

    2016-12-01

    Microcavity supported lipid bilayers, MSLBs, were applied to an electrochemical investigation of ionophore mediated ion transport. The arrays comprise of a 1cm(2) gold electrode imprinted with an ordered array of uniform spherical-cap pores of 2.8μm diameter prepared by gold electrodeposition through polystyrene templating spheres. The pores were pre-filled with aqueous buffer prior to Langmuir-Blodgett assembly of a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer. Fluorescence lifetime correlation spectroscopy enabled by the micron dimensions of the pores permitted study of lipid diffusion across single apertures, yielding a diffusion coefficient of 12.58±1.28μm(2)s(-1) and anomalous exponent of 1.03±0.02, consistent with Brownian motion. From FLCS, the MSLBs were stable over 3days and electrochemical impedance spectroscopy of the membrane with and without ionic gradient over experimental windows of 6h showed excellent stability. Two ionophores were studied at the MSLBs; Valinomycin, a K(+) uniporter and Nigericin, a K(+)/H(+) antiporter. Ionophore reconstituted into the DOPC bilayer resulted in a decrease and increase in membrane resistance and capacitance respectively. Significant increases in Valinomycin and Nigericin activity were observed, reflected in large decreases in membrane resistance when K(+) was present in the contacting buffer and in the presence of H(+) ionic gradient across the membrane respectively.

  14. Proteome scale census of major facilitator superfamily transporters in Trichoderma reesei using protein sequence and structure based classification enhanced ranking.

    Science.gov (United States)

    Chaudhary, Nitika; Kumari, Indu; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2016-07-01

    Trichoderma spp. have been acknowledged as potent bio-control agents against microbial pathogens and also as plant growth promoters. Various secondary metabolites are attributed for these beneficial activities. Major facilitator superfamily (MFS) includes the large proportion of efflux-pumps which are linked with membrane transport of these secondary metabolites. We have carried out a proteome-wide identification of MFS transporters using protein sequence and structure based hierarchical method in Trichoderma reesei. 448 proteins out of 9115 were detected to carry transmembrane helices. MFS specific intragenic gene duplication and its context with transport function have been presented. Finally, using homology based techniques, domains and motifs of MFS families have been identified and utilized to classify them. From query dataset of 448 transmembrane proteins, 148 proteins are identified as potential MFS transporters. Sugar porter, drug: H(+) antiporter-1, monocarboxylate porter and anion: cation symporter emerged as major MFS families with 51, 35, 17 and 11 members respectively. Representative protein tertiary structures of these families are homology modeled for structure-function analysis. This study may help to understand the molecular basis of secretion and transport of agriculturally valuable secondary metabolites produced by these bio-control fungal agents which may be exploited in future for enhancing its biotechnological applications in eco-friendly sustainable development. PMID:27041239

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Tohru Minamino

    2016-03-01

    Full Text Available 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.

  17. Essential role of mitochondrial Ca2+ uniporter in the generation of mitochondrial pH gradient and metabolism-secretion coupling in insulin-releasing cells.

    Science.gov (United States)

    Quan, Xianglan; Nguyen, Tuyet Thi; Choi, Seong-Kyung; Xu, Shanhua; Das, Ranjan; Cha, Seung-Kuy; Kim, Nari; Han, Jin; Wiederkehr, Andreas; Wollheim, Claes B; Park, Kyu-Sang

    2015-02-13

    In pancreatic β-cells, ATP acts as a signaling molecule initiating plasma membrane electrical activity linked to Ca(2+) influx, which triggers insulin exocytosis. The mitochondrial Ca(2+) uniporter (MCU) mediates Ca(2+) uptake into the organelle, where energy metabolism is further stimulated for sustained second phase insulin secretion. Here, we have studied the contribution of the MCU to the regulation of oxidative phosphorylation and metabolism-secretion coupling in intact and permeabilized clonal β-cells as well as rat pancreatic islets. Knockdown of MCU with siRNA transfection blunted matrix Ca(2+) rises, decreased nutrient-stimulated ATP production as well as insulin secretion. Furthermore, MCU knockdown lowered the expression of respiratory chain complexes, mitochondrial metabolic activity, and oxygen consumption. The pH gradient formed across the inner mitochondrial membrane following nutrient stimulation was markedly lowered in MCU-silenced cells. In contrast, nutrient-induced hyperpolarization of the electrical gradient was not altered. In permeabilized cells, knockdown of MCU ablated matrix acidification in response to extramitochondrial Ca(2+). Suppression of the putative Ca(2+)/H(+) antiporter leucine zipper-EF hand-containing transmembrane protein 1 (LETM1) also abolished Ca(2+)-induced matrix acidification. These results demonstrate that MCU-mediated Ca(2+) uptake is essential to establish a nutrient-induced mitochondrial pH gradient which is critical for sustained ATP synthesis and metabolism-secretion coupling in insulin-releasing cells.

  18. Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.

    Science.gov (United States)

    Wang, Weimin; Kryczek, Ilona; Dostál, Lubomír; Lin, Heng; Tan, Lijun; Zhao, Lili; Lu, Fujia; Wei, Shuang; Maj, Tomasz; Peng, Dongjun; He, Gong; Vatan, Linda; Szeliga, Wojciech; Kuick, Rork; Kotarski, Jan; Tarkowski, Rafał; Dou, Yali; Rattan, Ramandeep; Munkarah, Adnan; Liu, J Rebecca; Zou, Weiping

    2016-05-19

    Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

  19. [Genetic transformation of buckwheat ( Fagopyrum esculentum Moench ) with AtNHX1 gene and regeneration of salt-tolerant transgenic plants].

    Science.gov (United States)

    Cheng, Li-Hong; Zhang, Bo; Xu, Zi-Qin

    2007-01-01

    The Arabidopsis thaliana tonoplast Na+ /H+ antiporter gene, AtNHX1, was transferred into buckwheat by Agrobacterium-mediated method. Transgenic buckwheat plants were regenerated and selected on MS basal medium supplemented with 2.0mg/L 6-BA, 1.0mg/L KT, 0.lmg/L IAA, 50mg/L kanamycin and 500mg/L carbenicillin. 426 seedlings from 36 resistant calli originated from 864 explants (transformed about at 4.17 percentage) exhibited resistance to kanamycin. The transformants were confirmed by PCR, Southern blotting, RT-PCR and Northern blotting analysis. After stress treatment for 6 weeks with 200mmol/L NaCl, transgenic plants survived, while wild-type plants did not. After 3 days of stress treatment through different concentrations of NaCl, transgenic plants accumulated higher concentration of Na+ and proline than the control plants. However, the K+ concentration of transgenic plants declined in comparison with the control plants. Moreover, the rutin content of the roots, stems and leaves of transgenic buckwheat increased than those of the control plants. These results showed that it could be possible to improve the salt-tolerance of crops with genetic technology.

  20. Evidence that the Entamoeba histolytica Mitochondrial Carrier Family Links Mitosomal and Cytosolic Pathways through Exchange of 3'-Phosphoadenosine 5'-Phosphosulfate and ATP.

    Science.gov (United States)

    Mi-ichi, Fumika; Nozawa, Akira; Yoshida, Hiroki; Tozawa, Yuzuru; Nozaki, Tomoyoshi

    2015-11-01

    Entamoeba histolytica, a microaerophilic protozoan parasite, possesses mitosomes. Mitosomes are mitochondrion-related organelles that have largely lost typical mitochondrial functions, such as those involved in the tricarboxylic acid cycle and oxidative phosphorylation. The biological roles of Entamoeba mitosomes have been a long-standing enigma. We previously demonstrated that sulfate activation, which is not generally compartmentalized to mitochondria, is a major function of E. histolytica mitosomes. Sulfate activation cooperates with cytosolic enzymes, i.e., sulfotransferases (SULTs), for the synthesis of sulfolipids, one of which is cholesteryl sulfate. Notably, cholesteryl sulfate plays an important role in encystation, an essential process in the Entamoeba life cycle. These findings identified a biological role for Entamoeba mitosomes; however, they simultaneously raised a new issue concerning how the reactions of the pathway, separated by the mitosomal membranes, cooperate. Here, we demonstrated that the E. histolytica mitochondrial carrier family (EhMCF) has the capacity to exchange 3'-phosphoadenosine 5'-phosphosulfate (PAPS) with ATP. We also confirmed the cytosolic localization of all the E. histolytica SULTs, suggesting that in Entamoeba, PAPS, which is produced through mitosomal sulfate activation, is translocated to the cytosol and becomes a substrate for SULTs. In contrast, ATP, which is produced through cytosolic pathways, is translocated into the mitosomes and is a necessary substrate for sulfate activation. Taking our findings collectively, we suggest that EhMCF functions as a PAPS/ATP antiporter and plays a crucial role in linking the mitosomal sulfate activation pathway to cytosolic SULTs for the production of sulfolipids.

  1. Introduction of AtNHX1 into beet improved salt-tolerance of transgenic plants

    Institute of Scientific and Technical Information of China (English)

    Yang Aifang; Zhao Shilan; Yin Xiaoyan; Gao Feng; Zhang Juren

    2006-01-01

    AtNHX1 gene encoding the Na+/H+ antiport on the vacuole membrane of Arabidopsis was transferred into small bud tips of 1-3mm in length derived from immature inflorescence cultures of six genotypes of beet ( Beta vulgaris L. ) by the infection of Agrobacterium tumefaciens and transgenic plants with improved salt-tolerance were obtained. When transgenic plants at 5-leaf stage were potted in sand and irrigated with solutions containing a range of concentrations of NaCl (171-513mM), they showed minor symptoms of damage from salinity and better tolerance than the controls. There were considerable discrepancies of salt-tolerance between transgenic plants originated from the same genotype and also between different genotypes. After vernalization, bolting transgenic plants were enveloped with two layers of gauzes for self-pollination. T1 seedlings tolerant to 342-427mM NaCl were obtained respectively. These results revealed that it was feasible to improve salt-tolerance of beets by the introduction of AtNHX1 gene into cultured bud cells.

  2. Rice Phospholipase Dα is Involved in Salt Tolerance by the Mediation of H+-ATPase Activity and Transcription

    Institute of Scientific and Technical Information of China (English)

    Peng Shen; Rong Wang; Wen Jing; Wenhua Zhang

    2011-01-01

    Phospholipase Dα (PLDα) is involved in plant response to salt stress, but the mechanisms remain unclear.We investigated rice PLDα (OsPLDα) localization and its effect on tonoplast (TP) and plasma membrane (PM) H+-ATPase activity and transcription in response to NaCl. When rice suspension-cultured cells were treated with 100 mM NaCI, PLDα activity in cell extracts showed a transient activation with a threefold increase at 1 h. The amount of OsPLDα protein decreased slightly in the cytosolic fractions, whereas it increased significantly in the TP after NaCI treatment. OsPLDα1 knockdown cells were developed using RNA interference (RNAi) methods. The increase in TP and PM H+-ATPase activity induced by NaCl was significantly inhibited in OsPLDα1-RNAi cells. Knockdown of OsPLDα1 prevented the NaCl-induced increase in the transcript level of OsVHA-A (encodes TP H+-ATPase) and OSA2 (encodes PM H+-ATPase),as well as OsNHX1 (encodes TP Na+/H+ antiporter). The cells died more in OsPLDα1-RNAi mutant than in wild type when they were treated with NaCl. These results suggest that OsPLDα is involved in salt tolerance in rice through the mediation of H+-ATPase activity and transcription.

  3. A Cytotoxic, Co-operative Interaction Between Energy Deprivation and Glutamate Release From System xc- Mediates Aglycemic Neuronal Cell Death.

    Science.gov (United States)

    Thorn, Trista L; He, Yan; Jackman, Nicole A; Lobner, Doug; Hewett, James A; Hewett, Sandra J

    2015-01-01

    The astrocyte cystine/glutamate antiporter (system xc(-)) contributes substantially to the excitotoxic neuronal cell death facilitated by glucose deprivation. The purpose of this study was to determine the mechanism by which this occurred. Using pure astrocyte cultures, as well as, mixed cortical cell cultures containing both neurons and astrocytes, we found that neither an enhancement in system xc(-) expression nor activity underlies the excitotoxic effects of aglycemia. In addition, using three separate bioassays, we demonstrate no change in the ability of glucose-deprived astrocytes--either cultured alone or with neurons--to remove glutamate from the extracellular space. Instead, we demonstrate that glucose-deprived cultures are 2 to 3 times more sensitive to the killing effects of glutamate or N-methyl-D-aspartate when compared with their glucose-containing controls. Hence, our results are consistent with the weak excitotoxic hypothesis such that a bioenergetic deficiency, which is measureable in our mixed but not astrocyte cultures, allows normally innocuous concentrations of glutamate to become excitotoxic. Adding to the burgeoning literature detailing the contribution of astrocytes to neuronal injury, we conclude that under our experimental paradigm, a cytotoxic, co-operative interaction between energy deprivation and glutamate release from astrocyte system xc(-) mediates aglycemic neuronal cell death.

  4. PET Imaging with [(18)F]FSPG Evidences the Role of System xc(-) on Brain Inflammation Following Cerebral Ischemia in Rats.

    Science.gov (United States)

    Domercq, Maria; Szczupak, Boguslaw; Gejo, Jon; Gómez-Vallejo, Vanessa; Padro, Daniel; Gona, Kiran Babu; Dollé, Frédéric; Higuchi, Makoto; Matute, Carlos; Llop, Jordi; Martín, Abraham

    2016-01-01

    In vivo Positron Emission Tomography (PET) imaging of the cystine-glutamate antiporter (system xc(-)) activity with [(18)F]FSPG is meant to be an attractive tool for the diagnosis and therapy evaluation of brain diseases. However, the role of system xc(-) in cerebral ischemia and its involvement in inflammatory reaction has been scarcely explored. In this work, we report the longitudinal investigation of the neuroinflammatory process following transient middle cerebral artery occlusion (MCAO) in rats using PET with [(18)F]FSPG and the translocator protein (TSPO) ligand [(18)F]DPA-714. In the ischemic territory, [(18)F]FSPG showed a progressive binding increase that peaked at days 3 to 7 and was followed by a progressive decrease from days 14 to 28 after reperfusion. In contrast, [(18)F]DPA-714 evidenced maximum binding uptake values over day 7 after reperfusion. Ex vivo immnunohistochemistry confirmed the up-regulation of system xc(-) in microglial cells and marginally in astrocytes. Inhibition of system xc(-) with sulfasalazine and S-4-CPG resulted in increased arginase (anti-inflammatory M2 marker) expression at day 7 after ischemia, together with a decrease in TSPO and microglial M1 proinflammatory markers (CCL2, TNF and iNOS) expression. Taken together, these results suggest that system xc(-) plays a key role in the inflammatory reaction underlying experimental stroke.

  5. Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc-, leading to glutathione depletion.

    Science.gov (United States)

    Sleire, L; Skeie, B S; Netland, I A; Førde, H E; Dodoo, E; Selheim, F; Leiss, L; Heggdal, J I; Pedersen, P-H; Wang, J; Enger, P Ø

    2015-12-01

    Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, mainly provided by the system X(c)(-) antiporter, is a requirement for glioma cell synthesis of glutathione (GSH) which has a critical role in scavenging free radicals, for example, after radiotherapy. Thus, we hypothesized that the X(c)(-)-inhibitor sulfasalazine (SAS) could potentiate the efficacy of radiotherapy against gliomas. Here, we show that the catalytic subunit of system X(c)(-), xCT, was uniformly expressed in a panel of 30 human GBM biopsies. SAS treatment significantly reduced cystine uptake and GSH levels, whereas it significantly increased the levels of reactive oxygen species (ROS) in glioma cells in vitro. Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the antioxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and gamma knife radiosurgery (GKRS) synergistically prolonged survival in nude rats harboring human GBM xenografts, compared with controls or either treatment alone. In conclusion, SAS effectively blocks cystine uptake in glioma cells in vitro, leading to GSH depletion and increased ROS levels, DNA damage and cell death. Moreover, it potentiates the anti-tumor efficacy of GKRS in rats with human GBM xenografts, providing a survival benefit. Thus, SAS may have a role as a radiosensitizer to enhance the efficacy of current radiotherapies for glioma patients.

  6. The EGF Receptor Promotes the Malignant Potential of Glioma by Regulating Amino Acid Transport System xc(-).

    Science.gov (United States)

    Tsuchihashi, Kenji; Okazaki, Shogo; Ohmura, Mitsuyo; Ishikawa, Miyuki; Sampetrean, Oltea; Onishi, Nobuyuki; Wakimoto, Hiroaki; Yoshikawa, Momoko; Seishima, Ryo; Iwasaki, Yoshimi; Morikawa, Takayuki; Abe, Shinya; Takao, Ayumi; Shimizu, Misato; Masuko, Takashi; Nagane, Motoo; Furnari, Frank B; Akiyama, Tetsu; Suematsu, Makoto; Baba, Eishi; Akashi, Koichi; Saya, Hideyuki; Nagano, Osamu

    2016-05-15

    Extracellular free amino acids contribute to the interaction between a tumor and its microenvironment through effects on cellular metabolism and malignant behavior. System xc(-) is composed of xCT and CD98hc subunits and functions as a plasma membrane antiporter for the uptake of extracellular cystine in exchange for intracellular glutamate. Here, we show that the EGFR interacts with xCT and thereby promotes its cell surface expression and function in human glioma cells. EGFR-expressing glioma cells manifested both enhanced antioxidant capacity as a result of increased cystine uptake, as well as increased glutamate, which promotes matrix invasion. Imaging mass spectrometry also revealed that brain tumors formed in mice by human glioma cells stably overexpressing EGFR contained higher levels of reduced glutathione compared with those formed by parental cells. Targeted inhibition of xCT suppressed the EGFR-dependent enhancement of antioxidant capacity in glioma cells, as well as tumor growth and invasiveness. Our findings establish a new functional role for EGFR in promoting the malignant potential of glioma cells through interaction with xCT at the cell surface. Cancer Res; 76(10); 2954-63. ©2016 AACR.

  7. Lack of effect of Theiler's murine encephalomyelitis virus infection on system xc⁻.

    Science.gov (United States)

    Merckx, Ellen; Demuyser, Thomas; Bentea, Eduard; Van Liefferinge, Joeri; Albertini, Giulia; Deneyer, Lauren; Michiels, Thomas; Massie, Ann

    2015-04-23

    Changes in the expression of xCT, the specific subunit of system xc(-) or the cystine/glutamate antiporter, have been associated with several neurological disorders and system xc(-) was recently proposed as a potential target for the development of new treatment strategies for multiple sclerosis (MS). In this study we used Theiler's murine encephalomyelitis virus (TMEV) infection, both in vitro and in vivo, as a model to further evaluate the involvement of system xc(-) in MS. Protein levels of xCT, as well as activity of system xc(-) were unaffected in RAW264.7 macrophages after infection with the demyelinating DA strain of TMEV. Also, protein expression of xCT remained stable in spinal cord and brain of FVB mice 1-2 and 6 weeks after intracranial injection of the DA strain of TMEV. These results demonstrate that TMEV infection of macrophages or FVB mice has no effect on system xc(-) and as such cannot be used as a model to study the involvement of system xc(-) in MS.

  8. A Cytotoxic, Co-operative Interaction Between Energy Deprivation and Glutamate Release From System xc− Mediates Aglycemic Neuronal Cell Death

    Directory of Open Access Journals (Sweden)

    Trista L. Thorn

    2015-11-01

    Full Text Available The astrocyte cystine/glutamate antiporter (system xc− contributes substantially to the excitotoxic neuronal cell death facilitated by glucose deprivation. The purpose of this study was to determine the mechanism by which this occurred. Using pure astrocyte cultures, as well as, mixed cortical cell cultures containing both neurons and astrocytes, we found that neither an enhancement in system xc− expression nor activity underlies the excitotoxic effects of aglycemia. In addition, using three separate bioassays, we demonstrate no change in the ability of glucose-deprived astrocytes—either cultured alone or with neurons—to remove glutamate from the extracellular space. Instead, we demonstrate that glucose-deprived cultures are 2 to 3 times more sensitive to the killing effects of glutamate or N-methyl-D-aspartate when compared with their glucose-containing controls. Hence, our results are consistent with the weak excitotoxic hypothesis such that a bioenergetic deficiency, which is measureable in our mixed but not astrocyte cultures, allows normally innocuous concentrations of glutamate to become excitotoxic. Adding to the burgeoning literature detailing the contribution of astrocytes to neuronal injury, we conclude that under our experimental paradigm, a cytotoxic, co-operative interaction between energy deprivation and glutamate release from astrocyte system xc− mediates aglycemic neuronal cell death.

  9. Comparative Study on Growth Performance of Transgenic (Over-ExpressedOsNHX1) and Wild-Type Nipponbare under Different Salinity Regimes

    Institute of Scientific and Technical Information of China (English)

    Nurul Kahrani ISHAK; Zohrah SULAIMAN; Kushan U TENNAKOON

    2015-01-01

    Transgenic Nipponbarewhich over-expressed a Na+/H+ antiporter geneOsNHX1 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.

  10. Transient removal of alkaline zones after excitation of Chara cells is associated with inactivation of high conductance in the plasmalemma.

    Science.gov (United States)

    Bulychev, Alexander A; Krupenina, Natalia A

    2009-08-01

    The action potential (AP) of excitable plant cells is a multifunctional physiological signal. Its generation in characean algae suppresses the pH banding for 15-30 min and enhances the heterogeneity of spatial distribution of photosynthetic activity. This suppression is largely due to the cessation of H(+) influx (OH(-) efflux) in the alkaline cell regions. Measurements of local pH and membrane conductance in individual space-clamped alkaline zones (small cell areas bathed in an isolated pool of external medium) showed that the AP generation is followed by the transient disappearance of alkaline zone in parallel with a large decrease in membrane conductance. These changes, specific to alkaline zones, were only observed under continuous illumination following a relaxation period of at least 15 min after previous excitation. The excitation of dark-adapted cells produced no conductance changes in the post-excitation period. The results indicate that the origin of alkaline zones in characean cells is not due to operation of electroneutral H(+)/HCO(3)(-) symport or OH(-)/HCO(3)(-) antiport. It is concluded that the membrane excitation is associated with inactivation of plasmalemma high conductance in the alkaline cell regions. PMID:19820298

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

    International Nuclear Information System (INIS)

    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

  12. Molecular characterization of the PR-toxin gene cluster in Penicillium roqueforti and Penicillium chrysogenum: cross talk of secondary metabolite pathways.

    Science.gov (United States)

    Hidalgo, Pedro I; Ullán, Ricardo V; Albillos, Silvia M; Montero, Olimpio; Fernández-Bodega, María Ángeles; García-Estrada, Carlos; Fernández-Aguado, Marta; Martín, Juan-Francisco

    2014-01-01

    The PR-toxin is a potent mycotoxin produced by Penicillium roqueforti in moulded grains and grass silages and may contaminate blue-veined cheese. The PR-toxin derives from the 15 carbon atoms sesquiterpene aristolochene formed by the aristolochene synthase (encoded by ari1). We have cloned and sequenced a four gene cluster that includes the ari1 gene from P. roqueforti. Gene silencing of each of the four genes (named prx1 to prx4) resulted in a reduction of 65-75% in the production of PR-toxin indicating that the four genes encode enzymes involved in PR-toxin biosynthesis. Interestingly the four silenced mutants overproduce large amounts of mycophenolic acid, an antitumor compound formed by an unrelated pathway suggesting a cross-talk of PR-toxin and mycophenolic acid production. An eleven gene cluster that includes the above mentioned four prx genes and a 14-TMS drug/H(+) antiporter was found in the genome of Penicillium chrysogenum. This eleven gene cluster has been reported to be very poorly expressed in a transcriptomic study of P. chrysogenum genes under conditions of penicillin production (strongly aerated cultures). We found that this apparently silent gene cluster is able to produce PR-toxin in P. chrysogenum under static culture conditions on hydrated rice medium. Noteworthily, the production of PR-toxin was 2.6-fold higher in P. chrysogenum npe10, a strain deleted in the 56.8kb amplifiable region containing the pen gene cluster, than in the parental strain Wisconsin 54-1255 providing another example of cross-talk between secondary metabolite pathways in this fungus. A detailed PR-toxin biosynthesis pathway is proposed based on all available evidence.

  13. 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. PMID:3543375

  14. Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

    Science.gov (United States)

    Reynafarje, B; Brand, M D; Lehninger, A L

    1976-12-10

    The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0.

  15. Microelectrode study of intracellular pH in frog skin: dependence on serosal Cl-

    International Nuclear Information System (INIS)

    Replacing external Cl- reduces Na+ transport across frog skin, but the sidedness and mechanisms have been unclear. We have monitored current (l/sub T/), resistance (R/sub T/) and basolateral membrane potential, both with reference micropipettes (psi/sup sc/) and pH-selective microelectrodes (E/sub H//sup sc/), in short-circuited epithelial sheets isolated from frog skins; removal of the dermis facilitates basolateral exchange. Intracellular pH was 7.25 +- 0.03 (mean +- SE) when the pH of the external Cl- Ringer's solution was 7.60 +- 0.01, in reasonable agreement with estimates from 31P and 19F NMR analyses. Complete mucosal replacement of Cl- by gluconate had variable effects on l/sub T/ and R/sub T/. However, serosal Cl- substitution uniformly increased R/sub T/ and markedly decreased l/sub T/, absolute value phi/sup sc/ and absolute value E/sub A//sup sc/. The membrane depolarization was usually preceded by a small hyperpolarization (0.5-3.5 mV). The serosal Cl- replacement also produced an intracellular alkalinization of 0.4 +- 0.1 U. These data suggest that: (1) serosal Cl- substitution alkalinizes the cells by either enhancing HCO- entry or blocking HCO- loss through a basolateral Cl/HCO antiport, and (2) the fall in absolute value phi/sup sc/ and l/sub T/ may partly reflect inhibition of apical Na+ entry, produced indirectly by membrane depolarization resulting from altered basolateral ionic conductances

  16. Chloride dependence of intracellular pH in frog skin: a 31P NMR study

    International Nuclear Information System (INIS)

    Single frog skins from Northern Variety Rana pipiens were analyzed by 31P NMR spectroscopy during superfusion alternately with control and experimental Ringer's solutions, permitting each preparation to serve as its own control. The spectral positions of intracellular inorganic phosphate and extracellular methylphosphonate permitted continuous monitoring of intracellular (pH/sub c/) and extracellular (pH0) pH, respectively. Acute and steady state measurements suggested that pH/sub c/ is well regulated at about 7.29 +- 0.05 over an external pH range of about 7.25-7.65. Below this range, pH/sub c/ decreased markedly when the external pH was reduced with nonvolatile acid. In the presence of 2.5 mM HCO3- and 1% CO2, total substitution of external Cl- by gluconate reversibly increased pH/sub c/ by 0.34 +- 0.05 U (mean +- SE). Replacing external Cl- by SO2-4 increased pH/sub c/ by 0.12 +- 0.01 in the presence of external HCO-3, but only by 0.05 +- 0.02 in its absence. SITS (1 mM) at a pH0 of 6.95 +- 0.05 did not significantly alter pH/sub c/, but entirely prevented the steady state alkalinization characteristically induced by gluconate substitution for external Cl-. The results document that: (1) intracellular pH is maintained relatively constant when the external pH is varied over the physiologic range by adding fixed acid or base, and (2) this regulation is (at least in part) a reflection of Cl/HCO3 antiport activity

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

    Directory of Open Access Journals (Sweden)

    Mathilde Lescat

    Full Text Available 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.

  18. Potentiation of platinum antitumor effects in human lung tumor xenografts by the angiogenesis inhibitor squalamine: effects on tumor neovascularization.

    Science.gov (United States)

    Schiller, J H; Bittner, G

    1999-12-01

    Squalamine is a novel anti-angiogenic aminosterol that is postulated to inhibit neovascularization by selectively inhibiting the sodium-hydrogen antiporter exchanger. To determine how to most effectively use this agent in patients with cancer, we examined the antitumor effects of squalamine with or without cytotoxic agents in human lung cancer xenografts and correlated these observations with the degree of tumor neovascularization. No direct cytotoxic effects of squalamine against tumor cells were observed in vitro with or without cisplatin. Squalamine was effective in inhibiting the establishment of H460 human tumors in BALBc nude mice but was ineffective in inhibiting the growth of H460, CALU-6, or NL20T-A human tumor xenografts when administered i.p. to mice bearing established tumors. However, when combined with cisplatin or carboplatin, squalamine increased tumor growth delay by > or =1.5-fold in the three human lung carcinoma cell lines compared with cisplatin or carboplatin alone. No enhancement of antitumor activity was observed when squalamine was combined with paclitaxel, vinorelbine, gemcitabine, or docetaxel. Repeated cycles of squalamine plus cisplatin administration delayed H460 tumor growth >8.6-fold. Squalamine plus cisplatin reduced CD31 vessel formation by 25% compared with controls, squalamine alone, or cisplatin alone; however, no inhibition in CD31 vessel formation was observed when squalamine was combined with vinorelbine. These data demonstrate that the combination of squalamine and a platinum analog has significant preclinical antitumor activity against human lung cancer that is related to the anti-angiogenic effects of squalamine. PMID:10632372

  19. 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.

  20. 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 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. PMID:22384111

  1. 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.

  2. Arabidopsis thaliana AtUTr7 Encodes a Golgi-Localized UDP-Glucose/UDP-Galactose Transporter that Affects Lateral Root Emergence

    Institute of Scientific and Technical Information of China (English)

    Michael Handford; Cecilia Rodríguez-Furlán; Lorena Marchant; Marcelo Segura; Daniela Gómez; Elena Alvarez-Buyll; Guang-Yan Xiong; Markus Pauly; Ariel Orellana

    2012-01-01

    Nucleotide sugar transporters (NSTs) are antiporters comprising a gene family that plays a fundamental role in the biosynthesis of complex cell wall polysaccharides and glycoproteins in plants.However,due to the limited number of related mutants that have observable phenotypes,the biological function(s) of most NSTs in cell wall biosynthesis and assembly have remained elusive.Here,we report the characterization of AtUTr7 from Arabidopsis (Arabidopsis thaliana (L.) Heynh.),which is homologous to multi-specific UDP-sugar transporters from Drosophila melanogaster,humans,and Caenorhabditis elegans.We show that AtUTr7 possesses the common structural characteristics conserved among NSTs.Using a green fluorescent protein (GFP) tagged version,we demonstrate that AtUTr7 is localized in the Golgi apparatus.We also show that AtUTr7 is widely expressed,especially in the roots and in specific floral organs.Additionally,the results of an in vitro nucleotide sugar transport assay carried out with a tobacco and a yeast expression system suggest that AtUTr7 is capable of transferring UDP-Gal and UDP-GIc,but not a range of other UDP-and GDP-sugars,into the Golgi lumen.Mutants lacking expression of AtUTr7 exhibited an early proliferation of lateral roots as well as distorted root hairs when cultivated at high sucrose concentrations.Furthermore,the distribution of homogalacturonan with a low degree of methyl esterification differed in lateral root tips of the mutant compared to wild-type plants,although additional analytical procedures revealed no further differences in the composition of the root cell walls.This evidence suggests that the transport of UDP-Gal and UDP-GIc into the Golgi under conditions of high root biomass production plays a role in lateral root and root hair development.

  3. Sodium Stress in the Halophyte Thellungiella halophila and Transcriptional Changes in a thsos1-RNA Interference Line

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The plasma membrane Na+/H+-antiporter salt overly sensitive1 (SOS1) from the halophytic Arabidopsis-relative Thellunglella halophila (ThSOS1) shows conserved sequence and domain structure with the orthologous genes from Arabidopsis thaliana and other plants. When expression of ThSOS1 was reduced by RNA interference (RNAi), pronounced characteristics of salt-sensitivity were observed. We were interested in monitoring altered transcriptional responses between Thellungiella wild type and thsos1-4, a representative RNAI line with particular emphasis on root responses to salt stress at 350 mmol/L NaCl, a concentration that is only moderately stressful for mature wild type plants. Transcript profiling revealed several functional categories of genes that were differently affected in wild-type and RNAi plants. Down-regulation of SOS1 resulted In different gene expression even In the absence of stress. The pattern of gene induction in the RNAi plant under salt stress was similar to that of glycophytic Arabidopsis rather than that of wild type Thellungiella. The RNAi plants failed to down-regulate functions that are normally reduced in wild type Thellungiella upon stress and did not up-regulate functions that characterize the Thellungiella salt stress response. Metabolite changes observed in wild type Thellungiella after salt stress were less pronounced or absent in RNAi plants. Transcript and metabolite behavior suggested SOS1 functions including but also extending its established function as a sodium transporter. The down-regulation of ThSOS1 converted the halophyte Thellungiella into a salt-sensitive plant.

  4. 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

  5. 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

  6. Proton Sensing of CLC-0 Mutant E166D

    Science.gov (United States)

    Traverso, Sonia; Zifarelli, Giovanni; Aiello, Rita; Pusch, Michael

    2006-01-01

    CLC Cl− channels are homodimers in which each subunit has a proper pore and a (fast) gate. An additional slow gate acts on both pores. A conserved glutamate (E166 in CLC-0) is a major determinant of gating in CLC-0 and is crucially involved in Cl−/H+ antiport of CLC-ec1, a CLC of known structure. We constructed tandem dimers with one wild-type (WT) and one mutant subunit (E166A or E166D) to show that these mutations of E166 specifically alter the fast gate of the pore to which they belong without effect on the fast gate of the neighboring pore. In addition both mutations activate the common slow gate. E166A pores have a large, voltage-independent open probability of the fast gate (popen), whereas popen of E166D pores is dramatically reduced. Similar to WT, popen of E166D was increased by lowering pHint. At negative voltages, E166D presents a persistent inward current that is blocked by p-chlorophenoxy-acetic acid (CPA) and increased at low pHext. The pHext dependence of the persistent current is analogous to a similar steady inward current in WT CLC-0. Surprisingly, however, the underlying unitary conductance of the persistent current in E166D is about an order of magnitude smaller than that of the transient deactivating inward Cl− current. Collectively, our data support the possibility that the mutated CLC-0 channel E166D can assume two distinct open states. Voltage-independent protonation of D166 from the outside favors a low conductance state, whereas protonation from the inside favors the high conductance state. PMID:16380443

  7. 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.

  8. Chloride channels in stroke

    Institute of Scientific and Technical Information of China (English)

    Ya-ping ZHANG; Hao ZHANG; Dayue Darrel DUAN

    2013-01-01

    Vascular remodeling of cerebral arterioles,including proliferation,migration,and apoptosis of vascular smooth muscle cells (VSMCs),is the major cause of changes in the cross-sectional area and diameter of the arteries and sudden interruption of blood flow or hemorrhage in the brain,ie,stroke.Accumulating evidence strongly supports an important role for chloride (Clˉ) channels in vascular remodeling and stroke.At least three Clˉ channel genes are expressed in VSMCs:1) the TMEM16A (or Ano1),which may encode the calcium-activated Clˉ channels (CACCs); 2) the CLC-3 Clˉ channel and Clˉ/H+ antiporter,which is closely related to the volume-regulated Clˉ channels (VRCCs); and 3) the cystic fibrosis transmembrane conductance regulator (CFTR),which encodes the PKA-and PKC-activated Clˉ channels.Activation of the CACCs by agonist-induced increase in intracellular Ca2+ causes membrane depolarization,vasoconstriction,and inhibition of VSMC proliferation.Activation of VRCCs by cell volume increase or membrane stretch promotes the production of reactive oxygen species,induces proliferation and inhibits apoptosis of VSMCs.Activation of CFTR inhibits oxidative stress and may prevent the development of hypertension.In addition,Clˉ current mediated by gammaaminobutyric acid (GABA) receptor has also been implicated a role in ischemic neuron death.This review focuses on the functional roles of Clˉ channels in the development of stroke and provides a perspective on the future directions for research and the potential to develop Clˉ channels as new targets for the prevention and treatment of stroke.

  9. Protein architecture and core residues in unwound α-helices provide insights to the transport function of plant AtCHX17.

    Science.gov (United States)

    Czerny, Daniel D; Padmanaban, Senthilkumar; Anishkin, Andriy; Venema, Kees; Riaz, Zoya; Sze, Heven

    2016-09-01

    Using Arabidopsis thaliana AtCHX17 as an example, we combine structural modeling and mutagenesis to provide insights on its protein architecture and transport function which is poorly characterized. This approach is based on the observation that protein structures are significantly more conserved in evolution than linear sequences, and mechanistic similarities among diverse transporters are emerging. Two homology models of AtCHX17 were obtained that show a protein fold similar to known structures of bacterial Na(+)/H(+) antiporters, EcNhaA and TtNapA. The distinct secondary and tertiary structure models highlighted residues at positions potentially important for CHX17 activity. Mutagenesis showed that asparagine-N200 and aspartate-D201 inside transmembrane5 (TM5), and lysine-K355 inside TM10 are critical for AtCHX17 activity. We reveal previously unrecognized threonine-T170 and lysine-K383 as key residues at unwound regions in the middle of TM4 and TM11 α-helices, respectively. Mutation of glutamate-E111 located near the membrane surface inhibited AtCHX17 activity, suggesting a role in pH sensing. The long carboxylic tail of unknown purpose has an alternating β-sheet and α-helix secondary structure that is conserved in prokaryote universal stress proteins. These results support the overall architecture of AtCHX17 and identify D201, N200 and novel residues T170 and K383 at the functional core which likely participates in ion recognition, coordination and/or translocation, similar to characterized cation/H(+) exchangers. The core of AtCHX17 models according to EcNhaA and TtNapA templates faces inward and outward, respectively, which may reflect two conformational states of the alternating access transport mode for proteins belonging to the plant CHX family. PMID:27179641

  10. Roles of the sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) on vibrio cholerae metabolism, motility and osmotic stress resistance.

    Science.gov (United States)

    Minato, Yusuke; Fassio, Sara R; Kirkwood, Jay S; Halang, Petra; Quinn, Matthew J; Faulkner, Wyatt J; Aagesen, Alisha M; Steuber, Julia; Stevens, Jan F; Häse, Claudia C

    2014-01-01

    The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology.

  11. Genome Analysis of a New Rhodothermaceae Strain Isolated from a Hot Spring

    Science.gov (United States)

    Goh, Kian Mau; Chan, Kok-Gan; Lim, Soon Wee; Liew, Kok Jun; Chan, Chia Sing; Shamsir, Mohd Shahir; Ee, Robson; Adrian, Tan-Guan-Sheng

    2016-01-01

    A bacterial strain, designated RA, was isolated from water sample of a hot spring on Langkawi Island of Malaysia using marine agar. Strain RA is an aerophilic and thermophilic microorganism that grows optimally at 50–60°C and is capable of growing in marine broth containing 1–10% (w/v) NaCl. 16S rRNA gene sequence analysis demonstrated that this strain is most closely related (<90% sequence identity) to Rhodothermaceae, which currently comprises of six genera: Rhodothermus (two species), Salinibacter (three species), Salisaeta (one species), Rubricoccus (one species), Rubrivirga (one species), and Longimonas (one species). Notably, analysis of average nucleotide identity (ANI) values indicated that strain RA may represent the first member of a novel genus of Rhodothermaceae. The draft genome of strain RA is 4,616,094 bp with 3630 protein-coding gene sequences. Its GC content is 68.3%, which is higher than that of most other genomes of Rhodothermaceae. Strain RA has genes for sulfate permease and arylsulfatase to withstand the high sulfur and sulfate contents of the hot spring. Putative genes encoding proteins involved in adaptation to osmotic stress were identified which encode proteins namely Na+/H+ antiporters, a sodium/solute symporter, a sodium/glutamate symporter, trehalose synthase, malto-oligosyltrehalose synthase, choline-sulfatase, potassium uptake proteins (TrkA and TrkH), osmotically inducible protein C, and the K+ channel histidine kinase KdpD. Furthermore, genome description of strain RA and comparative genome studies in relation to other related genera provide an overview of the uniqueness of this bacterium. PMID:27471502

  12. A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut

    Energy Technology Data Exchange (ETDEWEB)

    Wieczorek, H.; Weerth, S.; Schindlbeck, M.; Klein, U.

    1989-07-05

    Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.

  13. Mycobacterial mutants with defective control of phagosomal acidification.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.

  14. 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.

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Guo-Qiang eWu

    2015-07-01

    Full Text Available 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.

  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

    2014-03-01

    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.

  18. A Thermodynamic Model of Monovalent Cation Homeostasis in the Yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Gerber, Susanne; Fröhlich, Martina; Lichtenberg-Fraté, Hella; Shabala, Sergey; Shabala, Lana; Klipp, Edda

    2016-01-01

    Cationic and heavy metal toxicity is involved in a substantial number of diseases in mammals and crop plants. Therefore, the understanding of tightly regulated transporter activities, as well as conceiving the interplay of regulatory mechanisms, is of substantial interest. A generalized thermodynamic description is developed for the complex interplay of the plasma membrane ion transporters, membrane potential and the consumption of energy for maintaining and restoring specific intracellular cation concentrations. This concept is applied to the homeostasis of cation concentrations in the yeast cells of S. cerevisiae. The thermodynamic approach allows to model passive ion fluxes driven by the electrochemical potential differences, but also primary or secondary active transport processes driven by the inter- play of different ions (symport, antiport) or by ATP consumption (ATPases). The model-confronted with experimental data-reproduces the experimentally observed potassium and proton fluxes induced by the external stimuli KCl and glucose. The estimated phenomenological constants combine kinetic parameters and transport coefficients. These are in good agreement with the biological understanding of the transporters thus providing a better understanding of the control exerted by the coupled fluxes. The model predicts the flux of additional ion species, like e.g. chloride, as a potential candidate for counterbalancing positive charges. Furthermore, the effect of a second KCl stimulus is simulated, predicting a reduced cellular response for cells that were first exposed to a high KCl stimulus compared to cells pretreated with a mild KCl stimulus. By describing the generalized forces that are responsible for a given flow, the model provides information and suggestions for new experiments. Furthermore, it can be extended to other systems such as e.g. Candida albicans, or selected plant cells.

  19. A Thermodynamic Model of Monovalent Cation Homeostasis in the Yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Susanne Gerber

    2016-01-01

    Full Text Available Cationic and heavy metal toxicity is involved in a substantial number of diseases in mammals and crop plants. Therefore, the understanding of tightly regulated transporter activities, as well as conceiving the interplay of regulatory mechanisms, is of substantial interest. A generalized thermodynamic description is developed for the complex interplay of the plasma membrane ion transporters, membrane potential and the consumption of energy for maintaining and restoring specific intracellular cation concentrations. This concept is applied to the homeostasis of cation concentrations in the yeast cells of S. cerevisiae. The thermodynamic approach allows to model passive ion fluxes driven by the electrochemical potential differences, but also primary or secondary active transport processes driven by the inter- play of different ions (symport, antiport or by ATP consumption (ATPases. The model-confronted with experimental data-reproduces the experimentally observed potassium and proton fluxes induced by the external stimuli KCl and glucose. The estimated phenomenological constants combine kinetic parameters and transport coefficients. These are in good agreement with the biological understanding of the transporters thus providing a better understanding of the control exerted by the coupled fluxes. The model predicts the flux of additional ion species, like e.g. chloride, as a potential candidate for counterbalancing positive charges. Furthermore, the effect of a second KCl stimulus is simulated, predicting a reduced cellular response for cells that were first exposed to a high KCl stimulus compared to cells pretreated with a mild KCl stimulus. By describing the generalized forces that are responsible for a given flow, the model provides information and suggestions for new experiments. Furthermore, it can be extended to other systems such as e.g. Candida albicans, or selected plant cells.

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

    Science.gov (United States)

    Spencer, T L; Lehninger, A L

    1976-02-15

    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

  1. Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

    Science.gov (United States)

    Reynafarje, B; Brand, M D; Lehninger, A L

    1976-12-10

    The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0. PMID:12164

  2. A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

    Science.gov (United States)

    Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

    2000-01-01

    The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

  3. Increased abscisic acid levels in transgenic maize overexpressing AtLOS5 mediated root ion fluxes and leaf water status under salt stress.

    Science.gov (United States)

    Zhang, Juan; Yu, Haiyue; Zhang, Yushi; Wang, Yubing; Li, Maoying; Zhang, Jiachang; Duan, Liusheng; Zhang, Mingcai; Li, Zhaohu

    2016-03-01

    Abscisic acid (ABA) is a vital cellular signal in plants, and effective ABA signalling is pivotal for stress tolerance. AtLOS5 encoding molybdenum cofactor sulphurase is a key regulator of ABA biosynthesis. Here, transgenic AtLOS5 plants were generated to explore the role of AtLOS5 in salt tolerance in maize. AtLOS5 overexpression significantly up-regulated the expression of ZmVp14-2, ZmAO, and ZmMOCO, and increased aldehyde oxidase activities, which enhanced ABA accumulation in transgenic plants under salt stress. Concurrently, AtLOS5 overexpression induced the expression of ZmNHX1, ZmCBL4, and ZmCIPK16, and enhanced the root net Na(+) efflux and H(+) influx, but decreased net K(+) efflux, which maintained a high cytosolic K(+)/Na(+) ratio in transgenic plants under salt stress. However, amiloride or sodium orthovanadate could significantly elevate K(+) effluxes and decrease Na(+) efflux and H(+) influx in salt-treated transgenic roots, but the K(+) effluxes were inhibited by TEA, suggesting that ion fluxes regulated by AtLOS5 overexpression were possibly due to activation of Na(+)/H(+) antiport and K(+) channels across the plasma membrane. Moreover, AtLOS5 overexpression could up-regulate the transcripts of ZmPIP1:1, ZmPIP1:5, and ZmPIP2:4, and enhance root hydraulic conductivity. Thus transgenic plants had higher leaf water potential and turgor, which was correlated with greater biomass accumulation under salt stress. Thus AtLOS5 overexpression induced the expression of ABA biosynthetic genes to promote ABA accumulation, which activated ion transporter and PIP aquaporin gene expression to regulate root ion fluxes and water uptake, thus maintaining high cytosolic K(+) and Na(+) homeostasis and better water status in maize exposed to salt stress. PMID:26743432

  4. Growth at high pH and sodium and potassium tolerance in media above the cytoplasmic pH depend on ENA ATPases in Ustilago maydis.

    Science.gov (United States)

    Benito, Begoña; Garciadeblás, Blanca; Pérez-Martín, José; Rodríguez-Navarro, Alonso

    2009-06-01

    Potassium and Na(+) effluxes across the plasma membrane are crucial processes for the ionic homeostasis of cells. In fungal cells, these effluxes are mediated by cation/H(+) antiporters and ENA ATPases. We have cloned and studied the functions of the two ENA ATPases of Ustilago maydis, U. maydis Ena1 (UmEna1) and UmEna2. UmEna1 is a typical K(+) or Na(+) efflux ATPase whose function is indispensable for growth at pH 9.0 and for even modest Na(+) or K(+) tolerances above pH 8.0. UmEna1 locates to the plasma membrane and has the characteristics of the low-Na(+)/K(+)-discrimination ENA ATPases. However, it still protects U. maydis cells in high-Na(+) media because Na(+) showed a low cytoplasmic toxicity. The UmEna2 ATPase is phylogenetically distant from UmEna1 and is located mainly at the endoplasmic reticulum. The function of UmEna2 is not clear, but we found that it shares several similarities with Neurospora crassa ENA2, which suggests that endomembrane ENA ATPases may exist in many fungi. The expression of ena1 and ena2 transcripts in U. maydis was enhanced at high pH and at high K(+) and Na(+) concentrations. We discuss that there are two modes of Na(+) tolerance in fungi: the high-Na(+)-content mode, involving ENA ATPases with low Na(+)/K(+) discrimination, as described here for U. maydis, and the low-Na(+)-content mode, involving Na(+)-specific ENA ATPases, as in Neurospora crassa.

  5. SLC9A3R1 stimulates autophagy via BECN1 stabilization in breast cancer cells.

    Science.gov (United States)

    Liu, Hong; Ma, Yan; He, Hong-Wei; Wang, Jia-Ping; Jiang, Jian-Dong; Shao, Rong-Guang

    2015-01-01

    Autophagy, a self-catabolic process, has been found to be involved in abrogating the proliferation and metastasis of breast cancer. SLC9A3R1 (solute carrier family 9, subfamily A [NHE3, cation proton antiporter 3], member 3 regulator 1), a multifunctional scaffold protein, is involved in suppressing breast cancer cells proliferation and the SLC9A3R1-related signaling pathway regulates the activation of autophagy processes. However, the precise regulatory mechanism and signaling pathway of SLC9A3R1 in the regulation of autophagy processes in breast cancer cells remains unknown. Here, we report that the stability of BECN1, the major component of the autophagic core lipid kinase complex, is augmented in SLC9A3R1-overexpressing breast cancer MDA-MB-231 cells, subsequently stimulating autophagy by attenuating the interaction between BECN1 and BCL2. Initially, we found that SLC9A3R1 partially stimulated autophagy through the PTEN-PI3K-AKT1 signaling cascade in MDA-MB-231 cells. SLC9A3R1 then attenuated the interaction between BECN1 and BCL2 to stimulate the autophagic core lipid kinase complex. Further findings revealed that SLC9A3R1 bound to BECN1 and subsequently blocked ubiquitin-dependent BECN1 degradation. And the deletion of the C-terminal domain of SLC9A3R1 resulted in significantly reduced binding to BECN1. Moreover, the lack of C-terminal of SLC9A3R1 neither reduced the ubiquitination of BECN1 nor induced autophagy in breast cancer cells. The decrease in BECN1 degradation induced by SLC9A3R1 resulted in the activity of autophagy stimulation in breast cancer cells. These findings indicate that the SLC9A3R1-BECN1 signaling pathway participates in the activation of autophagy processes in breast cancer cells.

  6. Components of calcium homeostasis in Archaeon Methanobacterium thermoautotrophicum

    International Nuclear Information System (INIS)

    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 Ca2+ 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 Ca2+ transport in Halobacterium halobium membrane vesicles, the first complex description of processes of Ca2+ homeostasis in Archaea. The Ca2+ influx and efflux was measured using radionuclide 45Ca2+. The experiment were performed under strictly anaerobic conditions. The measurement of the membrane potential by means of 3H-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 Ca2+ homeostasis may be involved in controlling these cellular functions. The results indicate the presence of three components involved in establishing the Ca2+ homeostasis in cell of M. thermoautotrophicum. The first is the Ca2+-carrier mediating the CA2+ influx driven by the proton motive force or the membrane potential. The Ca2+ efflux is mediated by two transport systems, Na+/Ca2+ and H+/Ca2+ anti-porters. The evidence for the presence of the Ca2+-transporting ATPase was not obtained so far. (authors)

  7. 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.

  8. Cadmium uptake and interaction with phytochelatins in wheat protoplasts.

    Science.gov (United States)

    Lindberg, Sylvia; Landberg, Tommy; Greger, Maria

    2007-01-01

    In order to investigate the role of phytochelatins in short-time uptake of Cd(2+) into the cytosol of wheat protoplasts, a new method was applied, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. The uptake of Cd(2+) into protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum, L. cv. Kadett) was lower in protoplasts from seedlings raised in the presence of 1 microM CdCl(2), than in the absence. Presence of CdCl(2) in the cultivation medium increased the content of phytochelatins (PCs) in the protoplasts. When seedlings were raised in the presence of both Cd(2+) and buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, only little PC was found in the protoplasts. Pre-treatment with BSO alone did not affect the content of PC, but inhibited that of GSH. The inhibition of GSH was independent of pre-treatment with Cd(2+). Unidirectional flux analyses, using (109)Cd(2+), showed approximately the same uptake pattern of Cd(2+) as did the fluorescence experiments showing the cytosolic uptake of Cd(2+). Thus, the diminished uptake of Cd(2+) into protoplasts from cadmium-pre-treated plants was not depending on PCs. Instead, it is likely that pre-treatment with Cd(2+) causes a down-regulation of the short-term Cd(2+) uptake, or an up-regulation of the Cd(2+) extrusion. Moreover, since addition of Cd(2+) to protoplasts from control plants caused a cytosol acidification, it is likely that a Cd(2+/)H(+)-antiport mechanism is involved in the extrusion of Cd(2+) from these protoplasts.

  9. Global transcriptional response of the alkali-tolerant cyanobacterium Synechocystis sp. strain PCC 6803 to a pH 10 environment.

    Science.gov (United States)

    Summerfield, Tina C; Sherman, Louis A

    2008-09-01

    Many cyanobacterial strains are able to grow at a pH range from neutral to pH 10 or 11. Such alkaline conditions favor cyanobacterial growth (e.g., bloom formation), and cyanobacteria must have developed strategies to adjust to changes in CO2 concentration and ion availability. Synechocystis sp. strain PCC 6803 exhibits similar photoautotrophic growth characteristics at pH 10 and pH 7.5, and we examined global gene expression following transfer from pH 7.5 to pH 10 to determine cellular adaptations at an elevated pH. The strategies used to develop homeostasis at alkaline pH had elements similar to those of many bacteria, as well as components unique to phototrophic microbes. Some of the response mechanisms previously identified in other bacteria included upregulation of Na+/H+ antiporters, deaminases, and ATP synthase. In addition, upregulated genes encoded transporters with the potential to contribute to osmotic, pH, and ion homeostasis (e.g., a water channel protein, a large-conductance mechanosensitive channel, a putative anion efflux transporter, a hexose/proton symporter, and ABC transporters of unidentified substrates). Transcriptional changes specific to photosynthetic microbes involved NADH dehydrogenases and CO2 fixation. The pH transition altered the CO2/HCO3(-) ratio within the cell, and the upregulation of three inducible bicarbonate transporters (BCT1, SbtA, and NDH-1S) likely reflected a response to this perturbed ratio. Consistent with this was increased transcript abundance of genes encoding carboxysome structural proteins and carbonic anhydrase. Interestingly, the transition to pH 10 resulted in increased abundance of transcripts of photosystem II genes encoding extrinsic and low-molecular-weight polypeptides, although there was little change in photosystem I gene transcripts. PMID:18606800

  10. Unravelling the complete genome sequence of Advenella mimigardefordensis strain DPN7T and novel insights in the catabolism of the xenobiotic polythioester precursor 3,3'-dithiodipropionate.

    Science.gov (United States)

    Wübbeler, Jan Hendrik; Hiessl, Sebastian; Schuldes, Jörg; Thürmer, Andrea; Daniel, Rolf; Steinbüchel, Alexander

    2014-07-01

    Advenella mimigardefordensis strain DPN7(T) is a remarkable betaproteobacterium because of its extraordinary ability to use the synthetic disulfide 3,3'-dithiodipropionic acid (DTDP) as the sole carbon source and electron donor for aerobic growth. One application of DTDP is as a precursor substrate for biotechnically synthesized polythioesters (PTEs), which are interesting non-degradable biopolymers applicable for plastics materials. Metabolic engineering for optimization of PTE production requires an understanding of DTDP conversion. The genome of A. mimigardefordensis strain DPN7(T) was sequenced and annotated. The circular chromosome was found to be composed of 4,740,516 bp and 4112 predicted ORFs, whereas the circular plasmid consisted of 23,610 bp and 24 predicted ORFs. The genes participating in DTDP catabolism had been characterized in detail previously, but knowing the complete genome sequence and with support of Tn5: :mob-induced mutants, putatively involved transporter proteins and a transcriptional regulator were also identified. Most probably, DTDP is transported into the cell by a specific tripartite tricarboxylate transport system and is then cleaved by the disulfide reductase LpdA, sulfoxygenated by the 3-mercaptopropionate dioxygenase Mdo, activated by the CoA ligase SucCD and desulfinated by the acyl-CoA dehydrogenase-like desulfinase AcdA. Regulation of this pathway is presumably performed by a transcriptional regulator of the xenobiotic response element family. The excessive sulfate that is inevitably produced is secreted by the cells by a unique sulfate exporter of the CPA (cation : proton antiporter) superfamily.

  11. 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.

  12. Mutations associated with Dent’s disease affect gating and voltage dependence of the human anion/proton exchanger ClC-5

    Directory of Open Access Journals (Sweden)

    Alexi eAlekov

    2015-05-01

    Full Text Available Dent’s disease is associated with impaired renal endocytosis and endosomal acidification. It is linked to mutations in the membrane chloride/proton exchanger ClC-5, however, a direct link between localization in the protein and functional phenotype of the mutants has not been established until now. Here, two Dent’s disease mutations, G212A and E267A, were investigated using heterologous expression in HEK293T cells, patch-clamp measurements and confocal imaging. WT and, mutant ClC-5 exhibited mixed cell membrane and vesicular distribution. Reduced ion currents were measured for both mutants and both exhibited reduced capability to support endosomal acidification. Functionally, mutation G212A was capable of mediating anion/proton antiport but dramatically shifted the activation of ClC-5 towards more depolarized potentials. The shift can be explained by impeded movements of the neighboring gating glutamate Gluext, a residue that confers major part of the voltage dependence of ClC-5 and serves as a gate at the extracellular entrance of the anion transport pathway. Cell surface abundance of E267A was reduced by ~50% but also dramatically increased gating currents were detected for this mutant and accordingly reduced probability to undergoing cycles associated with electrogenic ion transport. Structurally, the gating alternations correlate to the proximity of E267A to the proton glutamate Gluin that serves as intracellular gate in the proton transport pathway and regulates the open probability of ClC-5. Remarkably, two other mammalian isoforms, ClC-3 and ClC-4, also differ from ClC-5 in gating characteristics affected by the here investigated disease-causing mutations. This evolutionary specialization, together with the functional defects arising from mutations G212A and E267A, demonstrate that the complex gating behavior exhibited by most of the mammalian CLC transporters is an important determinant of their cellular function.

  13. CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp.

    Science.gov (United States)

    Baliardini, Cecilia; Meyer, Claire-Lise; Salis, Pietrino; Saumitou-Laprade, Pierre; Verbruggen, Nathalie

    2015-09-01

    Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium(2+)/hydrogen(+) antiporter, cation/hydrogen(+) exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants. PMID:26162428

  14. CATION EXCHANGER1 Cosegregates with Cadmium Tolerance in the Metal Hyperaccumulator Arabidopsis halleri and Plays a Role in Limiting Oxidative Stress in Arabidopsis Spp.1[OPEN

    Science.gov (United States)

    Baliardini, Cecilia; Meyer, Claire-Lise; Salis, Pietrino; Saumitou-Laprade, Pierre; Verbruggen, Nathalie

    2015-01-01

    Arabidopsis halleri is a model species for the study of plant adaptation to extreme metallic conditions. In this species, cadmium (Cd) tolerance seems to be constitutive, and the mechanisms underlying the trait are still poorly understood. A previous quantitative trait loci (QTL) analysis performed on A. halleri × Arabidopsis lyrata backcross population1 identified the metal-pump gene Heavy Metal ATPase4 as the major genetic determinant for Cd tolerance. However, although necessary, Heavy Metal ATPase4 alone is not sufficient for determining this trait. After fine mapping, a gene encoding a calcium2+/hydrogen+ antiporter, cation/hydrogen+ exchanger1 (CAX1), was identified as a candidate gene for the second QTL of Cd tolerance in A. halleri. Backcross population1 individuals displaying the A. halleri allele for the CAX1 locus exhibited significantly higher CAX1 expression levels compared with the ones with the A. lyrata allele, and a positive correlation between CAX1 expression and Cd tolerance was observed. Here, we show that this QTL is conditional and that it is only detectable at low external Ca concentration. CAX1 expression in both roots and shoots was higher in A. halleri than in the close Cd-sensitive relative species A. lyrata and Arabidopsis thaliana. Moreover, CAX1 loss of function in A. thaliana led to higher Cd sensitivity at low concentration of Ca, higher sensitivity to methylviologen, and stronger accumulation of reactive oxygen species after Cd treatment. Overall, this study identifies a unique genetic determinant of Cd tolerance in the metal hyperaccumulator A. halleri and offers a new twist for the function of CAX1 in plants. PMID:26162428

  15. Complete Sequences of Four Plasmids of Lactococcus lactis subsp. cremoris SK11 Reveal Extensive Adaptation to the Dairy Environment†

    Science.gov (United States)

    Siezen, Roland J.; Renckens, Bernadet; van Swam, Iris; Peters, Sander; van Kranenburg, Richard; Kleerebezem, Michiel; de Vos, Willem M.

    2005-01-01

    Lactococcus lactis strains are known to carry plasmids encoding industrially important traits. L. lactis subsp. cremoris SK11 is widely used by the dairy industry in cheese making. Its complete plasmid complement was sequenced and found to contain the plasmids pSK11A (10,372 bp), pSK11B (13,332 bp), pSK11L (47,165 bp), and pSK11P (75,814 bp). Six highly homologous repB-containing replicons were found, all belonging to the family of lactococcal theta-type replicons. Twenty-three complete insertion sequence elements segment the plasmids into numerous modules, many of which can be identified as functional units or containing functionally related genes. Plasmid-encoded functions previously known to reside on L. lactis SK11 plasmids were now mapped in detail, e.g., lactose utilization (lacR-lacABCDFEGX), the proteolytic system (prtM-prtP, pepO, pepF), and the oligopeptide permease system (oppDFBCA). Newly identified plasmid-encoded functions could facilitate the uptake of various cations, while the pabA and pabB genes could be essential for folate biosynthesis. A competitive advantage could be obtained by using the putative flavin adenine dinucleotide-dependent d-lactate dehydrogenase and oxalate:formate antiporter for enhanced ATP synthesis, while the activity of the predicted α-acetolactate decarboxylase may contribute to the formation of an additional electron sink. Various stress response proteins are plasmid encoded, which could enhance strain robustness. A substantial number of these “adaptation” genes have not been described before on L. lactis plasmids. Moreover, several genes were identified for the first time in L. lactis, possibly reflecting horizontal gene transfer. PMID:16332824

  16. Increased plasma citrulline in mice marks diet-induced obesity and may predict the development of the metabolic syndrome.

    Directory of Open Access Journals (Sweden)

    Manuela Sailer

    Full Text Available 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

  17. Expression of major photosynthetic and salt-resistance genes in invasive reed lineages grown under elevated CO2 and temperature

    Science.gov (United States)

    Eller, Franziska; Lambertini, Carla; Nielsen, Mette W; Radutoiu, Simona; Brix, Hans

    2014-01-01

    It is important to investigate the molecular causes of the variation in ecologically important traits to fully understand phenotypic responses to climate change. In the Mississippi River Delta, two distinct, sympatric invasive lineages of common reed (Phragmites australis) are known to differ in several ecophysiological characteristics and are expected to become more salt resistant due to increasing atmospheric CO2 and temperature. We investigated whether different patterns of gene expression can explain their ecophysiological differences and increased vigor under future climatic conditions. We compared the transcript abundance of photosynthetic genes of the Calvin cycle (Rubisco small subunit, RbcS; Phosphoglycerate kinase, PGK; Phosphoribulokinase, PRK), genes related with salt transport (Na+/H+ antiporter, PhaNHA) and oxidative stress response genes (Manganese Superoxide dismutase, MnSOD; Glutathione peroxidase, GPX), and the total aboveground biomass production between two genotypes representing the two lineages. The two genotypes (Delta-type, Mediterranean lineage, and EU-type, Eurasian lineage) were grown under an ambient and a future climate scenario with simultaneously elevated CO2 and temperature, and under two different soil salinities (0‰ or 20‰). We found neither differences in the aboveground biomass production nor the transcript abundances of the two genotypes, but soil salinity significantly affected all the investigated parameters, often interacting with the climatic conditions. At 20‰ salinity, most genes were higher expressed in the future than in the ambient climatic conditions. Higher transcription of the genes suggests higher abundance of the protein they code for, and consequently increased photosynthate production, improved stress responses, and salt exclusion. Therefore, the higher expression of these genes most likely contributed to the significantly ameliorated salinity impact on the aboveground biomass production of both P

  18. The sensor kinase DcuS of Escherichia coli: two stimulus input sites and a merged signal pathway in the DctA/DcuS sensor unit.

    Science.gov (United States)

    Witan, Julian; Monzel, Christian; Scheu, Patrick D; Unden, Gottfried

    2012-11-01

    The membrane-integral sensor kinase DcuS of Escherichia coli consists of a periplasmically located sensory PAS(P) domain, transmembrane helices TM1 and TM2, a cytoplasmic PAS(C) domain and the kinase domain. Stimulus (C(4)-dicarboxylate) binding at PAS(P) is required to stimulate phosphorylation of the kinase domain, resulting in phosphoryl transfer to the response regulator DcuR. PAS(C) functions as a signaling device or a relay in signal transfer from TM2 to the kinase. Phosphorylated DcuR induces the expression of the target genes. Sensing by DcuS requires the presence of the C(4)-dicarboxylate transporter DctA during aerobic growth. DctA forms a sensor unit with DcuS, and a short C-terminal sequence of DctA forming the putative helix 8b is required for interaction with DcuS. Helix 8b contains a LDXXXLXXXL motif that is essential for function and interaction. DcuS requires the PAS(C) domain for signal perception from DctA. Thus, DcuS and DctA form a DctA/DcuS sensory unit, and DcuS perceives stimuli from two different sites (PAS(P) and DctA). The signal transfer pathways are supposed to merge at PAS(C). The fumarate/succinate antiporter DcuB takes over the role as a co-sensor of DcuS under anaerobic growth conditions. PMID:23109544

  19. Suggested guidelines for the diagnosis and management of urea cycle disorders

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    Häberle Johannes

    2012-05-01

    Full Text Available Abstract Urea cycle disorders (UCDs are inborn errors of ammonia detoxification/arginine synthesis due to defects affecting the catalysts of the Krebs-Henseleit cycle (five core enzymes, one activating enzyme and one mitochondrial ornithine/citrulline antiporter with an estimated incidence of 1:8.000. Patients present with hyperammonemia either shortly after birth (~50% or, later at any age, leading to death or to severe neurological handicap in many survivors. Despite the existence of effective therapy with alternative pathway therapy and liver transplantation, outcomes remain poor. This may be related to underrecognition and delayed diagnosis due to the nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity. These guidelines aim at providing a trans-European consensus to: guide practitioners, set standards of care and help awareness campaigns. To achieve these goals, the guidelines were developed using a Delphi methodology, by having professionals on UCDs across seven European countries to gather all the existing evidence, score it according to the SIGN evidence level system and draw a series of statements supported by an associated level of evidence. The guidelines were revised by external specialist consultants, unrelated authorities in the field of UCDs and practicing pediatricians in training. Although the evidence degree did hardly ever exceed level C (evidence from non-analytical studies like case reports and series, it was sufficient to guide practice on both acute and chronic presentations, address diagnosis, management, monitoring, outcomes, and psychosocial and ethical issues. Also, it identified knowledge voids that must be filled by future research. We believe these guidelines will help to: harmonise practice, set common standards and spread good practices with a positive impact on the outcomes of UCD patients.

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

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    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.

  1. Rapid hyperosmotic-induced Ca2+ responses in Arabidopsis thaliana exhibit sensory potentiation and involvement of plastidial KEA transporters.

    Science.gov (United States)

    Stephan, Aaron B; Kunz, Hans-Henning; Yang, Eric; Schroeder, Julian I

    2016-08-30

    Plants experience hyperosmotic stress when faced with saline soils and possibly with drought stress, but it is currently unclear how plant roots perceive this stress in an environment of dynamic water availabilities. Hyperosmotic stress induces a rapid rise in intracellular Ca(2+) concentrations ([Ca(2+)]i) in plants, and this Ca(2+) response may reflect the activities of osmo-sensory components. Here, we find in the reference plant Arabidopsis thaliana that the rapid hyperosmotic-induced Ca(2+) response exhibited enhanced response magnitudes after preexposure to an intermediate hyperosmotic stress. We term this phenomenon "osmo-sensory potentiation." The initial sensing and potentiation occurred in intact plants as well as in roots. Having established a quantitative understanding of wild-type responses, we investigated effects of pharmacological inhibitors and candidate channel/transporter mutants. Quintuple mechano-sensitive channels of small conductance-like (MSL) plasma membrane-targeted channel mutants as well as double mid1-complementing activity (MCA) channel mutants did not affect the response. Interestingly, however, double mutations in the plastid K(+) exchange antiporter (KEA) transporters kea1kea2 and a single mutation that does not visibly affect chloroplast structure, kea3, impaired the rapid hyperosmotic-induced Ca(2+) responses. These mutations did not significantly affect sensory potentiation of the response. These findings suggest that plastids may play an important role in early steps mediating the response to hyperosmotic stimuli. Together, these findings demonstrate that the plant osmo-sensory components necessary to generate rapid osmotic-induced Ca(2+) responses remain responsive under varying osmolarities, endowing plants with the ability to perceive the dynamic intensities of water limitation imposed by osmotic stress. PMID:27528686

  2. Role for Na/sup +/, H/sup +/, and Ca/sup 2 +/ during (/sup 3/H)-serotonin release from rat basophilic leukemia cells

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    Stump, R.F.; Oliver, J.M.; Deanin, G.G.

    1986-03-05

    The authors have investigated the roles of Na/sup +/, pH, and Ca/sup 2 +/ in the release of (/sup 3/H)-serotonin from RBL-2H3 cells. The importance of extracellular Ca/sup 2 +/ for antigen-induced mediator release is well known. The authors report that mediator release also depends on extracellular Na/sup +/ and that the Na/sup +/ ionophore, monensin, like the Ca/sup 2 +/ ionophores A23187 and ionomycin, mimics antigen in causing release. Amiloride suppresses serotonin release, indicating that antigen activates the Na/sup +//H/sup +/ antiport. Antigen-stimulated Na/sup +//H/sup +/ exchange (and/or the resulting cytoplasmic alkalinization) may affect mediator release in part by controlling cytoplasmic free Ca/sup 2 +/ levels. The authors report that antigen normally causes a spike followed by a plateau of Ca/sup 2 +/-Quin 2 fluorescence. Only the spike occurs when cells are incubated with antigen in low Na/sup +/ medium. Conversely, monensin produces a Ca/sup 2 +/ plateau without a spike phase. In addition, cytoplasmic alkalinization due to increased Na/sup +//H/sup +/ exchange may directly cause secretion. Both NH/sub 4/Cl and monensin cause mediator release in Ca/sup 2 +/-free medium: these reagents increase pH by about 0.1 units as measured by the fluorescent dye, BCECF. TPA that stimulates Na/sup +//H/sup +/ exchange in other cells does not cause release directly but it potentiates both antigen and Ca/sup 2 +/ ionophore-induced release in RBL-2h3 cells. This further suggests synergistic roles for Na/sup +//H/sup +/ exchange and Ca/sup 2 +/ mobilization in the control of mediator release.

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

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    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.

  4. Mode of action and resistance studies unveil new roles for tropodithietic acid as an anticancer agent and the γ-glutamyl cycle as a proton sink.

    Science.gov (United States)

    Wilson, Maxwell Z; Wang, Rurun; Gitai, Zemer; Seyedsayamdost, Mohammad R

    2016-02-01

    While we have come to appreciate the architectural complexity of microbially synthesized secondary metabolites, far less attention has been paid to linking their structural features with possible modes of action. This is certainly the case with tropodithietic acid (TDA), a broad-spectrum antibiotic generated by marine bacteria that engage in dynamic symbioses with microscopic algae. TDA promotes algal health by killing unwanted marine pathogens; however, its mode of action (MoA) and significance for the survival of an algal-bacterial miniecosystem remains unknown. Using cytological profiling, we herein determine the MoA of TDA and surprisingly find that it acts by a mechanism similar to polyether antibiotics, which are structurally highly divergent. We show that like polyether drugs, TDA collapses the proton motive force by a proton antiport mechanism, in which extracellular protons are exchanged for cytoplasmic cations. The α-carboxy-tropone substructure is ideal for this purpose as the proton can be carried on the carboxyl group, whereas the basicity of the tropylium ion facilitates cation export. Based on similarities to polyether anticancer agents we have further examined TDA's cytotoxicity and find it to exhibit potent, broad-spectrum anticancer activities. These results highlight the power of MoA-profiling technologies in repurposing old drugs for new targets. In addition, we identify an operon that confers TDA resistance to the producing marine bacteria. Bioinformatic and biochemical analyses of these genes lead to a previously unknown metabolic link between TDA/acid resistance and the γ-glutamyl cycle. The implications of this resistance mechanism in the context of the algal-bacterial symbiosis are discussed. PMID:26802120

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

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    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.

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

    Science.gov (United States)

    Schut, Gerrit J.; Lipscomb, Gina L.; Nguyen, Diep M. N.; Kelly, Robert M.; Adams, Michael W. W.

    2016-01-01

    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 CO 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. PMID:26858706

  7. Absence of system xc- in mice decreases anxiety and depressive-like behavior without affecting sensorimotor function or spatial vision.

    Science.gov (United States)

    Bentea, Eduard; Demuyser, Thomas; Van Liefferinge, Joeri; Albertini, Giulia; Deneyer, Lauren; Nys, Julie; Merckx, Ellen; Michotte, Yvette; Sato, Hideyo; Arckens, Lutgarde; Massie, Ann; Smolders, Ilse

    2015-06-01

    There is considerable preclinical and clinical evidence indicating that abnormal changes in glutamatergic signaling underlie the development of mood disorders. Astrocytic glutamate dysfunction, in particular, has been recently linked with the pathogenesis and treatment of mood disorders, including anxiety and depression. System xc- is a glial cystine/glutamate antiporter that is responsible for nonvesicular glutamate release in various regions of the brain. Although system xc- is involved in glutamate signal transduction, its possible role in mediating anxiety or depressive-like behaviors is currently unknown. In the present study, we phenotyped adult and aged system xc- deficient mice in a battery of tests for anxiety and depressive-like behavior (open field, light/dark test, elevated plus maze, novelty suppressed feeding, forced swim test, tail suspension test). Concomitantly, we evaluated the sensorimotor function of system xc- deficient mice, using motor and sensorimotor based tests (rotarod, adhesive removal test, nest building test). Finally, due to the presence and potential functional relevance of system xc- in the eye, we investigated the visual acuity of system xc- deficient mice (optomotor test). Our results indicate that loss of system xc- does not affect motor or sensorimotor function, in either adult or aged mice, in any of the paradigms investigated. Similarly, loss of system xc- does not affect basic visual acuity, in either adult or aged mice. On the other hand, in the open field and light/dark tests, and forced swim and tail suspension tests respectively, we could observe significant anxiolytic and antidepressive-like effects in system xc- deficient mice that in certain cases (light/dark, forced swim) were age-dependent. These findings indicate that, under physiological conditions, nonvesicular glutamate release via system xc- mediates aspects of higher brain function related to anxiety and depression, but does not influence sensorimotor function

  8. Novel di-aryl-substituted isoxazoles act as noncompetitive inhibitors of the system Xc(-) cystine/glutamate exchanger.

    Science.gov (United States)

    Newell, J L; Keyari, C M; McDaniel, S W; Diaz, P J; Natale, N R; Patel, S A; Bridges, R J

    2014-07-01

    The system xc(-) antiporter is a plasma membrane transporter that mediates the exchange of extracellular l-cystine with intracellular l-glutamate. This exchange is significant within the context of the CNS because the import of l-cystine is required for the synthesis of the antioxidant glutathione, while the efflux of l-glutamate has the potential to contribute to either excitatory signaling or excitotoxic pathology. Changes in the activity of the transport system have been linked to the underlying pathological mechanisms of a variety of CNS disorders, one of the most prominent of which is its highly enriched expression in glial brain tumors. In an effort to produce more potent system xc(-) blockers, we have been using amino-3-carboxy-5-methylisoxazole propionic acid (ACPA) as a scaffold for inhibitor development. We previously demonstrated that the addition of lipophilic aryl groups to either the #4 or #5 position on the isoxazole ring markedly increased the inhibitory activity at system xc(-). In the present work a novel series of analogues has been prepared in which aryl groups have been introduced at both the #4 and #5 positions. In contrast to the competitive action of the mono-substituted analogues, kinetic analyses indicate that the di-substituted isoxazoles block system xc(-)-mediated uptake of (3)H-l-glutamate into SNB-19 cells by a noncompetitive mechanism. These new analogues appear to be the first noncompetitive inhibitors identified for this transport system, as well as being among the most potent blockers identified to date. These diaryl-isoxazoles should be of value in assessing the physiological roles and molecular pharmacology of system xc(-).

  9. Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed.

    Science.gov (United States)

    Van Liefferinge, Joeri; Bentea, Eduard; Demuyser, Thomas; Albertini, Giulia; Follin-Arbelet, Virginie; Holmseth, Silvia; Merckx, Ellen; Sato, Hideyo; Aerts, Joeri L; Smolders, Ilse; Arckens, Lutgarde; Danbolt, Niels C; Massie, Ann

    2016-04-01

    The cystine/glutamate antiporter or system Xc- exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system Xc- in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti-xCT (the specific subunit of system Xc-) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT(-/-)) mice as negative controls, we show that xCT migrates as a 35-kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in-house-developed anti-xCT antibodies by comparing their immunoreactivity in brain tissue of xCT(+/+) and xCT(-/-) mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti-xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in-house-developed antibodies could reveal specific xCT labeling and exclusively on acetone-postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges.

  10. Functional upregulation of system xc- by fibroblast growth factor-2.

    Science.gov (United States)

    Liu, Xiaoqian; Resch, Jon; Rush, Travis; Lobner, Doug

    2012-02-01

    The cystine/glutamate antiporter (system xc-) is a Na(+)-independent amino acid transport system. Disruption of this system may lead to multiple effects in the CNS including decreased cellular glutathione. Since multiple neurological diseases involve glutathione depletion, and disruption of growth factor signaling has also been implicated in these diseases, it is possible that some growth factors effects are mediated by regulation of system xc-. We tested the growth factors fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), neuregulin-1 (NRG), neurotrophin-4 (NT-4), and brain derived neurotrophic factor (BDNF) on system xc- mediated 14C-cystine uptake in mixed neuronal and glial cortical cultures. Only FGF-2 significantly increased cystine uptake. The effect was observed in astrocyte-enriched cultures, but not in cultures of neurons or microglia. The increase was blocked by the system xc- inhibitor (s)-4-carboxyphenylglycine, required at least 12 h FGF-2 treatment, and was prevented by the protein synthesis inhibitor cycloheximide. Kinetic analysis indicated FGF-2 treatment increased the V(max) for cystine uptake while the K(m) remained the same. Quantitative PCR showed an increase in mRNA for xCT, the functional subunit of system xc-, beginning at 3 h of FGF-2 treatment, with a dramatic increase after 12 h. Blocking FGFR1 with PD 166866 blocked the FGF-2 effect. Treatment with a PI3-kinase inhibitor (LY-294002) or a MEK/ERK inhibitor (U0126) for 1 h prior to and during the FGF-2 treatment, each partially blocked the increased cystine uptake. The upregulation of system xc- by FGF-2 may be responsible for some of the known physiological actions of FGF-2. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

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

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    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.

  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.

  13. Non-cell autonomous influence of the astrocyte system xc− on hypoglycaemic neuronal cell death

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    Sandra J Hewett

    2012-02-01

    Full Text Available Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation is initiated by glutamate extruded from astrocytes via system xc− – an amino acid transporter that imports l-cystine and exports l-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents – whereas addition of l-cystine restores – GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc−. Indeed, drugs known to inhibit system xc− ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11 that encodes the substrate-specific light chain of system xc− (xCT. Finally, enhancement of astrocytic system xc− expression and function via IL-1β (interleukin-1β exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc− inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc−, have a direct, non-cell autonomous effect on cortical neuron survival.

  14. Expression of xCT and activity of system xc(-) are regulated by NRF2 in human breast cancer cells in response to oxidative stress.

    Science.gov (United States)

    Habib, Eric; Linher-Melville, Katja; Lin, Han-Xin; Singh, Gurmit

    2015-08-01

    Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc(-), an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc(-), is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc(-). We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.

  15. Non-cell autonomous influence of the astrocyte system xc- on hypoglycaemic neuronal cell death.

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    Jackman, Nicole A; Melchior, Shannon E; Hewett, James A; Hewett, Sandra J

    2012-02-08

    Despite longstanding evidence that hypoglycaemic neuronal injury is mediated by glutamate excitotoxicity, the cellular and molecular mechanisms involved remain incompletely defined. Here, we demonstrate that the excitotoxic neuronal death that follows GD (glucose deprivation) is initiated by glutamate extruded from astrocytes via system xc---an amino acid transporter that imports L-cystine and exports L-glutamate. Specifically, we find that depriving mixed cortical cell cultures of glucose for up to 8 h injures neurons, but not astrocytes. Neuronal death is prevented by ionotropic glutamate receptor antagonism and is partially sensitive to tetanus toxin. Removal of amino acids during the deprivation period prevents--whereas addition of L-cystine restores--GD-induced neuronal death, implicating the cystine/glutamate antiporter, system xc-. Indeed, drugs known to inhibit system xc- ameliorate GD-induced neuronal death. Further, a dramatic reduction in neuronal death is observed in chimaeric cultures consisting of neurons derived from WT (wild-type) mice plated on top of astrocytes derived from sut mice, which harbour a naturally occurring null mutation in the gene (Slc7a11) that encodes the substrate-specific light chain of system xc- (xCT). Finally, enhancement of astrocytic system xc- expression and function via IL-1β (interleukin-1β) exposure potentiates hypoglycaemic neuronal death, the process of which is prevented by removal of l-cystine and/or addition of system xc- inhibitors. Thus, under the conditions of GD, our studies demonstrate that astrocytes, via system xc-, have a direct, non-cell autonomous effect on cortical neuron survival.

  16. Interleukin 1β Regulation of the System xc- Substrate-specific Subunit, xCT, in Primary Mouse Astrocytes Involves the RNA-binding Protein HuR.

    Science.gov (United States)

    Shi, Jingxue; He, Yan; Hewett, Sandra J; Hewett, James A

    2016-01-22

    System xc(-) is a heteromeric amino acid cystine/glutamate antiporter that is constitutively expressed by cells of the CNS, where it functions in the maintenance of intracellular glutathione and extracellular glutamate levels. We recently determined that the cytokine, IL-1β, increases the activity of system xc(-) in CNS astrocytes secondary to an up-regulation of its substrate-specific light chain, xCT, and that this occurs, in part, at the level of transcription. However, an in silico analysis of the murine xCT 3'-UTR identified numerous copies of adenine- and uridine-rich elements, raising the possibility that undefined trans-acting factors governing mRNA stability and translation may also contribute to xCT expression. Here we show that IL-1β increases the level of mRNA encoding xCT in primary cultures of astrocytes isolated from mouse cortex in association with an increase in xCT mRNA half-life. Additionally, IL-1β induces HuR translocation from the nucleus to the cytoplasm. RNA immunoprecipitation analysis reveals that HuR binds directly to the 3'-UTR of xCT in an IL-1β-dependent manner. Knockdown of endogenous HuR protein abrogates the IL-1β-mediated increase in xCT mRNA half-life, whereas overexpression of HuR in unstimulated primary mouse astrocytes doubles the half-life of constitutive xCT mRNA. This latter effect is accompanied by an increase in xCT protein levels, as well as a functional increase in system xc(-) activity. Altogether, these data support a critical role for HuR in mediating the IL-1β-induced stabilization of astrocyte xCT mRNA.

  17. FGF-2 induces neuronal death through upregulation of system xc-.

    Science.gov (United States)

    Liu, Xiaoqian; Albano, Rebecca; Lobner, Doug

    2014-02-14

    The cystine/glutamate antiporter (system xc-) transports cystine into cell in exchange for glutamate. Fibroblast growth factor-2 (FGF-2) upregulates system xc- selectively on astrocytes, which leads to increased cystine uptake, the substrate for glutathione production, and increased glutamate release. While increased intracellular glutathione can limit oxidative stress, the increased glutamate release can potentially lead to excitotoxicity to neurons. To test this hypothesis, mixed neuronal and glial cortical cultures were treated with FGF-2. Treatment with FGF-2 for 48 h caused a significant neuronal death in these cultures. Cell death was not observed in neuronal-enriched cultures, or astrocyte-enriched cultures, suggesting the toxicity was the result of neuron-glia interaction. Blocking system xc- eliminated the neuronal death as did the AMPA/kainate receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX), but not the NMDA receptor antagonist memantine. When cultures were exposed directly to glutamate, both NBQX and memantine blocked the neuronal toxicity. The mechanism of this altered profile of glutamate receptor mediated toxicity by FGF-2 is unclear. The selective calcium permeable AMPA receptor antagonist 1-naphthyl acetyl spermine (NASPM) failed to offer protection. The most likely explanation for the results is that 48 h FGF-2 treatment induces AMPA/kainate receptor toxicity through increased system xc- function resulting in increased release of glutamate. At the same time, FGF-2 alters the sensitivity of the neurons to glutamate toxicity in a manner that promotes selective AMPA/kainate receptor mediated toxicity.

  18. High Throughput Sequencing of Small RNAs in the Two Cucurbita Germplasm with Different Sodium Accumulation Patterns Identifies Novel MicroRNAs Involved in Salt Stress Response.

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    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

  19. 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

  20. The SbSOS1 gene from the extreme halophyte Salicornia brachiata enhances Na+ loading in xylem and confers salt tolerance in transgenic tobacco

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    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

  1. Estradiol-17beta-BSA stimulates Ca(2+) uptake through nongenomic pathways in primary rabbit kidney proximal tubule cells: involvement of cAMP and PKC.

    Science.gov (United States)

    Han, H J; Lee, Y H; Park, S H

    2000-04-01

    The effect of estradiol-17beta-BSA (E(2)-BSA) on Ca(2+) uptake and its related signal pathways were examined in the primary cultured rabbit kidney proximal tubule cells. E(2)-BSA (10(-9) M) significantly stimulated Ca(2+) uptake from 2 h by 13% and at 8 h by 35% as compared to control, respectively. This stimulatory effect of E(2)-BSA was not inhibited by tamoxifen (10(-8) M, an intracellular estrogen receptor antagonist), actinomycin D (10(-7) M, a transcription inhibitor), and cycloheximide (4 x 10(-5) M, a protein synthesis inhibitor). However, E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by methoxyverapamil (10(-6) M, an L-type calcium channel blocker) and 5-(N-ethyl-N-isopropyl)-amiloride (10(-5) M, a Na(+)/H(+) antiporter blocker). These results suggest that E(2)-BSA stimulates Ca(2+) uptake through nongenomic pathways. Thus, we investigated which signal pathways were related to E(2)-BSA-induced stimulation of Ca(2+) uptake. 8-Br-cAMP (10(-6) M) alone increased Ca(2+) uptake by 22% compared to control. When E(2)-BSA combined with 8-Br-cAMP, Ca(2+) uptake was not significantly stimulated compared to E(2)-BSA. SQ 22536 (10(-6) M, an adenylate cyclase inhibitor) and myristoylated protein kinase A inhibitor amide 14-22 (10(-6) M, a protein kinase A inhibitor) blocked E(2)-BSA-induced stimulation of Ca(2+) uptake and E(2)-BSA also increased cAMP generation by 26% of that of control. In addition, TPA (0.02 ng/ml, an artificial PKC promoter) stimulated the Ca(2+) uptake by 14%, and the cotreatment of TPA and E(2)-BSA did not significantly stimulate Ca(2+) uptake compared to E(2)-BSA. E(2)-BSA-induced stimulation of Ca(2+) uptake was blocked by U 73122 (10(-6) M, a phospholipase C inhibitor) or bisindolylmaleimide I (10(-6) M, a protein kinase C inhibitor). Indeed, E(2)-BSA stimulated PKC activity by 26%. In conclusion, E(2)-BSA (10(-9) M) stimulated Ca(2+) uptake by nongenomic action, which is mediated by cAMP and PKC pathways.

  2. Complete genome sequence of Francisella tularensis subspecies holarctica FTNF002-00.

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    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.

  3. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

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    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

  4. Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria - An amazing defence tool against hyperosmotic stress

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    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

  5. Genetically modified plants for salinity stress tolerance (abstract)

    International Nuclear Information System (INIS)

    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

  6. The electrochemical transmission in I-Band segments of the mitochondrial reticulum.

    Science.gov (United States)

    Patel, Keval D; Glancy, Brian; Balaban, Robert S

    2016-08-01

    propose that the abundant cation-proton antiporter in skeletal muscle mitochondria operates in opposite directions in the IBS and PS to permit local recycling of H(+) at each site driven by cooperative gradients in H(+) and Na(+)/K(+) which favor H(+) entry in the PS and H(+) efflux in the IBS. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016,' edited by Prof. Paolo Bernardi. PMID:26921810

  7. Molecular pharmacology of kidney and inner ear CLC-K chloride channels

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    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

  8. Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. II. Changes in Na/sup +/ and Ca/sup 2 +/ fluxes, Na/sup +//K/sup +/ pump activity, and intracellular pH

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

    1986-06-01

    The amphibian tetradecapeptide, bombesin, and structurally related peptides caused a marked increase in ouabain-sensitive /sup 86/Rb/sup +/ uptake (a measure of Na/sup +//K/sup +/ 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/sup +/ entry into the cells. The effect of bombesin on Na/sup +/ entry and Na/sup +//K/sup +/ 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 /sup 86/Rb/sup +/ uptake; the relative potencies of these peptides in stimulating the Na/sup +//K/sup +/ pump were comparable to their potencies in increasing DNA synthesis. Bombesin increased Na/sup +/ influx, at least in part, through an Na/sup +//H/sup +/ antiport. The peptide augmented intracellular pH and this effect was abolished in the absence of extracellular Na/sup +/. In addition to monovalent ion transport, bombesin and the structurally related peptides rapidly increased the efflux of /sup 45/Ca/sup 2 +/ from quiescent Swiss 3T3 cells. This Ca/sup 2 +/ came from an intracellular pool and the efflux was associated with a 50% decrease in total intracellular Ca/sup 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 /sup 86/Rb/sup +/ uptake and Na/sup +/ 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.

  9. Involvement of Calmodulin and Calmodulin-like Proteins in Plant Responses to Abiotic Stresses

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    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. System xC- is a mediator of microglial function and its deletion slows symptoms in amyotrophic lateral sclerosis mice.

    Science.gov (United States)

    Mesci, Pinar; Zaïdi, Sakina; Lobsiger, Christian S; Millecamps, Stéphanie; Escartin, Carole; Seilhean, Danielle; Sato, Hideyo; Mallat, Michel; Boillée, Séverine

    2015-01-01

    Amyotrophic lateral sclerosis is the most common adult-onset motor neuron disease and evidence from mice expressing amyotrophic lateral sclerosis-causing SOD1 mutations suggest that neurodegeneration is a non-cell autonomous process where microglial cells influence disease progression. However, microglial-derived neurotoxic factors still remain largely unidentified in amyotrophic lateral sclerosis. With excitotoxicity being a major mechanism proposed to cause motor neuron death in amyotrophic lateral sclerosis, our hypothesis was that excessive glutamate release by activated microglia through their system [Formula: see text] (a cystine/glutamate antiporter with the specific subunit xCT/Slc7a11) could contribute to neurodegeneration. Here we show that xCT expression is enriched in microglia compared to total mouse spinal cord and absent from motor neurons. Activated microglia induced xCT expression and during disease, xCT levels were increased in both spinal cord and isolated microglia from mutant SOD1 amyotrophic lateral sclerosis mice. Expression of xCT was also detectable in spinal cord post-mortem tissues of patients with amyotrophic lateral sclerosis and correlated with increased inflammation. Genetic deletion of xCT in mice demonstrated that activated microglia released glutamate mainly through system [Formula: see text]. Interestingly, xCT deletion also led to decreased production of specific microglial pro-inflammatory/neurotoxic factors including nitric oxide, TNFa and IL6, whereas expression of anti-inflammatory/neuroprotective markers such as Ym1/Chil3 were increased, indicating that xCT regulates microglial functions. In amyotrophic lateral sclerosis mice, xCT deletion surprisingly led to earlier symptom onset but, importantly, this was followed by a significantly slowed progressive disease phase, which resulted in more surviving motor neurons. These results are consistent with a deleterious contribution of microglial-derived glutamate during symptomatic

  11. 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.

  12. 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

  13. Reduction of intracellular pH by tenidap. Involvement of cellular anion transporters in the pH change.

    Science.gov (United States)

    McNiff, P; Robinson, R P; Gabel, C A

    1995-10-26

    Tenidap [5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H- indole-1-carboxamide], a novel antirheumatic agent, produces a rapid and sustained intracellular acidification when applied to cells in culture. To investigate the mechanism by which this change in ionic homeostasis is achieved, the acidification activities of structural analogs of tenidap were determined, and the movements of [14C]tenidap into and out of cells were explored. The acidification activity of tenidap was enhanced by lowering extracellular pH, suggesting that the free acid species was required for this process. Consistent with this requirement, a non-acidic analog of tenidap did not produce a change in intracellular pH (pHi). In contrast, multihalogenated derivatives of tenidap produced greater changes in pHi than did tenidap, and one analog produced a transient acidification from which the cell recovered; this recovery, however, was blocked by an inhibitor of the Na+/H+ antiporter. Fibroblasts incubated with [14C]tenidap achieved within 5 min a level of cell-associated drug that remained constant during longer incubations. Simultaneous addition of the electrogenic ionophore valinomycin or the P-glycoprotein inhibitor 4-(3,4-dihydro-6,7-dimethoxy-2(1H)-isoquinolinyl)-N-[2-(3,4-dimethoxyphe nyl) ethyl]-6,7-dimethoxy-2-quinazolinamine (CP-100,356) caused a time- and concentration-dependent increase in the level of cell-associated [14C]tenidap; other agents tested did not promote this enhanced cellular accumulation. [14C]Tenidap accumulated by fibroblasts in the presence of CP-100,356 subsequently was released when these cells were placed in a tenidap- and CP-100,356-free medium. Importantly, several agents that are known to inhibit anion transport processes, including alpha-cyano-beta-(1-phenylindol-3-yl) acrylate, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and meclofenamic acid, inhibited efflux of [14C]tenidap. In contrast, ethacrynic acid and 4,4'-diisothiocyanatostilbene-2

  14. 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

  15. Phylogenetic Evidence for H2 based Electron Bifurcation In Early Life

    Science.gov (United States)

    Adams, M. W.; Boyd, E. S.; Schut, G.; Peters, J.

    2012-12-01

    Energy conservation is a fundamental underpinning of all life and is accomplished by electron transport phosphorylation and/or substrate level phosphorylation. A third mechanism, known as flavin-based electron bifurcation, has recently been established as a mechanism by which life can conserve energy. In this mechanism, a flavin-containing multienzyme complex catalyzes the thermodynamically unfavorable reduction of low potential ferredoxin using electron donors with higher potentials, such as NAD(P)H or H2. Such endergonic reactions are driven forward through the simultaneous oxidation of the electron donor with higher potential acceptors such as NAD+ or heterodisulfide. Membrane associated energy converting [NiFe]hydrogenases (Ech, Eha) link the oxidation of ferredoxin with the production of H2 and in the process conserve energy in the form of an ion (Na+/H+) gradient. Ech/Eha exhibit a modular composition represented by a Na+/H+ antiporter domain and a [NiFe] hydrogenase domain. In addition, Ech/Eha can be accompanied by a formate dehyrogenase, carbon monoxide dehydrogenase, or an FAD/NAD(P)H module that enables coupling with these substrates. Representatives of Ech/Eha have been identified among anaerobic Archaea and Bacteria, including deeply rooted methanogens, sulfur-reducing Crenarcheota/Euryarchaeota as well as Thermotogae. Ech exhibit extensive homology to a number of subunits within the NADH quinone oxidoreductase or complex I family (Nuo, Fpo). Metabolically, Ech generally couple the oxidation of carbon monooxide, formate or ferredoxin to the production of H2. In contrast, the Eha complex couples the translocation of Na+ and the oxidation of H2 to the reduction of ferredoxin, which is then available for the reduction of CO2 in methanogens. In the case of Eha, the gradient of Na+/H+ produced through translocation coupled to ferredoxin oxidation can in be used to drive the phosphorylation of ADP via an ATP synthase complex, thereby representing one of the

  16. Establishment and Optimization of Puna Chicory Genetic Transformation System with Agrobacterium-mediated Method%农杆菌介导普那菊苣遗传转化体系的建立

    Institute of Scientific and Technical Information of China (English)

    张丽君; 程林梅; 杜建中; 李贵全; 孙毅

    2011-01-01

    以普那菊苣(Cichorium intybus L.cv.Puna)叶片为试验材料,接种于含不同激素浓度配比的MS培养基上进行愈伤组织、芽分化以及根再生的诱导,分析了不同激素浓度及其配比对愈伤组织诱导和芽分化以及根再生效果的影响.以已经建立的再生体系为基础,以农杆菌菌株LBA4404(含质粒pBin438- TaNHX2)侵染转化普那菊苣,探索普那菊苣高效遗传转化体系.结果表明:对外植体适宜的预培养时间为2~3 d,与农杆菌的共培养时间也应控制在2~3 d;侵染时间控制在8 min左右;卡那霉素(Km)阳性筛选的适宜选择浓度为60mg·L-1.乙酰丁香酮(AS)200 μmol·L-1是促进农杆菌转化的最佳浓度,200 W超声波处理、20次负压处理也可提高农杆菌转化率效果.26 mg·L- 1 Km是野生型普那菊苣苗能够存活的上限,头孢唑林钠和头孢噻肟钠在500~1000 nmg·L-1浓度范围内、羧苄青霉素300 mg·L-1和氨苄青霉素在40~60 mg·L-1浓度范围内均能较好的诱导出愈伤组织和芽.将来自小麦(Triticum aestivum)的Na+/H+逆向转运蛋白(vacuolar Na+/H+ exchanger or antiporter,简称NHX,NHE或NHA)导入普那菊苣;经抗生素筛选以及针对TaNHX2基因的PCR检测和Southern杂交分析,证明获得了28株转TaNHX2基因的普那菊苣植株.%Chicory (Cichorium intybus L. Cv. Puna) leaf segments from aseptic seedlings were used as experimental materials. The explants were inoculated onto the MS medium with various phytohormone combinations to induce callus formation, and bud and root regeneration. Effects of phytohormone concentrations and combinations on the induction of callus, buds and roots were analyzed. Agrobacterium tumefa-ciens LBA4404 (harboring plasmid pBin438-TaNHX2) was used to infect Puna Chicory explants based on the regeneration system that had been established for the high efficiency transformation of the cultivar. Result showed that both suitable pre-culture time and co

  17. Effects of arbuscular mycorrhizal fungus on net ion fluxes in the roots of trifoliate orange(Poncirus trifoliata) and mineral nutrition in seedlings under zinc contamination%丛枝菌根真菌对枳根净离子流及锌污染下枳苗矿质营养的影响

    Institute of Scientific and Technical Information of China (English)

    肖家欣; 杨慧; 张绍铃

    2012-01-01

    concentrations in the roots of plants in medium with 600 mg/kg of added zinc and phosphorus concentrations in roots of plants in medium with 300 mg/kg added zinc were higher in arbuscular myeorrhizal seedlings. Arbuscular myeorrhizal colonization had no significant effects on calcium concentrations in seedlings. Copper and phosphorus concentrations gradually decreased in both arbuscular mycorrhizal and non-arbuscular mycorrhizal seedlings with increasing zinc levels, which demonstrated that zinc levels in seedlings are negatively correlated with copper or phosphorus. With no added zinc, phosphorus, potassium, magnesium and copper uptake was promoted by arbuscular mycorrhizal fungus infection. Under zinc contamination, phosphorus, and copper uptake was still accelerated by mycorrhizal colonization. Thus, the effects of mycorrhizal colonization were not only related to the degree of zinc pollution, but were also correlated with the species of fungi and host plants. Additionally, net Ca2+ efflux atO ujn and 600 pjn, net H+ influx at 600 jjum, and net NO3 influx at 2400 u.m from the root tip of arbuscular mycorrhizal seedlings in medium with no added zinc were significantly higher than those of non-arbuscular mycorrhizal seedlings. These results suggest that mycorrhizal symbiosis activates Ca2+-ATPase, Ca2+/H+ antiporters and Noj/rT symporters in root plasma membranes. Nutrient uptake and stimulation of growth are mediated by arbuscular mycorrhizal fungi. Furthermore, the variations detected in arbuscular mycorrhizal roots for Ca2+, H+ and NO3 fluxes point to a significant involvement of the fungus.%盆栽实验研究了不同施Zn水平(0、300 mg/kg和600 mg/kg)下,接种丛枝菌根真菌Glomus intraradices对枳苗生长、Zn、Cu、P、K、Ca、Mg分布的影响,并采用非损伤微测技术测定分析了菌根化与非菌根化枳根净Ca2+、H+、NO3-离子流动态.结果表明:(1)在不同施Zn水平下,接种菌根真菌显著提高了枳苗地上部及根部鲜重;

  18. Uptake and Transport of Calcium in Plants%植物钙素吸收和运转

    Institute of Scientific and Technical Information of China (English)

    杨洪强; 接玉玲

    2005-01-01

    近年来,钙素在植物体内的吸收和运输研究主要集中在细胞和分子水平,但整株水平上的研究也同样重要.整株水平上的钙吸收和运输包括根细胞的钙吸收、钙离子横向穿过根系并进入木质部、在木质部运输、从木质部移出并进入叶片或果实及在叶片或果实中运转分配等环节,既经过质外体也穿越共质体.钙离子通道、Ca2+-ATP酶和Ca2+/H+反向转运器等参与根细胞的钙吸收.在钙离子横向穿根进入木质部的过程中,需要穿越内皮层和木质部薄壁细胞组织.根系内皮层凯氏带阻挡了Ca2+沿质外体途径由内皮层外侧向内侧的移动,部分Ca2+由此通过离子通道流进内皮层细胞而转入共质体并到达木质部薄壁细胞组织,而由木质部薄壁细胞组织进入中柱质外体可能需要Ca2+-ATP酶驱动;还有一些Ca2+由内皮层细胞运出,沿内皮层内侧的质外体途径进入木质部导管,并通过导管运向枝干.钙离子以螯合态的形式在枝干导管运输;水流速率是影响钙离子沿导管运输的关键因子.钙离子在果实和叶片中的运输和分配不仅通过质外体途径也通过共质体途径.%Recently, research on Ca2+ transport in plants has been focused on cellular and molecular level.But the uptake, transport and distribution are also very important for calcium to accomplish its function at whole plant level. There are many cells along the way of transport of Ca2+ from root to shoot, and Ca2+ passes either through the cytoplasm of cells linked by plasmodesmata (the symplast) or through the spaces between cells (the apoplast), which include Ca2+ uptake by root cells, Ca2+ transport from root cortex to and through the xylem, and then out of it into leaves or fruits. Ca2+ channels, Ca2+/H+ antiporter and Ca2+-ATPase play roles in the uptake and transport of Ca2+ in root cells. To be transported from root surface to xylem,Ca2+ needs to traverse endodermal cells and

  19. Introduction of TaNHX2 gene enhanced salt tolerance of transgenic puna chicory plants%导入TaNHX2基因提高了转基因普那菊苣的耐盐性

    Institute of Scientific and Technical Information of China (English)

    张丽君; 程林梅; 杜建中; 郝曜山; 王亦学; 李贵全; 孙毅

    2011-01-01

    transgenic puna chicory explants tolerated certain concentrations of NaCl up to 500 mmol/L, which was much higher than that of the wild type. Under 300 mmol/L NaCl stress , the transgenic seeds germinating rate, callus induction rate and bud regeneration rate were 2-4 times higher than the wide type. NaCl concentration of 500 mmol/L was the maximum amount for the survival of wide type puna chicory plantlets, under which transgenic explants could form calli, buds, and roots, and grow normally but the wild type explants could not.. We also measured the contents of malonaldehyde ( MDA) , and activities of peroxidase ( POD) and superoxide dismutase (SOD) in transgenic puna chicory seedlings and its wild counterpart. Under the stress of 500 mmol/L NaCl the MDA content was decreased by 1 -3 times, superoxide dismutase ( SOD) activity was increased by 2-3 times and peroxidase (POD) activity was increased by 1-3 times compared with those in wide type plants. The decrease of MDA content in transgenic puna chicory seedlings was negatively correlated and the increases of the enzyme activities in them were positively correlated with their tolerance to NaCl. Above all, we can make a conclusion that salt-tolerant transgenic puna chicory plants, could be obtained by introducing wheat vacuolar NaVH+ exchanger gene into by plant engineering approaches.%我国部分地区土地盐碱化的日益严重,对作物的生长和生态环境产生了显著影响,因此通过植物基因工程手段培育耐盐碱的转基因作物品种对改善作物的生存能力和生态环境,提高作物产量具有重要的意义.采用农杆菌介导法将来自小麦(Triticum aestivum Linn)的Na+/H+逆向转运蛋白的基因(vacuolar Na+/H+exchanger or antiporter,简称NHX,NHE或NHA),对普那菊苣(Cichorium intybus L.cv.Puna)植株进行了遗传转化.经抗生素筛选以及针对TaNHX2基因的PCR检测和Southern杂交分析,证明获得了28株转TaNHX2基因的普那菊苣植株.用不同浓度Na

  20. 大豆耐盐相关基因GmNcl1的序列单倍型及表达分析%Haplotypes and Expression Analysis of Salt-Tolerant Gene GmNcl1 in Soybean

    Institute of Scientific and Technical Information of China (English)

    赫卫; 刘林; 关荣霞; 邱丽娟

    2014-01-01

    [Objective]The objective of the study is to confirm sequence polymorphisms of GmNcl1 gene and its differential pattern in stress. [Method]Amino acid sequence of GmNCl1 was compared by BLASTP to find homologous genes. Fifty soybean varieties were used for the sequencing of GmNcl1 promoter and gene, and indicating of salt tolerance, and haplotype cluster and phylogenetic tree were analyzed by MAGE, looking for the correlation between salt-tolerance and sequence of GmNcl1. The expression pattern of GmNcl1 by real time PCR analysis in roots of salt-tolerant cultivar Tiefeng 8 and salt-sensitive cultivar 85-140 under a series of treatments including the protein inhibitors, pH, ABA treatments and salt, alkali and drought stress. [Result]GmNcl1 was homologous with Na +(K+)/H+exchanger CHX. Sixteen single nucleotide polymorphism loci and two InDel loci were found in GmNcl1 gene sequence, including one G/A (salt-sensitive/salt-tolerant) base change in exon 3, which caused the nonsynonymous mutation from alanine to threonine. Eighteen polymorphism loci composed into fourteen haplotypes, of which there were three salt-tolerant haplotypes and eleven salt-sensitive haplotypes. The haplotypes, GAGATATTC (salt-tolerant)/TTT---- CT (salt-sensitive) composed of six loci were high-effectively distinguished between the salt tolerance and sensitivity. In the roots of soybean seedling, the expression of GmNcl1 gene was increased under the Na+/H+ antiporter inhibitor, amiloride, and unchanged under Ca2+-ATPase inhibitor, sodium vanadate. The expression of GmNcl1 was increased under alkaline pH, and showed fluctuations under acidic pH. The expression of GmNcl1 was also induced by ABA, and up-regulated by salt, alkali and drought stress. The greater stress, the more up-regulated expression of GmNcl1. Expression of GmNcl1 showed a similar trend in Tiefeng 8 and 85-140 under salt stress, but there was the greater increase in 85-140 than Tiefeng 8.[Conclusion]GmNcl1 was highly similar with